Lack of anti-tumor activity with the β-catenin expression inhibitor EZN-3892 in the C57BL/6J Min/+ model of intestinal carcinogenesis

Background

Previously, we showed that short-term inhibition of β-catenin expression and reversal of aberrant β-catenin subcellular localization by the selective COX-2 inhibitor celecoxib is associated with adenoma regression in the C57BL/6J Min/+ mouse. Conversly, long-term administration resulted in tumor resistance, leading us to investigate alternative methods for selective β-catenin chemoprevention. In this study, we hypothesized that disruption of β-catenin expression by EZN-3892, a selective locked nucleic acid (LNA)-based β-catenin inhibitor, would counteract the tumorigenic effect of Apc loss in Min/+ adenomas while preserving normal intestinal function.

Materials and methods

C57BL/6J Apc^+/+ wild-type (WT) and Min/+ mice were treated with the maximum tolerated dose (MTD) of EZN-3892 (30 mg/kg). Drug effect on tumor numbers, β-catenin protein expression, and nuclear β-catenin localization were determined.

Results

Although the tumor phenotype and β-catenin nuclear localization in Min/+ mice did not change following drug administration, we observed a decrease in β-catenin expression levels in the mature intestinal tissue of treated Min/+ and WT mice, providing proof of principle regarding successful delivery of the LNA-based antisense vehicle. Higher doses of EZN-3892 resulted in fatal outcomes in Min/+ mice, likely due to β-catenin ablation in the intestinal tissue and loss of function.

Conclusions

Our data support the critical role of Wnt/β-catenin signaling in maintaining intestinal homeostasis and highlight the challenges of effective drug delivery to target disease without permanent toxicity to normal cellular function.     

Toll‐like receptor 4 regulates spontaneous intestinal tumorigenesis by up‐regulating IL‐6 and GM‐CSF

Inflammation is as an important component of intestinal tumorigenesis. The activation of Toll‐like receptor 4 (TLR4) signalling promotes inflammation in colitis of mice, but the role of TLR4 in intestinal tumorigenesis is not yet clear. About 80%–90% of colorectal tumours contain inactivating mutations in the adenomatous polyposis coli (Apc) tumour suppressor, and intestinal adenoma carcinogenesis in familial adenomatous polyposis (FAP) is also closely related to the germline mutations in Apc. The Apc^Min/+ (multiple intestinal neoplasia) model mouse is a well‐utilized model of FAP, an inherited form of intestinal cancer. In this study, Apc^Min/+ intestinal adenoma mice were generated on TLR4‐sufficient and TLR4‐deficient backgrounds to investigate the carcinogenic effect of TLR4 in mouse gut by comparing mice survival, peripheral blood cells, bone marrow haematopoietic precursor cells and numbers of polyps in the guts of Apc^Min/+ WT and Apc^Min/+ TLR4^?/? mice. The results revealed that TLR4 had a critical role in promoting spontaneous intestinal tumorigenesis. Significant differential genes were screened out by the high‐throughput RNA‐Seq method. After combining these results with KEGG enrichment data, it was determined that TLR4 might promote intestinal tumorigenesis by activating cytokine‐cytokine receptor interaction and pathways in cancer signalling pathways. After a series of validation experiments for the concerned genes, it was found that IL6, GM‐CSF (CSF2), IL11, CCL3, S100A8 and S100A9 were significantly decreased in gut tumours of Apc^Min/+ TLR4^?/? mice compared with Apc^Min/+ WT mice. In the functional study of core down‐regulation factors, it was found that IL6, GM‐CSF, IL11, CCL3 and S100A8/9 increased the viability of colon cancer cell lines and decreased the apoptosis rate of colon cancer cells with irradiation and chemical treatment.     

