Intestinal Adenomas Research Articles

Abstract 4092: Long-lived Dclk1+ cells serve as colon cancer initiating cells

Abstract Introduction: In the rapidly proliferating gastrointestinal epithelium, long-lived tissue stem cells, characterized by multipotentiality and self-renewing ability, remain the most likely cellular origin for cancer. Previous studies have suggested actively cycling Lgr5+ stem cells are one cellular origin for intestinal adenomas. However, it has also recently been suggested that non-Lgr5+ cells may also contribute to the cellular origin of colorectal cancer. Doublecortin-like kinase 1 (Dclk1) protein is a gastrointestinal tuft cell marker that has been proposed to identify quiescent stem cells and cancer stem cells that sustain tumor growth. The role of Dclk1+ tuft cells within the gastrointestinal epithelium and their potential to function as cancer-initiating cells, however, remain poorly understood. Here, we used Dclk1(BAC)-CreERT;ROSA26rLacZ mice crossed to APCff mice to examine whether Dclk1+ cells contribute to colonic tumor formation. Methods: To recapitulate the endogenous expression pattern of Dclk1, we used a BAC strategy and generated a transgenic mouse with a Tamoxifen inducible Cre under the control of the Dclk1 promoter (Dclk1-BAC-Cre-ERT). Dclk1-CreERT mice were crossed to both ROSA26rLacZ and APCff mice and treated with tamoxifen (6 mg p.o.). Dclk1+ lineage tracing was assessed by X-gal staining. To examine the contribution of the Dclk1+ cells to colonic tumorigenesis, we treated Dclk1(BAC)-CreERT;ROSA26rLacZ; APCff mice with DSS (3% in drinking water) to induce colitis. Mice were sacrificed 3-4 months after DSS weeks to assess for tumor formation and X-gal staining performed to stain for the Dclk1+ cell lineage. Results: Dclk1-BAC-CreERT genetic lineage tracing demonstrated that a subpopulation of Dclk1+ cells is extremely long-lived and shows rare stem cell abilities. Moreover, genetic ablation reveals a pivotal role for Dclk1+ tuft cells in the response to intestinal and colonic injury. Surprisingly, conditional loss of APC in Dclk1+ cells is not sufficient to drive colonic carcinogenesis, whereas induction of DSS colitis in Dclk1-CreERT; APCflox/flox mice leads to the development of poorly differentiated colonic adenocarcinoma. Importantly, colonic tumor formation occurs even when the onset of colitis is delayed for up to 3 months after APC loss in Dclk1+ cells. Conclusions. Thus, our data define a novel intestinal Dclk1+ tuft cell population that is long-lived, quiescent and important for intestinal homeostasis and regeneration. Long-lived Dclk1+ cells maintain quiescence even following oncogenic mutation, but are activated by tissue injury and can serve as a potent cellular origin of colon cancer. Citation Format: Samuel Asfaha, Christoph Benedikt Westphalen, Yoku Hayakawa, Yoshihiro Takemoto, Dana J. Lukin, Wanda Setlik, Helen Remotti, Ashlesha Muley, Xiaowei Chen, Randal May, Courtney W. Houchen, James G. Fox, Michael D. Gershon, Michael Quante, Timothy Wang. Long-lived Dclk1+ cells serve as colon cancer initiating cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4092. doi:10.1158/1538-7445.AM2014-4092

Abstract 2975: The pluripotency factor LIN28 promotes colorectal tumorigenesis and tumor progression

