The epithelium and immune compartment in the intestine are constantly exposed to a fluctuating external environment. Defective communication between these compartments at this barrier surface underlies susceptibility to infections and chronic inflammation. Environmental factors play a significant, but mechanistically poorly understood, role in intestinal homeostasis. We found that regeneration of intestinal epithelial cells (IECs) upon injury through infection or chemical insults was profoundly influenced by the environmental sensor aryl hydrocarbon receptor (AHR). IEC-specific deletion of Ahr resulted in failure to control C. rodentium infection due to unrestricted intestinal stem cell (ISC) proliferation and impaired differentiation, culminating in malignant transformation. AHR activation by dietary ligands restored barrier homeostasis, protected the stem cell niche, and prevented tumorigenesis via transcriptional regulation of of Rnf43 and Znrf3, E3 ubiquitin ligases that inhibit Wnt-β-catenin signaling and restrict ISC proliferation. Thus, activation of the AHR pathway in IECs guards the stem cell niche to maintain intestinal barrier integrity.

Utilizing mouse models as well as intestinal organoid cultures, we found that AHR acts directly on IECs to restrict excessive proliferation of ISCs through regulation of Rnf43 and Znrf3 expression. As a consequence, Ahr deficiency in IECs compromised the ability of intestinal stem cells to repair and differentiate in response to tissue damage, leading to profound effects on resistance to infection and formation of colorectal cancer. These defects could be repaired by exposure to dietary AHR ligands in Villin Cre R26 LSL-Cyp1a1 mice, which have an intact Ahr, whereas Villin Cre Ahr fl/fl mice lacking Ahr in IECs could not be rescued.

The rapid regeneration of the intestinal epithelium is a highly coordinated process that is fueled by the proliferation of LGR5-expressing intestinal stem cells (ISCs) located at the bottom of each crypt (). The Wnt-β-catenin pathway is crucial for the proliferation and maintenance of ISCs and is tightly regulated by E3 ubiquitin ligases RNF43 and ZNRF3, which target WNT receptors for degradation (). Aberrant Wnt-β-catenin activation is a hallmark of colorectal cancers, highlighting the importance of this pathway in intestinal homeostasis ().

The environmental sensor AHR is highly expressed at barrier sites such as the skin, lung, and gut. Although the AHR was originally described as a receptor for dioxin and other xenobiotics, it is now clear that physiological AHR ligands such as dietary components and tryptophan metabolites () serve to drive beneficial functions of AHR in the immune system as well as in non-hematopoietic cells. In the context of intestinal homeostasis, Ahr deficiency has detrimental consequences associated with loss of intraepithelial lymphocytes and ILC3 and absence of IL-22 production (). An important aspect of AHR activation is the necessity for negative feedback regulation as prolonged stimulation has detrimental effects (). AHR activation induces expression of a family of cytochrome P450 enzymes (CYP1 family), which metabolize AHR ligands, thereby terminating the stimulus (). In support of this, we recently showed that selective overexpression of CYP1A1 in IECs (VillinR26mice) acts as a metabolic roadblock leading to insufficient AHR ligand supply to mucosal immune cells, thereby compromising ILC3- and Th17 cell-mediated immunity to enteric infection (). However, the expression of CYP1A1 along the crypt-villus axis in response to dietary AHR ligand exposure strongly suggests a role for AHR in IEC function beyond regulation of ligand supply to the host.

The intestinal epithelium constitutes a single-layer barrier that separates the mucosal immune system from trillions of commensal bacteria. Interactions between intestinal epithelial cells (IECs), immune cells, and the microbiota underlie the maintenance of intestinal homeostasis in steady state as well as upon perturbation by infection. The integrity of the intestinal barrier has substantial implications for health even beyond the intestine. Numerous genetic loci are known to contribute to the development of inflammatory bowel diseases such as Crohn’s disease or ulcerative colitis and the genetic susceptibility for disease is well documented (). However, environmental factors including smoking, diet, and use of antibiotics play a significant role in the etiology of intestinal disorders, and the molecular mechanisms underlying their impact remain poorly defined.