Tissue-Dependent Consequences of Apc Inactivation on Proliferation and Differentiation of Ciliated Cell Progenitors via Wnt and Notch Signaling

The molecular signals that control decisions regarding progenitor/stem cell proliferation versus differentiation are not fully understood. Differentiation of motile cilia from progenitor/stem cells may offer a simple tractable model to investigate this process. Wnt and Notch represent two key signaling pathways in progenitor/stem cell behavior in a number of tissues. Adenomatous Polyposis Coli, Apc is a negative regulator of the Wnt pathway and a well known multifunctional protein. Using the cre-LoxP system we inactivated the Apc locus via Foxj1-cre, which is expressed in cells committed to ciliated cell lineage. We then characterized the consequent phenotype in two select tissues that bear motile cilia, the lung and the testis. In the lung, Apc deletion induced β-catenin accumulation and Jag1 expression in ciliated cells and by lateral induction, triggered Notch signaling in adjacent Clara cells. In the bronchiolar epithelium, absence of Apc blocked the differentiation of a subpopulation of cells committed to the ciliogenesis program. In the human pulmonary adenocarcinoma cells, Apc over-expression inhibited Jag1 expression and promoted motile ciliogenic gene expression program including Foxj1, revealing the potential mechanism. In the testis, Apc inactivation induced β-catenin accumulation in the spermatogonia, but silenced Notch signaling and depleted spermatogonial stem cells, associated with reduced proliferation, resulting in male infertility. In sum, the present comparative analysis reveals the tissue-dependent consequences of Apc inactivation on proliferation and differentiation of ciliated cell progenitors by coordinating Wnt and Notch signaling.     

Post-translational Glycoprotein Modifications Regulate Colon Cancer Stem Cells and Colon Adenoma Progression in Apcmin/+ Mice through Altered Wnt Receptor Signaling

Deletion of GnT-V (MGAT5), which synthesizes N-glycans with β(1,6)-branched glycans, reduced the compartment of cancer stem cells (CSC) in the her-2 mouse model of breast cancer, leading to delay of tumor onset. Because GnT-V levels are also commonly up-regulated in colon cancer, we investigated their regulation of colon CSC and adenoma development. Anchorage-independent cell growth and tumor formation induced by injection of colon tumor cells into NOD/SCID mice were positively associated with GnT-V levels, indicating regulation of proliferation and tumorigenicity. Using Apc^min/+ mice with different GnT-V backgrounds, knock-out of GnT-V had no significant effect on the number of adenoma/mouse, but adenoma size was significantly reduced and accompanied increased survival of Apc^min/+ mice with GnT-V deletion (p < 0.01), suggesting an inhibition in the progression of colon adenoma caused by deletion of GnT-V. Decreased expression levels of GnT-V down-regulated the population of colon (intestine) CSC, affecting their ability for self-renewal and tumorigenicity in NOD/SCID mice. Furthermore, altered nuclear translocation of β-catenin and expression of Wnt target genes were positively associated with expression levels of GnT-V, indicating the regulation of canonical Wnt/β-catenin signaling. By overexpressing the Wnt receptor, FZD-7, in colon cancer cells, we found that FZD-7 receptors expressed N-linked β(1,6) branching, indicating that FZD-7 can be modified by GnT-V. The aberrant Wnt signaling observed after modulating GnT-V levels is likely to result from altered N-linked β(1,6) branching on FZD-7, thereby affecting Wnt signaling, the compartment of CSC, and tumor progression.     

The Somatic Mutation Hit on Top of Genetic APC mutations Cause Skin Tumor

Inactivation of the adenomatous polyposis coli (APC) gene is the initiating event in familial adenomatous polyposis (FAP) patients. Up to 90% of FAP patients show intestinal tumors and other extracolonic malignancies including hepatoblastomas, desmoid tumors, and brain cancer. APC mutation mice (Apc^Min/+ mice) develop benign polyps in the intestinal tract. It has been reported that small numbers of Apc^Min/+ mice develop breast carcinomas. Here, we found that approximately 1.6% of Apc^Min/+ mice suffered skin neoplasm. The results demonstrated that these skin tumors are not derived from intestinal adenomas. Sequencing of skin tumors of Apc^Min/+ mice and Apc^Min/+ mice skin. The data showed that somatic mutations and gene expression levels changed greatly in skin tumors compared to control. Similarly, APC mutation accounts for 27% in the patients of nonmelanoma skin carcinomas in cancer database, and two above genes mutation coexist was observed in all patients. Furthermore, using gene mutation reagent (DMBA)–treated Apc^Min/+ mice skin, the skin epithelium and glandular begin hyperplasia in Apc^Min/+ mice. These findings revealed that the somatic mutation hit on the germline mutation increase the tumor incidence, suggesting that the somatic mutation should be avoided if the germline mutation exists in one body.     