Abstract LIN28A and LIN28B are RNA-binding protein paralogs expressed in embryonic stem cells and in early developing tissues. Through blocking the terminal processing of let-7 miRNAs and regulating the translation of a number of mRNA binding targets, LIN28 controls an array of targets that mediate proliferation, pluripotency, differentiation and metabolism. While highly expressed in the early embryos, the levels of LIN28A/B are downregulated as differentiation and development proceed. However, aberrant overexpression of LIN28 is often observed in human malignancies especially those with poor prognosis. We have demonstrated that LIN28 promotes cellular transformation in vitro, yet it is unknown how LIN28 regulates tumorigenesis in vivo. Here, we investigate the role of LIN28 in colorectal cancer (CRC) in humans and in mouse models. We first investigated the status of LIN28A/B in 1000 CRC patients and observed 35-60% of the cancer samples express LIN28A and/or LIN28B. Furthermore, we generated intestinal-specific LIN28 overexpressing mice, and observed intestinal adenoma as well as invasive adenocarcinoma that highly resemble the histology of human CRCs. Moreover, in Apcmin/+ animals, additional LIN28 expression significantly increased tumor incidence and accelerated disease onset, as their tumors are poorly differentiated and more invasive compared with Apcmin/+ tumors. Together, our study shows that LIN28 drives intestinal tumorigenesis and further promotes tumor progression in vivo. CRC is one of the major contributors to cancer-related mortality; however, current mouse models seldom progress to later stages of the disease, which greatly impedes efforts to developing more effective therapies. Based on the knowledge gained from this research, we hope to provide novel insights into designing therapeutics targeting late-stage CRC. Citation Format: Sarah Schwitalla, Ho-Chou Tu, Srinivas Viswanathan, Hao Zhu, Zhirong Qian, Samar Shah, Shuji Ogino, George Q. Daley. The pluripotency factor LIN28 promotes colorectal tumorigenesis and tumor progression. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2975. doi:10.1158/1538-7445.AM2014-2975

Tripartite interactions between Wnt signaling, Notch and Myb for stem/progenitor cell functions during intestinal tumorigenesis

Deletion studies confirm Wnt, Notch and Myb transcriptional pathway engagement in intestinal tumorigenesis. Nevertheless, their contrasting and combined roles when activated have not been elucidated. This is important as these pathways are not ablated but rather are aberrantly activated during carcinogenesis. Using ApcMin/+ mice as a source of organoids we documented their transition, on a clone-by-clone basis, to cyst-like spheres with constitutively activated Wnt pathway, increased self-renewal and growth and reduced differentiation. We then looked at this transition when Myb and/or Notch1 are activated. Activated Notch promoted cyst-like organoids. Conversely growth and propagation of cyst-like, but not normal organoids were Notch-independent. Activated Myb promoted normal, but not cyst-like organoids. Interestingly the Wnt, Notch and Myb pathways were all involved in regulating the expression of the intestinal stem cell (ISC) gene Lgr5 in organoids, while ISC gene and Notch target Olfm4 was dominantly repressed by Wnt. These findings parallel mouse intestinal adenoma formation where Notch promoted the initiation, but not growth, of Wnt-driven Olfm4-repressed colon tumors. Also Myb was essential for colon tumor initiation and collateral mouse pathologies. These data reveal the complex interplay and hierarchy of transcriptional networks that operate in ISCs and uncover a shift in pathway-dependencies during tumor initiation.

DCLK1 facilitates intestinal tumor growth via enhancing pluripotency and epithelial mesenchymal transition.

Doublecortin-like kinase 1 (Dclk1) is overexpressed in many cancers including colorectal cancer (CRC) andit specifically marks intestinal tumor stem cells. However, the role of Dclk1 in intestinal tumorigenesis in Apc mutant conditions is still poorly understood. We demonstrate that Dclk1 expression and Dclk1+ cells are significantly increased in the intestinal epithelium of elderly ApcMin/+ mice compared to young ApcMin/+ mice and wild type mice. Intestinal epithelial cells of ApcMin/+ mice demonstrate increased pluripotency, self-renewing ability, and EMT. Furthermore, miRNAs are dysregulated, expression of onco-miRNAs are significantly increased with decreased tumor suppressor miRNAs. In support of these findings, knockdown of Dclk1 in elderly ApcMin/+ mice attenuates intestinal adenomas and adenocarcinoma by decreasing pluripotency, EMT and onco-miRNAs indicating that Dclk1 overexpression facilitates intestinal tumorigenesis. Knocking down Dclk1 weakens Dclk1-dependent intestinal processes for tumorigenesis. This study demonstrates that Dclk1 is critically involved in facilitating intestinal tumorigenesis by enhancing pluripotency and EMT factors in Apc mutant intestinal tumors and it also provides a potential therapeutic target for the treatment of colorectal cancer.