We next investigated whether dietary supplementation with AHR ligands after the onset of tumorigenesis affects tumor progression. VillinR26mice were subjected to AOM followed by DSS on standard chow diet. Four weeks later when four out of five mice had developed at least one tumor (data not shown), mice received the second dose of DSS and were placed on either purified control diet or purified diet supplemented with I3C ( Figure 6 A); tumors were assessed 6 weeks later. Whereas all mice on the purified control diet developed tumors ( Figures 6 B and 6C), two out of nine mice on the I3C diet did not develop tumors ( Figure 6 D) and the remaining mice had reduced numbers of tumors—mainly low-grade adenomas ( Figure 6 E). Thus, normalization of AHR signaling even after tumor induction has beneficial effects in reducing tumor load and severity, indicating the therapeutic potential of dietary AHR ligand I3C.

(A) Villin-creR26Cyp1a1 were injected with 10 mg/kg of AOM followed with one cycle of 1% DSS on standard chow diet. For the second cycle of DSS, mice were put either on a purified diet or I3C diet until the end of the experiment.

Thus, normalization of AHR signaling in response to dietary or natural ligands restored the regulation of the Wnt-β-catenin pathway, allowing repair of DSS-induced tissue damage by supporting epithelial cell differentiation and protection from inflammation-induced tumorigenesis.

Here we fed VillinR26mice with a purified control diet or purified diet supplemented with I3C for 2 weeks prior to as well as during treatment with AOM/DSS. Exposure to I3C diet for 2 weeks prior to AOM/DSS treatment sufficed to correct the expression of Znrf3 and Rnf43 ( Figure 5 A) in an AHR-dependent manner, as mice lacking Ahr in IEC did not upregulate these tumor suppressors ( Figure S3 B). Importantly, wild-type mice also expressed increased levels of Znrf3 and Rnf43 when exposed to I3C diet compared to control diet ( Figure S3 A). In line with reinstatement of negative feedback control of Wnt-β-catenin signaling by enhanced expression of Znrf3 and Rnf43 upon I3C diet exposure, we observed decreased expression of WNT target genes Axin2, cMyc, and Ephb2 in VillinR26mice. Wild-type littermate control mice fed with control purified diet showed a low level of tumor burden ( Figure 5 B) in contrast to what we had seen on our standard chow diet where wild-type mice never developed tumors ( Figure 4 A). This probably reflects the reduced availability of AHR ligands in the purified control diet as compared to standard chow diet. Strikingly, addition of I3C to purified diet reduced tumor formation to levels seen in wild-type mice and decreased β-catenin expression in the intestine and the tumor ( Figure 5 C). This effect was dependent on AHR activation in IECs as I3C diet did not reduce tumor formation in VillinAhrmice ( Figure 5 B). Furthermore, dietary substitution with AHR ligands restored the defect in epithelial cell differentiation ( Figure 5 D) and reduced the hyperproliferation of crypt stem cells ( Figure 5 E) in line with our findings in organoid cultures ( Figures 2 B and 2C).

Error bars, mean + SEM.p < 0.05,p < 0.01,p < 0.0001, as calculated by unpaired t test and two-way ANOVA with Tukey post-test. See also Figures S3 A and S3B.

(C) Representative images of hematoxylin and eosin (H&E) and β-catenin of colon tumors in Villin Cre R26 LSL-Cyp1a1 fed purified or I3C diet. Scale bars, 50 μm.

(A) qPCR analysis of WNT negative regulators (Znrf3 and Rnf43) and WNT target genes (Axin2, cMyc, and Ephb2) in the colon of Villin Cre R26 LSL-Cyp1a1 mice fed purified or I3C diet (n = 5).

The notion that a diet enriched in green vegetables reduces the risk of cancer formation is widely distributed, although scientific evidence from several clinical trials remains inconsistent (). In particular, there is no consensus for a molecular mechanism explaining the phenomenon of an inverse relationship between plant-derived food intake and cancer development. Some of the strongest endogenous AHR ligands are derived from phytochemicals such as indole-3-carbinol (I3C) that is converted to the high-affinity AHR ligand ICZ by exposure to stomach acid (). We previously showed that dietary application of I3C could cure the extreme sensitivity to enteric infection of VillinR26mice ().