Genetic Dissection of Differential Signaling Threshold Requirements for the Wnt/β-Catenin Pathway In Vivo

Contributions of null and hypomorphic alleles of Apc in mice produce both developmental and pathophysiological phenotypes. To ascribe the resulting genotype-to-phenotype relationship unambiguously to the Wnt/β-catenin pathway, we challenged the allele combinations by genetically restricting intracellular β-catenin expression in the corresponding compound mutant mice. Subsequent evaluation of the extent of resulting Tcf4-reporter activity in mouse embryo fibroblasts enabled genetic measurement of Wnt/β-catenin signaling in the form of an allelic series of mouse mutants. Different permissive Wnt signaling thresholds appear to be required for the embryonic development of head structures, adult intestinal polyposis, hepatocellular carcinomas, liver zonation, and the development of natural killer cells. Furthermore, we identify a homozygous Apc allele combination with Wnt/β-catenin signaling capacity similar to that in the germline of the Apc^min mice, where somatic Apc loss-of-heterozygosity triggers intestinal polyposis, to distinguish whether co-morbidities in Apc^min mice arise independently of intestinal tumorigenesis. Together, the present genotype–phenotype analysis suggests tissue-specific response levels for the Wnt/β-catenin pathway that regulate both physiological and pathophysiological conditions.     

Increased angiogenesis in Cdk4R24C/R24C:Apc+/Min intestinal tumors

Cyclin dependent kinase 4 (Cdk4) is a cell cycle regulator involved in early G1 cell cycle progression and has been indirectly implicated in angiogenesis in the Min mouse system, a mouse that harbors a mutation in the Apc gene. Apc^+/Min mice when crossed with Ink4a/arf-/- mice, exhibited increased angiogenesis of colorectal tumors suggesting that dysregulation of Cdk4 (due to loss of Ink4a-mediated suppression) may contribute to enhanced angiogenesis. To demonstrate a direct role for Cdk4 in angiogenesis, we crossed mice that have an activated Cdk4, Cdk4^R24C/R24C mice, with Apc^+/Min mice and examined levels of angiogenesis in intestinal tumors formed. Our results show an increase in the percentage of highly vascularized tumors in Cdk4^R24C/R24C:Apc^Min/+ and Cdk4^+/R24C:Apc^Min/+ mice compared to Cdk4^+/+:Apc^Min/+ mice. In addition immunohistochemical analysis showed an increase in CD-31 staining localized to endothelial cells of Cdk4^R24C/R24C:Apc^Min/+ mouse tumors, supporting the hypothesis of increased vasculature in these tumors. Further analysis showed an increase in the expression of the E2F1 target proteins Vegf-b and Cyclin A in Cdk4^R24C/R24C:Apc^+/Min intestinal tumors. Together these data suggest that the dysregulated Cdk4 gene plays an important role in angiogenesis during intestinal tumor formation and may in part act via increasing E2F1 target proteins. This is the first report to show that Cdk4 has a direct role in angiogenesis in vivo and may be an important drug target to reduce or prevent angiogenesis during intestinal tumor formation.     

ERG is required for the differentiation of embryonic stem cells along the endothelial lineage

Background

During skeletogenesis, protein levels of β-catenin in the canonical Wnt signaling pathway determine lineage commitment of skeletal precursor cells to osteoblasts and chondrocytes. Adenomatous polyposis coli (Apc) is a key controller of β-catenin turnover by down-regulating intracellular levels of β-catenin.