Prox1 Promotes Expansion of the Colorectal Cancer Stem Cell Population to Fuel Tumor Growth and Ischemia Resistance

Colorectal cancer (CRC) initiation and growth is often attributed to stem cells, yet little is known about the regulation of these cells. We show here that a subpopulation of Prox1-transcription-factor-expressing cells have stem cell activity in intestinal adenomas, but not in the normal intestine. Using in vivo models and 3D ex vivo organoid cultures of mouse adenomas and human CRC, we found that Prox1 deletion reduced the number of stem cells and cell proliferation and decreased intestinal tumor growth via induction of annexin A1 and reduction of the actin-binding protein filamin A, which has been implicated as a prognostic marker in CRC. Loss of Prox1 also decreased autophagy and the survival of hypoxic tumor cells in tumor transplants. Thus, Prox1 is essential for the expansion of the stem cell pool in intestinal adenomas and CRC without being critical for the normal functions of the gut.

Intestinal PTGS2 mRNA levels, PTGS2 gene polymorphisms, and colorectal carcinogenesis.

Background & AimsInflammation is a major risk factor for development of colorectal cancer (CRC). Prostaglandin synthase cyclooxygenase-2 (COX-2) encoded by the PTGS2 gene is the rate limiting enzyme in prostaglandin synthesis and therefore plays a distinct role as regulator of inflammation.Methods PTGS2 mRNA levels were determined in intestinal tissues from 85 intestinal adenoma cases, 115 CRC cases, and 17 healthy controls. The functional PTGS2 polymorphisms A-1195G (rs689466), G-765C (rs20417), T8473C (rs5275) were assessed in 200 CRC cases, 991 adenoma cases and 399 controls from the Norwegian KAM cohort.Results PTGS2 mRNA levels were higher in mild/moderate adenoma tissue compared to morphologically normal tissue from the same individual (P<0.0001) and (P<0.035) and compared to mucosa from healthy individuals (P<0.0039) and (P<0.0027), respectively. In CRC patients, PTGS2 mRNA levels were 8–9 times higher both in morphologically normal tissue and in cancer tissue, compared to healthy individuals (P<0.0001). PTGS2 A-1195G variant allele carriers were at reduced risk of CRC (odds ratio (OR) = 0.52, 95% confidence interval (95% CI): 0.28–0.99, P = 0.047). Homozygous carriers of the haplotype encompassing the A-1195G and G-765C wild type alleles and the T8473C variant allele (PTGS2 AGC) were at increased risk of CRC as compared to homozygous carriers of the PTGS2 AGT (A-1195G, G-765C, T8473C) haplotype (OR = 5.37, 95% CI: 1.40–20.5, P = 0.014). No association between the investigated polymorphisms and PTGS2 mRNA levels could be detected.ConclusionHigh intestinal PTGS2 mRNA level is an early event in colorectal cancer development as it occurs already in mild/moderate dysplasia. PTGS2 polymorphisms that have been associated with altered PTGS2 mRNA levels/COX-2 activity in some studies, although not the present study, were associated with colorectal cancer risk. Thus, both PTGS2 polymorphisms and PTGS2 mRNA levels may provide information regarding CRC risk.

The secondary bile acid, deoxycholate accelerates intestinal adenoma-adenocarcinoma sequence in Apc (min/+) mice through enhancing Wnt signaling.