The combination of enhanced stem cell proliferation and subclinical inflammation is commonly associated with malignant transformation and colorectal cancer. Indeed, VillinAhrand VillinR26mice, when exposed to the mutagen azoxymethane (AOM), developed large tumors within 4 months of AOM application, whereas no tumors were observed in wild-type mice ( Figure 3 F). In order to accelerate the process of tumorigenesis, we applied the widely used AOM/DSS (dextran sulfate sodium) model in which one dose of AOM was followed by two cycles of DSS. As Ahr-deficient mice are highly sensitive to DSS (), we had to reduce the dose to 1% DSS, which is suboptimal for wild-type mice. Consequently, no tumors were observed in wild-type mice under this treatment regime, while both VillinAhrand VillinR26mice developed numerous tumors throughout the colon ( Figures 4 A–4C, top row), ranging in severity from low-grade adenoma to adenocarcinoma ( Figure 4 D). In agreement with published data on increased levels of β-catenin in Ahrmice (), both strains of mice with dysregulated AHR in IECs had substantially increased β-catenin expression in the intestine ( Figure 4 E, bottom row). We next investigated the underlying mechanism for Wnt-β-catenin pathway dysregulation in our mice. We focused on the tumor suppressors RNF43 and ZNRF3, which are transmembrane E3 ubiquitin ligases expressed in LGR5stem cells that regulate WNT signals by targeting WNT receptors for degradation in an R-spondin-sensitive manner (). Both Rnf43 and Znrf3 were expressed at significantly lower levels in epithelium from VillinAhrand VillinR26mice, while WNT target genes such as Axin2, cMyc, and Ephb2 were expressed at higher levels in steady-state conditions ( Figure S1 A) and following AOM/DSS administration ( Figure 4 F). ChIP qPCR of intestinal epithelial cells established Rnf43 as a direct target of AHR, suggesting that this tumor suppressor, which acts in a complex with ZNRF3, is transcriptionally regulated by AHR ( Figure 4 G). Similar results were obtained in colon organoids ( Figure S2 B). Thus, dysregulated feedback signaling in the Wnt-β-catenin pathway causes increased expansion of crypt stem cells in mice with compromised physiological AHR signaling. In further corroboration of defective feedback regulation, organoids from Ahr-deficient strains grew and survived in the absence of R-Spondin1, as shown for Rnf45/Znrf3 mutants (), whereas wild-type organoids died upon withdrawal of R-spondin1 ( Figure S2 C).

(E) Representative images of hematoxylin and eosin (H&E) (top) and β-catenin staining of whole intestine (middle) and focus (bottom) of the indicated mice 60 days after injection of azoxymethane. Scale bars, 50 μm.

Proliferation of LGR5stem cells, measured by Ki67 expression, was already significantly increased in young Ahr-deficient mice compared to littermate controls ( Figure 3 A). However, a numeric increase in LGR5stem cells manifested only in older mice with a compromised AHR pathway ( Figure S1 ). Since we were not able to maintain Ahr-deficient Lgr5reporter mice viable for long enough, we used mice heterozygous for Ahr deletion, which were partially affected. With respect to epithelial differentiation, we did not observe any significant difference in young mice (aged between 5 and 8 weeks) ( Figure 3 B), while Muc2 and Car4 expression were strongly reduced in older mice aged between 14 and 16 weeks ( Figure 3 C). Likewise, IL-6 levels were not different at steady state in young mice, but were elevated in older mice ( Figures 3 D and 3E).

(F) Number of colon tumors in WT (n = 8), Villin Cre Ahr fl/fl (n = 8), and Villin Cre R26 LSL-Cyp1a1 (n = 8) mice injected with 10 mg/kg of azoxymethane once a week for 6 weeks. Representative image of colon in mice 22 weeks after the first azoxymethane injection.

(B and C) qPCR analysis of the goblet cell marker Muc2 and enterocyte marker Car4 from sorted EpCam + cells (WT, n = 6; Villin Cre Ahr fl/fl , n = 6; Villin Cre R26 LSL-Cyp1a1 , n = 6) in young (5 to 9 weeks) and old (14 to 16 weeks) mice.

(A) Flow cytometry analysis of Lgr5 and Ki-67 expression in EpCam + CD45 − cells and absolute number of Lgr5 + Ki-67 + cells at steady state from 5- to 8-week-old mice.