Results

To investigate whether Apc is involved in lineage commitment of skeletal precursor cells, we generated conditional knockout mice lacking functional Apc in Col2a1-expressing cells. In contrast to other models in which an oncogenic variant of β-catenin was used, our approach resulted in the accumulation of wild type β-catenin protein due to functional loss of Apc. Conditional homozygous Apc mutant mice died perinatally showing greatly impaired skeletogenesis. All endochondral bones were misshaped and lacked structural integrity. Lack of functional Apc resulted in a pleiotropic skeletal cell phenotype. The majority of the precursor cells lacking Apc failed to differentiate into chondrocytes or osteoblasts. However, skeletal precursor cells in the proximal ribs were able to escape the noxious effect of functional loss of Apc resulting in formation of highly active osteoblasts. Inactivation of Apc in chondrocytes was associated with dedifferentiation of these cells.

Conclusion

Our data indicate that a tight Apc-mediated control of β-catenin levels is essential for differentiation of skeletal precursors as well as for the maintenance of a chondrocytic phenotype in a spatio-temporal regulated manner.     

Deficiency of the LIM-Only Protein FHL2 Reduces Intestinal Tumorigenesis in Apc Mutant Mice

Background

The four and a half LIM-only protein 2 (FHL2) is capable of shuttling between focal adhesion and nucleus where it signals through direct interaction with a number of proteins including β-catenin. Although FHL2 activation has been found in various human cancers, evidence of its functional contribution to carcinogenesis has been lacking.

Methodology/Principal Findings

Here we have investigated the role of FHL2 in intestinal tumorigenesis in which activation of the Wnt pathway by mutations in the adenomatous polyposis coli gene (Apc) or in β-catenin constitutes the primary transforming event. In this murine model, introduction of a biallelic deletion of FHL2 into mutant Apc^Δ14/+ mice substantially reduces the number of intestinal adenomas but not tumor growth, suggesting a role of FHL2 in the initial steps of tumorigenesis. In the lesions, Wnt signalling is not affected by FHL2 deficiency, remaining constitutively active. Nevertheless, loss of FHL2 activity is associated with increased epithelial cell migration in intestinal epithelium, which might allow to eliminate more efficiently deleterious cells and reduce the risk of tumorigenesis. This finding may provide a mechanistic basis for tumor suppression by FHL2 deficiency. In human colorectal carcinoma but not in low-grade dysplasia, we detected up-regulation and enhanced nuclear localization of FHL2, indicating the activation of FHL2 during the development of malignancy.

Conclusions/Significance

Our data demonstrate that FHL2 represents a critical factor in intestinal tumorigenesis.     

Differential Effects of β-catenin and NF-κB Interplay in the Regulation of Cell Proliferation,Inflammation and Tumorigenesis in Response to Bacterial Infection

Both β-catenin and NF-κB have been implicated in our laboratory as candidate factors in driving proliferation in an in vivo model of Citrobacter rodentium (CR)-induced colonic crypt hyper-proliferation and hyperplasia. Herein, we test the hypothesis that β-catenin and not necessarily NF-κB regulates colonic crypt hyperplasia or tumorigenesis in response to CR infection. When C57Bl/6 wild type (WT) mice were infected with CR, sequential increases in proliferation at days 9 and 12 plateaued off at day 19 and paralleled increases in NF-κB signaling. In Tlr4^−/− (KO) mice, a sequential but sustained proliferation which tapered off only marginally at day 19, was associated with TLR4-dependent and independent increases in NF-κB signaling. Similarly, increases in either activated or total β-catenin in the colonic crypts of WT mice as early as day 3 post-infection coincided with cyclinD1 and c-myc expression and associated crypt hyperplasia. In KO mice, a delayed kinetics associated predominantly with increases in non-phosphorylated (active) β-catenin coincided with increases in cyclinD1, c-myc and crypt hyperplasia. Interestingly, PKCζ-catalyzed Ser-9 phosphorylation and inactivation of GSK-3β and not loss of wild type APC protein accounted for β-catenin accumulation and nuclear translocation in either strain. In vitro studies with Wnt2b and Wnt5a further validated the interplay between the Wnt/β-catenin and NF-κB pathways, respectively. When WT or KO mice were treated with nanoparticle-encapsulated siRNA to β-catenin (si- β-Cat), almost complete loss of nuclear β-catenin coincided with concomitant decreases in CD44 and crypt hyperplasia without defects in NF-κB signaling. si-β-Cat treatment to Apc ^Min/+ mice attenuated CR-induced increases in β-catenin and CD44 that halted the growth of mutated crypts without affecting NF-κB signaling. The predominant β-catenin-induced crypt proliferation was further validated in a Castaneus strain (B6.CAST.11M) that exhibited significant crypt hyperplasia despite an attenuated NF-κB signaling. Thus, β-catenin and not necessarily NF-κB regulates crypt hyperplasia in response to bacterial infection.     