Colorectal cancer is one of the leading causes of cancer deaths. It correlates to a high fat diet, which causes an increase of the secondary bile acids including deoxycholate (DOC) in the intestine. We aimed to determine the effects of DOC on intestinal carcinogenesis in Apc (min/+) mice, a model of spontaneous intestinal adenomas. Four-week old Apc (min/+) mice were treated with 0.2 % DOC in drinking water for 12 weeks. The number and size of tumors were measured, and tissue sections were prepared for the evaluation of intestinal carcinogenesis, cell proliferation, and apoptosis. The activation of Wnt signaling was detected in the intestinal tumor cells of the Apc (min/+) mice, and also in the human colon samples. DOC increased the number of intestine tumors by 165.1 % compared with that in untreated Apc (min/+) mice mainly in the middle and distal segments of the small intestine and colon. The numbers of all sizes of tumors in the small intestine were increased. Intestinal carcinogenesis was confirmed in 75 % mice in DOC treated-Apc (min/+) mice compared with 0 % in untreated mice. This was accompanied by promoting tumor cell proliferation and decreasing apoptosis, and increasing the percentage of β-catenin positive cells and its nuclear expression in intestinal tumor cells of Apc (min/+) mice, and also up-regulating the expression of cyclin D1. In addition, the activation of Wnt signaling also played in modulating human colon carcinogenesis. Our studies suggest that DOC enhances the multiplicity of intestinal tumor, and accelerates intestinal adenoma-adenocarcinoma sequence in Apc (min/+) mice mediated by stimulating tumor cell proliferation and decreasing apoptosis through enhancing Wnt signaling.

Low-grade neuroendocrine tumors arising in intestinal adenomas: evidence for alterations in the adenomatous polyposis coli/β-catenin pathway.

Low-grade neuroendocrine tumors (NETs) arising in intestinal adenomas are rare. They are occasionally observed in patients with familial adenomatous polyposis (FAP), suggesting a role for the adenomatous polyposis coli/β-catenin pathway. We identified 25 composite adenoma/low-grade NETs from colorectum (21) and duodenum (4) and evaluated their clinicopathological features, survival, and nuclear β-catenin expression by immunohistochemistry. β-catenin staining was scored as % positivity × intensity (weak, 1; moderate, 2; and strong, 3), for a total possible score of 300. Control groups included 1781 adenomas without NET, 63 composite adenoma/high-grade neuroendocrine carcinomas (NECs), and 32 sporadic NETs. Among 25 adenoma/low-grade NETs, 4 (16%) occurred in patients with FAP. Size of the NET component ranged from 0.01 to 0.9 cm (mean, 0.32 cm). Most (84%) arose in "advanced" adenomas (size >1 cm, villous architecture [72%], or high-grade dysplasia [56%]). In contrast, villous architecture and high-grade dysplasia were present in only 14% (P < .001) and 7% (P < .001), respectively, of adenomas without NET. Overall survival with adenoma/low-grade NET was significantly higher than adenoma/high-grade NEC but significantly lower than sporadic NET (P < .001). Higher β-catenin expression was seen in adenoma/low-grade NETs (mean score, 231) compared with sporadic NETs (mean score, 48; P < .0001) and adenoma/high-grade NEC (mean score, 173; P = .04). In summary, composite adenoma/low-grade NETs most commonly occur with advanced polyps, but the NET component itself is generally small and indolent. In contrast to sporadic NETs, the occurrence of these lesions in FAP and their high levels of nuclear β-catenin expression support a pathogenic role for the adenomatous polyposis coli/β-catenin pathway.

Repurposing the FDA-approved pinworm drug pyrvinium as a novel chemotherapeutic agent for intestinal polyposis.

Mutations in the WNT-pathway regulator ADENOMATOUS POLYPOSIS COLI (APC) promote aberrant activation of the WNT pathway that is responsible for APC-associated diseases such as Familial Adenomatous Polyposis (FAP) and 85% of spontaneous colorectal cancers (CRC). FAP is characterized by multiple intestinal adenomas, which inexorably result in CRC. Surprisingly, given their common occurrence, there are few effective chemotherapeutic drugs for FAP. Here we show that the FDA-approved, anti-helminthic drug Pyrvinium attenuates the growth of WNT-dependent CRC cells and does so via activation of CK1α. Furthermore, we show that Pyrvinium can function as an in vivo inhibitor of WNT-signaling and polyposis in a mouse model of FAP: APCmin mice. Oral administration of Pyrvinium, a CK1α agonist, attenuated the levels of WNT-driven biomarkers and inhibited adenoma formation in APCmin mice. Considering its well-documented safe use for treating enterobiasis in humans, our findings suggest that Pyrvinium could be repurposed for the clinical treatment of APC-associated polyposes.