For this we made use of a mouse strain in which LGR5crypt stem cells are marked with GFP, Lgr5).

Thus far, our data indicate that dysregulated AHR in IECs leads to aberrant inflammation and enhanced stem cell proliferation. To determine whether this phenotype is apparent under steady-state conditions, we compared stem cell proliferation, epithelial cell differentiation, and basic inflammatory tone in untreated Villin Cre Ahr fl/fl and Villin Cre R26 LSL-Cyp1a1 mice.

The intestinal epithelium continuously renews itself from crypt stem cells that differentiate into short-lived specialized epithelial subtypes (). This is a highly regulated process that depends on orchestrated Wnt-β-catenin signaling in crypt stem cells. In order to directly investigate the role of AHR in the process of crypt stem cell proliferation and differentiation, we generated colon organoids (). Comparing organoids from wild-type mice with mice exhibiting dysregulated AHR signaling (R26) and mice lacking Ahr (Ahr) allowed us to investigate the role of AHR pathway activation in stem cells. Colon organoids from Ahror R26crypts showed increased proliferation, as indicated by higher uptake of EdU ( Figures 2 A and 2B ). The proliferative response was normalized in R26but not Ahrorganoids by addition of AHR ligands FICZ (6-formylindolo[3,2-b]carbazole) or ICZ (indolo[3,2-b]carbazole) into the culture medium ( Figure 2 C). As expected, withdrawal of WNT in wild-type stem cell organoid cultures led to an increase in expression of the goblet cell marker Muc2 ( Figure 2 D, left, white bars) and enterocyte marker Car4 ( Figure 2 E, right, white bars). However, organoids with dysregulated AHR ( Figures 2 D and 2E, black and gray bars) had substantially lower expression of Muc2 and Car4, indicating compromised differentiation to goblet and enterocyte fate. Nevertheless, addition of AHR ligands restored goblet cell and enterocyte differentiation in organoids derived from R26(gray bars) but not of Ahr(black bars) mice to wild-type levels. This indicates that appropriate stimulation of the AHR pathway is required for epithelial cell differentiation from crypt stem cells.

Data represent pooled results of at least two independent experiments (n = 6). Error bars, mean + SEM. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001 as calculated by one-way and two-way ANOVA with Tukey post-test.

(D and E) qPCR analysis of the goblet cell marker Muc2 and enterocyte marker Car4 in SC or differentiated (diff) organoids treated or not with 5 nM FICZ for 4 days.

C. rodentium is an attaching effacing pathogen that causes IEC apoptosis () and necessitates replenishment of damaged IECs for maintenance of barrier integrity and repair processes. Resistance to C. rodentium infection varies between different strains of mice and susceptible strains are characterized by aberrant R-spondin 2 (RSPO2)-mediated Wnt-β-catenin activation which causes excessive ISC proliferation and poor differentiation into epithelial subtypes (). Compared with Ahrmice, infected VillinAhrmice had significantly lower expression of Muc2 and Car4 ( Figure 1 H), with corresponding reduction of goblet cells ( Figures 1 I and 1J). This indicates a defective repair process following infection, which is likely to contribute to the severe barrier defect that leads to dissemination of bacteria in this strain.

Given the profound impact of Ahr deficiency on intestinal homeostasis, we set out to define whether Ahr deficiency in hematopoietic versus non-hematopoietic cells affects mice differently during infection with the intestinal pathogen Citrobacter rodentium. For this purpose, we set up bone marrow chimeras, transferring bone marrow either from Ahr-deficient donors into wild-type hosts (Ahr→ WT) or from wild-type B6 donors into Ahr-deficient hosts (WT → Ahr). Although both types of chimeras eventually succumbed to infection, mice with Ahr deficiency in the non-hematopoietic compartment exhibited accelerated mortality ( Figure 1 A), suggesting that AHR function is particularly important in IECs. We subsequently crossed mice with a floxed Ahr locus to Villin-Cre mice to restrict Ahr deficiency to IECs (referred to as VillinAhr). These mice had an intact immune response to C. rodentium infection, with similar numbers of colonic ILC3 and Th17 cells as WT mice ( Figure 1 B) and comparable or even enhanced expression of IL-22 and its target genes Reg3g and S100a9 ( Figures 1 C and 1D). Nevertheless, infection of VillinAhrmice with C. rodentium led to deep penetration of bacteria to the intestinal crypts, bacterial dissemination to the liver and spleen ( Figures 1 E and 1F), and rapid onset of mortality ( Figure 1 G). This indicates that AHR activation in immune cells is not sufficient to protect against C. rodentium infection and that AHR signaling in IECs serves a cell-autonomous role in promoting epithelial barrier function in an IL-22-independent manner.