Cyclin D1 genetic heterozygosity regulates colonic epithelial cell differentiation and tumor number in ApcMin mice

Constitutive β-catenin/Tcf activity, the primary transforming events in colorectal carcinoma, occurs through induction of the Wnt pathway or APC gene mutations that cause familial adenomatous polyposis. Mice carrying Apc mutations in their germ line (Apc^Min) develop intestinal adenomas. Here, the crossing of Apc^Min with cyclin D1^−/− mice reduced the intestinal tumor number in animals genetically heterozygous or nullizygous for cyclin D1. Decreased tumor number in the duodenum, intestines, and colons of Apc^Min/cyclin D1^+/− mice correlated with reduced cellular proliferation and increased differentiation. Cyclin D1 deficiency reduced DNA synthesis and induced differentiation of colonic epithelial cells harboring mutant APC but not wild-type APC cells in vivo. In previous studies, the complete loss of cyclin D1 through homozygous genetic deletion conveyed breast tumor resistance. The protection of mice, genetically predisposed to intestinal tumorigenesis, through cyclin D1 heterozygosity suggests that modalities that reduce cyclin D1 abundance could provide chemoprotection.     

A Targeted Constitutive Mutation in the Apc Tumor Suppressor Gene Underlies Mammary But Not Intestinal Tumorigenesis

Germline mutations in the adenomatous polyposis coli (APC) gene are responsible for familial adenomatous polyposis (FAP), an autosomal dominant hereditary predisposition to the development of multiple colorectal adenomas and of a broad spectrum of extra-intestinal tumors. Moreover, somatic APC mutations play a rate-limiting and initiating role in the majority of sporadic colorectal cancers. Notwithstanding its multifunctional nature, the main tumor suppressing activity of the APC gene resides in its ability to regulate Wnt/β-catenin signaling. Notably, genotype–phenotype correlations have been established at the APC gene between the length and stability of the truncated proteins encoded by different mutant alleles, the corresponding levels of Wnt/β-catenin signaling activity they encode for, and the incidence and distribution of intestinal and extra-intestinal tumors. Here, we report a novel mouse model, Apc1572T, obtained by targeting a truncated mutation at codon 1572 in the endogenous Apc gene. This hypomorphic mutant allele results in intermediate levels of Wnt/β-catenin signaling activation when compared with other Apc mutations associated with multifocal intestinal tumors. Notwithstanding the constitutive nature of the mutation, Apc^+/1572T mice have no predisposition to intestinal cancer but develop multifocal mammary adenocarcinomas and subsequent pulmonary metastases in both genders. The histology of the Apc1572T primary mammary tumours is highly heterogeneous with luminal, myoepithelial, and squamous lineages and is reminiscent of metaplastic carcinoma of the breast in humans. The striking phenotype of Apc^+/1572T mice suggests that specific dosages of Wnt/β-catenin signaling activity differentially affect tissue homeostasis and initiate tumorigenesis in an organ-specific fashion.     

IL-17F deficiency inhibits small intestinal tumorigenesis in Apc mice

IL-17 plays an important role in gut homeostasis. However, the role of IL-17F in intestinal tumorigenesis has not been addressed. Here we demonstrate that ablation of IL-17F significantly inhibits spontaneous intestinal tumorigenesis in the small intestine of Apc^Min/+ mice. IL-17F ablation decreased IL-1β and Cox-2 expression as well as IL-17 receptor C (IL-17RC) expression, which were increased in tumors from Apc^Min/+ mice. Lack of IL-17F did not reverse the splenomegaly but partially restored thymic atrophy, suggesting a local effect of IL-17F in the intestine. IL-17F deficient Apc^Min/+ mice showed a significant decrease in immune cell infiltration in the lamina propria. Interestingly, the expression of IL-17A from CD4 T cells in the lamina propria remains unchanged in the absence of IL-17F. Collectively, our results suggest the proinflammatory and essential role of IL-17F to develop spontaneous intestinal tumorigenesis in Apc^Min/+ mice in the presence of IL-17A.     