OGA heterozygosity suppresses intestinal tumorigenesis in Apc(min/+) mice.

Emerging evidence suggests that aberrant O-GlcNAcylation is associated with tumorigenesis. Many oncogenic factors are O-GlcNAcylated, which modulates their functions. However, it remains unclear how O-GlcNAcylation and O-GlcNAc cycling enzymes, O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA), affect the development of cancer in animal models. In this study, we show that reduced level of OGA attenuates colorectal tumorigenesis induced by Adenomatous polyposis coli (Apc) mutation. The levels of O-GlcNAcylation and O-GlcNAc cycling enzymes were simultaneously upregulated in intestinal adenomas from mice, and in human patients. In two independent microarray data sets, the expression of OGA and OGT was significantly associated with poor cancer-specific survival of colorectal cancer (CRC) patients. In addition, OGA heterozygosity, which results in increased levels of O-GlcNAcylation, attenuated intestinal tumor formation in the Apcmin/+ background. Apcmin/+ OGA+/− mice exhibited a significantly increased survival rate compared with Apcmin/+ mice. Consistent with this, Apcmin/+ OGA+/− mice expressed lower levels of Wnt target genes than Apcmin/+. However, the knockout of OGA did not affect Wnt/β-catenin signaling. Overall, these findings suggest that OGA is crucial for tumor growth in CRC independently of Wnt/β-catenin signaling.

Paneth cells

Paneth cells

PWE-021 Mtorc1 Mediated Translational Elongation Is Limiting For Intestinal Tumour Initiation And Growth

IntroductionThe loss of Apc, causing Wnt-mediated epithelial proliferation, is an early event in colorectal cancer (CRC) development. This hyperproliferative state requires signalling though the mTOR pathway, with the current paradigm...

CCR6, the sole receptor for the chemokine CCL20, promotes spontaneous intestinal tumorigenesis.

Interactions between the inflammatory chemokine CCL20 and its receptor CCR6 have been associated with colorectal cancer growth and metastasis, however, a causal role for CCL20 signaling through CCR6 in promoting intestinal carcinogenesis has not been demonstrated in vivo. In this study, we aimed to determine the role of CCL20-CCR6 interactions in spontaneous intestinal tumorigenesis. CCR6-deficient mice were crossed with mice heterozygous for a mutation in the adenomatous polyposis coli (APC) gene (APCMIN/+ mice) to generate APCMIN/+ mice with CCR6 knocked out (CCR6KO-APCMIN/+ mice). CCR6KO-APCMIN/+ mice had diminished spontaneous intestinal tumorigenesis. CCR6KO-APCMIN/+ also had normal sized spleens as compared to the enlarged spleens found in APCMIN/+ mice. Decreased macrophage infiltration into intestinal adenomas and non-tumor epithelium was observed in CCR6KO-APCMIN/+ as compared to APCMIN/+ mice. CCL20 signaling through CCR6 caused increased production of CCL20 by colorectal cancer cell lines. Furthermore, CCL20 had a direct mitogenic effect on colorectal cancer cells. Thus, interactions between CCL20 and CCR6 promote intestinal carcinogenesis. Our results suggest that the intestinal tumorigenesis driven by CCL20-CCR6 interactions may be driven by macrophage recruitment into the intestine as well as proliferation of neoplastic epithelial cells. This interaction could be targeted for the treatment or prevention of malignancy.

Oncogenic mutations in intestinal adenomas regulate Bim-mediated apoptosis induced by TGF-β.