Discussion

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Bradfield C.A. Aryl hydrocarbon receptor-dependent liver development and hepatotoxicity are mediated by different cell types. b allele (data not shown). AHR is widely expressed in the intestine and absence of AHR has been shown to result in loss of several immune cell types or their functional activities (). However, as we showed here, Ahr deficiency profoundly affected intestinal homeostasis through direct activity on intestinal epithelial cells. Our previous research established an important role of the AHR pathway in IECs as “gatekeeper” for the supply of ligands to intestinal immune cells (). However, we now found additional IEC-intrinsic AHR functions that did not involve mucosal immune cells such as ILC3 or Th17 cells and were independent of their production of IL-22. This was borne out of experiments with mice lacking the AHR pathway in IECs (VillinAhr), which have a normal intestinal immune compartment and no impairment in IL-22 production, but nevertheless were as susceptible to infection with C. rodentium as mice with overactive CYP1A1 in IECs (VillinR26). Instead, we identified an important role for AHR in controlling the response of crypt stem cells to WNT signals that affect proliferation and the differentiation of epithelial subsets. Repair of damaged epithelium is a crucial feature for resistance to intestinal infection with pathogens that cause epithelial damage, such as C. rodentium and replacement of mucus-producing goblet cells is vital in order to prevent dissemination of bacteria. The failure to achieve this in a timely manner was clearly evident in VillinAhror VillinR26mice that both succumbed to severe bacterial dissemination and were also highly susceptible to chemically induced epithelial damage by DSS. A previous publication demonstrated a link between susceptibility to C. rodentium infection and RSPO2, a secreted G-coupled receptor that enhances Wnt-β-catenin signaling (), but the underlying mechanisms that regulate WNT signaling in susceptible strains remained uncharacterized. Interestingly, mouse strains that are susceptible to C. rodentium, such as FVB and AKR, express the low-affinity Ahrallele as do mice with a floxed Ahr locus () and these are substantially more affected by C. rodentium infection than C57BL/6 mice with the high-affinity Ahrallele (data not shown).

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Stockinger B. Feedback control of AHR signalling regulates intestinal immunity. CreAhrfl/fl mice. Thus, IL-6 rather than IL-22 is likely to provide the inflammatory setting in these mice. Aberrant stem cell proliferation was evident at steady state in older mice with a dysregulated AHR pathway, suggesting that the intestinal barrier is compromised to some extent prior to any challenge. This would explain why such mice expressed elevated levels of IL-6 that can promote malignant progression in colon cancer (). IL-22 that has also been linked to colon cancer in mice and humans () is elevated in VillinAhrmice, probably due to increased levels of AHR ligands accessible to mucosal immune cells, as Ahr-deficient IECs do not express CYP1A1. However, this cytokine is profoundly depleted in VillinR26mice () yet they are similarly prone to tumorigenesis as VillinAhrmice. Thus, IL-6 rather than IL-22 is likely to provide the inflammatory setting in these mice.

