Enapotamab Vedotin, an AXL-Specific Antibody-Drug Conjugate, Demonstrates Antitumor Efficacy in Patient-Derived Xenograft Models of Soft Tissue Sarcoma.

Doxorubicin (doxo) remains the standard of care for patients with advanced soft tissue sarcoma (STS), even though response rates to doxo are only around 14% to 18%. We evaluated enapotamab vedotin (EnaV), an AXL-specific antibody-drug conjugate (ADC), in a panel of STS patient-derived xenografts (PDX). Eight models representing multiple STS subtypes were selected from our STS PDX platform (n = 45) by AXL immunostaining on archived passages. Models were expanded by unilateral transplantation of tumor tissue into the left flank of 20 NMRI nu/nu mice. Once tumors were established, mice were randomized into an EnaV treatment group, or a group treated with isotype control ADC. Treatment efficacy was assessed by tumor volume evaluation, survival analysis, and histological evaluation of tumors, and associated with AXL expression. EnaV demonstrated significant tumor growth delay, regression, and/or prolonged survival compared to isotype control ADC in 5/8 STS PDX models investigated. Experimental passages of responding models were all found positive for AXL at varying levels, but no linear relationship could be identified between the level of expression and level of response to EnaV. One model was found negative for AXL on experimental passage and did not respond to EnaV. This study provides a preclinical rationale for the evaluation of AXL-targeting ADCs in the treatment of AXL-expressing sarcomas.

Cooperative Targeting of Immunotherapy-Resistant Melanoma and Lung Cancer by an AXL-Targeting Antibody-Drug Conjugate and Immune Checkpoint Blockade.

Although immune checkpoint blockade (ICB) has shown remarkable clinical benefit in a subset of patients with melanoma and lung cancer, most patients experience no durable benefit. The receptor tyrosine kinase AXL is commonly implicated in therapy resistance and may serve as a marker for therapy-refractory tumors, for example in melanoma, as we previously demonstrated. Here, we show that enapotamab vedotin (EnaV), an antibody-drug conjugate targeting AXL, effectively targets tumors that display insensitivity to immunotherapy or tumor-specific T cells in several melanoma and lung cancer models. In addition to its direct tumor cell killing activity, EnaV treatment induced an inflammatory response and immunogenic cell death in tumor cells and promoted the induction of a memory-like phenotype in cytotoxic T cells. Combining EnaV with tumor-specific T cells proved superior to either treatment alone in models of melanoma and lung cancer and induced ICB benefit in models otherwise insensitive to anti-PD-1 treatment. Our findings indicate that targeting AXL-expressing, immunotherapy-resistant tumors with EnaV causes an immune-stimulating tumor microenvironment and enhances sensitivity to ICB, warranting further investigation of this treatment combination. SIGNIFICANCE: These findings show that targeting AXL-positive tumor fractions with an antibody-drug conjugate enhances antitumor immunity in several humanized tumor models of melanoma and lung cancer.

The role of S100a4 (Mts1) in Apc- and Smad4-driven tumour onset and progression.

INTRODUCTION: S100a4 is a calcium-binding protein belonging to the family of S100-proteins, highly expressed in different stromal cell types. S100A4 has been reported as a prognostic marker in colorectal cancer in association with tumour progression and metastasis. METHODS: In this study, we analysed the in vivo role of S100a4 in intestinal tumour initiation and progression using different transgenic and knockout mouse models. RESULTS: We found that genetic ablation or overexpression of S100a4 in both Apc- and Smad4-mutant mice do not affect tumour initiation in the intestinal tract. In contrast, S100a4 epithelial overexpression in Apc1638N/+/KRASV12G mice increases the dissemination of intestinal tumour cells to the liver, in agreement with its role in tumour metastasis. Moreover, we report a novel role for S100a4 in desmoid formation where S100a4 deficiency results in a significant reduction of the tumour burden characteristic of the Apc1638N model. In agreement with these results, S100a4 appears to be co-expressed together with mesenchymal stem cell (MSC) markers in desmoid tumours from Apc1638N/+ mice, as well as from sporadic and hereditary human desmoids. CONCLUSION: Our data provide the first report on the in vivo role of S100a4 in intestinal tumourigenesis and describe a new role for S100a4 in the aetiology of desmoids formation.

Secreted Phospholipases A2 Are Intestinal Stem Cell Niche Factors with Distinct Roles in Homeostasis, Inflammation, and Cancer.