In the majority of microsatellite-stable colorectal cancers (CRCs), an initiating mutation occurs in the adenomatous polyposis coli (APC) or β-catenin gene, activating the β-catenin/TCF pathway. The progression of resulting adenomas is associated with oncogenic activation of KRas and inactivation of the p53 and TGF-β/Smad functions. Most established CRC cell lines contain mutations in the TGF-β/Smad pathway, but little is known about the function of TGF-β in the early phases of intestinal tumorigenesis. We used mouse and human ex vivo 3D intestinal organoid cultures and in vivo mouse models to study the effect of TGF-β on the Lgr5(+) intestinal stem cells and their progeny in intestinal adenomas. We found that the TGF-β-induced apoptosis in Apc-mutant organoids, including the Lgr5(+) stem cells, was mediated by up-regulation of the BH3-only proapoptotic protein Bcl-2-like protein 11 (Bim). BH3-mimetic compounds recapitulated the effect of Bim not only in the adenomas but also in human CRC organoids that had lost responsiveness to TGF-β-induced apoptosis. However, wild-type intestinal crypts were markedly less sensitive to TGF-β than Apc-mutant adenomas, whereas the KRas oncogene increased resistance to TGF-β via the activation of the Erk1/2 kinase pathway, leading to Bim down-regulation. Our studies identify Bim as a critical mediator of TGF-β-induced apoptosis in intestinal adenomas and show that the common progression mutations modify Bim levels and sensitivity to TGF-β during intestinal adenoma development.

Sa1958 Serrated Polyposis Syndrome: Pushing Through Prevalence and Clinicopathological Features

G A A b st ra ct s diet alone group (p<0.05). INCB3619 significantly inhibited pEGFR and pErbB2 signals in intestinal adenomas. Colon tumor incidence was 0.33 in the INCB3619-treated group, compared to 0.83 in Western diet alone group (p=0.10). Summary INCB3619 significantly reduced intestinal tumor multiplicity and tumor size in Apc Min mice. INCB3619 also concomitantly suppressed intestinal mucosal ADAM17 enzyme activity as well as EGFR signals in intestinal adenomas. Conclusions Our results show that targeting ADAM17 inhibits intestinal tumor promotion byWestern diet.We postulate thatWestern diet promotes tumorigenesis by activating ADAM17 and up-regulating EGFR signals. Our studies suggest that ADAM17 inhibitors might provide a novel strategy to reduce colon cancer risk in individuals consuming Western diets.

Tu1651 Indian Hedgehog Is Required for Intestinal Adenoma Formation

Introduction: Activating mutations in the Hedgehog pathway are found in basal cell carcinomas and medulloblastomas. The role of Hedgehog signaling in intestinal tumorigenesis has not yet been clarified. Hedgehog is expressed by differentiated enterocytes and signals in a paracrine manner from the epithelium to the mesenchyme. Hedgehog controls mesenchymal factors such as Bone Morphogenetic Proteins and Activins which negatively regulate the proliferation of precursor cells. Since Indian Hedgehog (Ihh) is the major Hedgehog expressed in the intestinal epithelium we decided to study the potential role of Ihh signaling in intestinal adenoma formation.Methods: In order to study the effect of loss of Ihh signaling in sporadic tumorigenesis we conditionally deleted Ihh in adult mice in the intestinal epithelium using the Cyp1a1Cre promoter. These compound mice were crossed with conditional mutant Apc mice to generate Cyp1a1Cre-Ihh-Apc mice. At 4-7 weeks of age mice were intraperitoneally injected with 80 mg/kg β-naphthoflavone for 5 days. Cyp1a1Cre-IhhApc littermates served as controls. Four months after recombination, we sacrificed and analyzed the mice (n=15). Results: Ihh expression was upregulated in polyps of Cyp1a1CreIhh-Apc mice as assessed by in situ hybridization. Deletion of Ihh resulted in a marked reduction of 90.3% in the number of polyps (17.2 ± 13.65 polyps per mouse vs. 1.7 ± 1.5, P < 0.01). The average polyp size did not differ between groups. Neither did the number of proliferating or apoptotic cells in the polyps, as assessed by immunostaining for BrdU and cleaved caspase 3. Hh responsive cells in the adenomawere restricted to themesenchyme. In those adenomas that did develop in Ihh mutant mice, we observed a remarkable loss of α-sma/desmin double positive smooth muscle cells. COX2 is expressed early in adenoma to carcinoma transition and believed to play an important role in adenoma formation. Stromal cells are known to be a source of COX2. In accordance with this, examination of Cox2 expression by Q-PCR and immunostaining showed up regulation in polyps ofCyp1a1Cre-Ihh -Apc mice in compared to normal intestinal epithelium (P < 0.01, resp. P < 0.05). In contrast, Cox2 expression in the polyps of Cyp1a1Cre-Ihh-Apc mice was not significantly up regulated. Conclusion: Surprisingly, in contrast to its role as an anti-proliferative signal in the normal epithelium, Ihh acts as a tumor promoter. Our data show that Ihh signaling is increased during intestinal adenoma formation and as in the normal intestine Ihh signaling is exclusively from the epithelium to the mesenchyme. Loss of Ihh signaling is associated with major changes in the composition of the adenoma mesenchyme and decreased Cox2 expression, which significantly impairs adenoma formation.