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Clevers H. Tumour suppressor RNF43 is a stem-cell E3 ligase that induces endocytosis of Wnt receptors. + stem cells. It is conceivable that the E3 ubiquitin ligase activity of AHR contributed to β-catenin accumulation in our study. However, we observed a selective defect in induction of Znrf3 and Rnf43 in IECs with dysregulated AHR, while expression of other WNT pathway targets such as Axin2, cMyc, and Ephb2 was enhanced. Thus, our findings strongly support a model by which AHR modulates β-catenin levels through transcriptional induction of negative regulators of the Wnt-β-catenin pathway. Our data further support the notion that WNT responsiveness of intestinal stem cells is normally restrained by physiological AHR signals, and restoration of the AHR response in VillinCreR26LSL-Cyp1a1 mice via dietary supplementation reset the feedback control of Wnt-β-catenin signaling in crypt stem cells and promoted their differentiation, resulting in protection from tumorigenesis. The progression from inflammation to tumorigenesis is well established () and our mice readily developed mixed colon adenomas/adenocarcinomas in the standard AOM/DSS model in line with previous data in global Ahrmice () or even with the mutagen AOM alone. In vitro studies on cell lines proposed that AHR functions as part of an E3 ubiquitin ligase complex that serves to ubiquinate β-catenin and target it for degradation (). Our results provide evidence that Wnt-β-catenin pathway dysregulation in Ahr-deficient IECs occurred upstream of β-catenin degradation, affecting the response to WNT signals themselves through the expression of the two related E3 ubiquitin ligases (ZNRF3 and RNF43) that degrade the Wnt frizzled receptors () in LGR5stem cells. It is conceivable that the E3 ubiquitin ligase activity of AHR contributed to β-catenin accumulation in our study. However, we observed a selective defect in induction of Znrf3 and Rnf43 in IECs with dysregulated AHR, while expression of other WNT pathway targets such as Axin2, cMyc, and Ephb2 was enhanced. Thus, our findings strongly support a model by which AHR modulates β-catenin levels through transcriptional induction of negative regulators of the Wnt-β-catenin pathway. Our data further support the notion that WNT responsiveness of intestinal stem cells is normally restrained by physiological AHR signals, and restoration of the AHR response in VillinR26mice via dietary supplementation reset the feedback control of Wnt-β-catenin signaling in crypt stem cells and promoted their differentiation, resulting in protection from tumorigenesis.

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et al. High-fat diet enhances stemness and tumorigenicity of intestinal progenitors. The impact of nutrition on tumorigenesis is widely documented () and in particular a diet high in fat was proposed to augment intestinal stem cell numbers and—in the context of Apcmutation—promote susceptibility to cancerogenesis (). Conversely, a diet rich in vegetables and phytochemicals is thought to be beneficial. It remains to be investigated whether experimental high-fat diets contain similar levels of phytochemicals generating AHR ligands as control diets and it might be worth considering that the deleterious effects of high-fat diet on cancerogenesis could have been partly due to lack of phytochemicals rather than increased fat content.

We found here that the purified control diet, which is used as comparison for I3C supplemented diet, appeared to contain reduced levels of AHR ligands and as a consequence produced some features of Ahr deficiency in wild-type mice. As an example, wild-type mice fed purified control diet developed some tumors within 10 weeks of AOM/DSS, whereas this was not observed when they were fed the standard chow diet.

Díaz-Díaz et al., 2016 Díaz-Díaz C.J.

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Chen C.S. Indole-3-carbinol as a chemopreventive and anti-cancer agent. CreR26LSL-Cyp1a1 mice corrected the expression of Rnf43 and Znrf3 in an AHR-dependent manner, thus normalizing the negative feedback regulation of the Wnt-β-catenin pathway, which resulted in significantly reduced or absent tumorigenesis. Furthermore, dietary supplementation with AHR ligands in mice with a dysregulated AHR pathway normalized crypt stem cell hyperproliferation and the differentiation process to goblet cells and enterocytes. Dietary supplementation with AHR ligands also worked in a therapeutic setting after onset of tumorigenesis, resulting in significantly reduced numbers of tumors with a more benign phenotype. There are numerous reports on I3C as a cancer-preventive agent (). However, consensus on the mode of action and mechanistic details of how nutrition interfaces with the inflammatory process are missing. We have provided evidence that supplementation with dietary I3C to VillinR26mice corrected the expression of Rnf43 and Znrf3 in an AHR-dependent manner, thus normalizing the negative feedback regulation of the Wnt-β-catenin pathway, which resulted in significantly reduced or absent tumorigenesis. Furthermore, dietary supplementation with AHR ligands in mice with a dysregulated AHR pathway normalized crypt stem cell hyperproliferation and the differentiation process to goblet cells and enterocytes. Dietary supplementation with AHR ligands also worked in a therapeutic setting after onset of tumorigenesis, resulting in significantly reduced numbers of tumors with a more benign phenotype.