Deficiency of Mbd2 attenuates Wnt signaling

We have previously shown that deficiency of the methyl binding domain protein Mbd2 dramatically reduces adenoma burden on an Apc^Min/+ background. To investigate the mechanism underlying this phenomenon, we have determined the effect of Mbd2 deficiency upon the phenotypes imposed by the conditional deletion of Apc in the small intestine. Microarray analysis demonstrated a partial suppression of the Wnt pathway in the absence of Mbd2. Mbd2 deficiency also influenced one immediate cellular consequence of Apc loss, with normalization of Paneth cell positioning. From a mechanistic perspective, we show that deficiency of Mbd2 elevates levels of the known Wnt target Lect2, and we confirm here that Mbd2 binds the Lect2 promoter in association with NuRD. Furthermore, we show that Lect2 is capable of functioning as a Wnt pathway repressor. These results therefore provide a mechanistic basis for the epigenetic control of adenoma formation mediated through Mbd2.     

Oncogenic mutations in adenomatous polyposis coli (Apc) activate mechanistic target of rapamycin complex 1 (mTORC1) in mice and zebrafish

Truncating mutations in adenomatous polyposis coli (APC) are strongly linked to colorectal cancers. APC is a negative regulator of the Wnt pathway and constitutive Wnt activation mediated by enhanced Wnt–β-catenin target gene activation is believed to be the predominant mechanism responsible for APC mutant phenotypes. However, recent evidence suggests that additional downstream effectors contribute to APC mutant phenotypes. We previously identified a mechanism in cultured human cells by which APC, acting through glycogen synthase kinase-3 (GSK-3), suppresses mTORC1, a nutrient sensor that regulates cell growth and proliferation. We hypothesized that truncating Apc mutations should activate mTORC1 in vivo and that mTORC1 plays an important role in Apc mutant phenotypes. We find that mTORC1 is strongly activated in apc mutant zebrafish and in intestinal polyps in Apc mutant mice. Furthermore, mTORC1 activation is essential downstream of APC as mTORC1 inhibition partially rescues Apc mutant phenotypes including early lethality, reduced circulation and liver hyperplasia. Importantly, combining mTORC1 and Wnt inhibition rescues defects in morphogenesis of the anterior-posterior axis that are not rescued by inhibition of either pathway alone. These data establish mTORC1 as a crucial, β-catenin independent effector of oncogenic Apc mutations and highlight the importance of mTORC1 regulation by APC during embryonic development. Our findings also suggest a new model of colorectal cancer pathogenesis in which mTORC1 is activated in parallel with Wnt/β-catenin signaling.KEY WORDS: APC, Wnt, mTOR, mTORC1, Zebrafish, Colon cancer, Polyposis, GSK-3     

Pathogenic Intestinal Bacteria Enhance Prostate Cancer Development via Systemic Activation of Immune Cells in Mice

A role for microbes has been suspected in prostate cancer but difficult to confirm in human patients. We show here that a gastrointestinal (GI) tract bacterial infection is sufficient to enhance prostate intraepithelial neoplasia (PIN) and microinvasive carcinoma in a mouse model. We found that animals with a genetic predilection for dysregulation of wnt signaling, Apc ^Min/+ mutant mice, were significantly susceptible to prostate cancer in an inflammation-dependent manner following infection with Helicobacter hepaticus. Further, early neoplasia observed in infected Apc ^Min/+ mice was transmissible to uninfected mice by intraperitoneal injection of mesenteric lymph node (MLN) cells alone from H. hepaticus-infected mutant mice. Transmissibility of neoplasia was preventable by prior neutralization of inflammation using anti-TNF-α antibody in infected MLN donor mice. Taken together, these data confirm that systemic inflammation triggered by GI tract bacteria plays a pivotal role in tumorigenesis of the prostate gland.