Tu1653 Farnesoid X Receptor (FXR) Represses Matrix Metalloproteinase 7 (MMP7) Expression in Intestinal Epithelial Cells

Introduction: Activating mutations in the Hedgehog pathway are found in basal cell carcinomas and medulloblastomas. The role of Hedgehog signaling in intestinal tumorigenesis has not yet been clarified. Hedgehog is expressed by differentiated enterocytes and signals in a paracrine manner from the epithelium to the mesenchyme. Hedgehog controls mesenchymal factors such as Bone Morphogenetic Proteins and Activins which negatively regulate the proliferation of precursor cells. Since Indian Hedgehog (Ihh) is the major Hedgehog expressed in the intestinal epithelium we decided to study the potential role of Ihh signaling in intestinal adenoma formation.Methods: In order to study the effect of loss of Ihh signaling in sporadic tumorigenesis we conditionally deleted Ihh in adult mice in the intestinal epithelium using the Cyp1a1Cre promoter. These compound mice were crossed with conditional mutant Apc mice to generate Cyp1a1Cre-Ihh-Apc mice. At 4-7 weeks of age mice were intraperitoneally injected with 80 mg/kg β-naphthoflavone for 5 days. Cyp1a1Cre-IhhApc littermates served as controls. Four months after recombination, we sacrificed and analyzed the mice (n=15). Results: Ihh expression was upregulated in polyps of Cyp1a1CreIhh-Apc mice as assessed by in situ hybridization. Deletion of Ihh resulted in a marked reduction of 90.3% in the number of polyps (17.2 ± 13.65 polyps per mouse vs. 1.7 ± 1.5, P < 0.01). The average polyp size did not differ between groups. Neither did the number of proliferating or apoptotic cells in the polyps, as assessed by immunostaining for BrdU and cleaved caspase 3. Hh responsive cells in the adenomawere restricted to themesenchyme. In those adenomas that did develop in Ihh mutant mice, we observed a remarkable loss of α-sma/desmin double positive smooth muscle cells. COX2 is expressed early in adenoma to carcinoma transition and believed to play an important role in adenoma formation. Stromal cells are known to be a source of COX2. In accordance with this, examination of Cox2 expression by Q-PCR and immunostaining showed up regulation in polyps ofCyp1a1Cre-Ihh -Apc mice in compared to normal intestinal epithelium (P < 0.01, resp. P < 0.05). In contrast, Cox2 expression in the polyps of Cyp1a1Cre-Ihh-Apc mice was not significantly up regulated. Conclusion: Surprisingly, in contrast to its role as an anti-proliferative signal in the normal epithelium, Ihh acts as a tumor promoter. Our data show that Ihh signaling is increased during intestinal adenoma formation and as in the normal intestine Ihh signaling is exclusively from the epithelium to the mesenchyme. Loss of Ihh signaling is associated with major changes in the composition of the adenoma mesenchyme and decreased Cox2 expression, which significantly impairs adenoma formation.

The Regulation of TIGAR

TIGAR (TP53-induced glycolysis and apoptosis regulator) functions to promote antioxidant defence, with a loss of TIGAR associated with a defect in a cell’s ability to control reactive oxygen species (ROS) and resultant oxidative damage. TIGAR can function as a fructose-2,6-bisphosphatase, lowering the levels of fructose-2,6- bisphosphate, which is an activator of phosphofructokinase-1. As a consequence, TIGAR activity results in a dampening of the glycolytic pathway and, by enhancing the pentose phosphate pathway, increases cellular antioxidant capacity by promoting the generation of NADPH and GSH. Although TIGAR is clearly a transcriptional target of the tumour suppressor p53 in human cells, the activation of TIGAR expression in mouse cells in vitro and in a mouse model of intestinal regeneration was not dependent on p53 or its family member TAp73. However, TIGAR expression was strongly induced in the mouse intestine during proliferation following damage or APC loss, suggesting a role for the Wnt signalling pathway. The increase in TIGAR expression seen in response to APC loss was lost after simultaneous deletion of c-Myc, suggesting that TIGAR responds to c-Myc activation downstream of the Wnt signalling pathway. While TIGAR may be a direct Myc target, Myc was shown to induce ROS, which were also found to regulate the expression of TIGAR. In order to further understand the function of TIGAR, a TIGAR-deficient mouse was generated and TIGAR was found to play a role in supporting intestinal regeneration by lowering oxidative stress in the small intestinal crypts following tissue damage by irradiation or cisplatin treatment. Moreover, TIGAR-null mice showed decreased tumour development in a model of intestinal adenoma. In particular, it was found that TIGAR acts to lower the damaging pool of ROS during oxidative stress. Through this, TIGAR can function to promote tumourigenesis and elevated TIGAR expression has been observed in various cancer types, independently of p53 status. This suggests that a deregulated expression of TIGAR may play a role in supporting rather than inhibiting cancer development. This study reveals p53-independent mechanisms by which TIGAR is regulated and how TIGAR can contribute to promote cell growth and tumourigenesis.

Tu1660 Intestine-Specific Deletion of KLF4 Renders Mice Susceptible to Inflammation-Associated Tumorigenesis and Increases Intestinal Adenoma Multiplicity and Colonic Adenoma Incidence in APCMIN/+ Mice

Tu1660 Intestine-Specific Deletion of KLF4 Renders Mice Susceptible to Inflammation-Associated Tumorigenesis and Increases Intestinal Adenoma Multiplicity and Colonic Adenoma Incidence in APCMIN/+ Mice

Altered chemokine production and accumulation of regulatory T cells in intestinal adenomas of APC(Min/+) mice.

Tumor progression in the colon moves from aberrant crypt foci to adenomatous polyps to invasive carcinomas. The composition of the tumor-infiltrating leukocyte population affects the ability of the immune system to fight the tumor. T cell infiltration into colorectal adenocarcinomas, particularly T helper 1 (Th1) type T cells as well as increased regulatory T cell (Treg) frequencies, is correlated with improved prognosis. However, whether Th1 cells and Tregs are already present at the adenoma stage is not known. In this study, the APC(Min/+) mouse model of intestinal adenomatous polyposis was used to investigate tumor-associated lymphocyte subsets and the mechanisms of their accumulation into gastrointestinal adenomas. Compared to unaffected tissue, adenomas accumulated CD4(+)FoxP3(+) putative Treg in parallel with lower frequencies of conventional T cells and B cells. The accumulation of Treg was also observed in human adenomatous polyps. Despite high Treg numbers, the function of conventional T cells present in the APC(Min/+) adenomas was not different from those in the unaffected tissue. Adenomas displayed an altered chemokine balance, with higher CCL17 and lower CXCL11 and CCL25 expression than in the unaffected tissue. In parallel, CXCR3(+) Tregs were largely absent from adenomas. The data indicate that already in early stages of tumor development, the balance of lymphocyte-recruiting chemokines is altered possibly contributing to the observed shift toward higher frequencies of Treg.