The intestinal stem cell niche provides cues that actively maintain gut homeostasis. Dysregulation of these cues may compromise intestinal regeneration upon tissue insult and/or promote tumor growth. Here, we identify secreted phospholipases A2 (sPLA2s) as stem cell niche factors with context-dependent functions in the digestive tract. We show that group IIA sPLA2, a known genetic modifier of mouse intestinal tumorigenesis, is expressed by Paneth cells in the small intestine, while group X sPLA2 is expressed by Paneth/goblet-like cells in the colon. During homeostasis, group IIA/X sPLA2s inhibit Wnt signaling through intracellular activation of Yap1. However, upon inflammation they are secreted into the intestinal lumen, where they promote prostaglandin synthesis and Wnt signaling. Genetic ablation of both sPLA2s improves recovery from inflammation but increases colon cancer susceptibility due to release of their homeostatic Wnt-inhibitory role. This "trade-off" effect suggests sPLA2s have important functions as genetic modifiers of inflammation and colon cancer.

Cancer stemness in Wnt-driven mammary tumorigenesis.

Wnt signaling plays a central role in mammary stem cell (MaSC) homeostasis and in breast cancer. In particular, epigenetic alterations at different members of the Wnt pathway have been identified among triple-negative, basal-like breast cancers. Previously, we developed a mouse model for metaplastic breast adenocarcinoma, a subtype of triple-negative breast cancer, by targeting a hypomorphic mutations in the endogenous Apc gene (Apc (1572T/+)). Here, by employing the CD24 and CD29 cell surface antigens, we have identified a subpopulation of mammary cancer stem cells (MaCSCs) from Apc (1572T/+) capable of self-renewal and differentiation both in vivo and in vitro. Moreover, immunohistochemical analysis of micro- and macrolung metastases and preliminary intravenous transplantation assays suggest that the MaCSCs underlie metastasis at distant organ sites. Expression profiling of the normal and tumor cell subpopulations encompassing MaSCs and CSCs revealed that the normal stem cell compartment is more similar to tumor cells than to their own differentiated progenies. Accordingly, Wnt signaling appears to be active in both the normal and cancer stem cell compartments, although at different levels. By comparing normal with cancer mouse mammary compartments, we identified a MaCSC gene signature able to predict outcome in breast cancer in man. Overall, our data indicate that constitutive Wnt signaling activation affects self-renewal and differentiation of MaSCs leading to metaplasia and basal-like adenocarcinomas.

Cancer stemness in Apc- vs. Apc/KRAS-driven intestinal tumorigenesis.

Constitutive activation of the Wnt pathway leads to adenoma formation, an obligatory step towards intestinal cancer. In view of the established role of Wnt in regulating stemness, we attempted the isolation of cancer stem cells (CSCs) from Apc- and Apc/KRAS-mutant intestinal tumours. Whereas CSCs are present in Apc/KRAS tumours, they appear to be very rare (<10(-6)) in the Apc-mutant adenomas. In contrast, the Lin(-)CD24(hi)CD29(+) subpopulation of adenocarcinoma cells appear to be enriched in CSCs with increased levels of active ß-catenin. Expression profiling analysis of the CSC-enriched subpopulation confirmed their enhanced Wnt activity and revealed additional differential expression of other signalling pathways, growth factor binding proteins, and extracellular matrix components. As expected, genes characteristic of the Paneth cell lineage (e.g. defensins) are co-expressed together with stem cell genes (e.g. Lgr5) within the CSC-enriched subpopulation. This is of interest as it may indicate a cancer stem cell niche role for tumor-derived Paneth-like cells, similar to their role in supporting Lgr5(+) stem cells in the normal intestinal crypt. Overall, our results indicate that oncogenic KRAS activation in Apc-driven tumours results in the expansion of the CSCs compartment by increasing ®-catenin intracellular stabilization.

Wnt5a promotes human colon cancer cell migration and invasion but does not augment intestinal tumorigenesis in Apc1638N mice.

Whereas aberrant activation of canonical Wnt/ß-catenin signaling underlies the majority of colorectal cancer cases, the contribution of non-canonical Wnt signaling is unclear. As enhanced expression of the most extensively studied non-canonical Wnt ligand WNT5A is observed in various diseases including colon cancer, WNT5A is gaining attention nowadays. Numerous in vitro studies suggest modulating capacities of WNT5A on proliferation, differentiation, migration and invasion, affecting tumor and non-mutant cells. However, a possible contribution of WNT5A to colorectal cancer remains to be elucidated. We have analyzed WNT5A expression in colorectal cancer profiling data sets, altered WNT5A expression in colon cancer cells and used our inducible Wnt5a transgenic mouse model to gain more insight into the role of WNT5A in intestinal cancer. We observed that increased WNT5A expression is associated with poor prognosis of colorectal cancer patients. WNT5A knockdown in human colon cancer cells caused reduced directional migration, deregulated focal adhesion site formation and reduced invasion, whereas Wnt5a administration promoted the directional migration of colon cancer cells. Despite these observed protumorigenic activities of WNT5A, the induction of Wnt5a expression in intestinal tumors of Apc1638N mice was not sufficient to augment malignancy or metastasis by itself. In conclusion, WNT5A promotes adhesion sites to form in a focal fashion and promotes the directional migration and invasion of colon cancer cells. Although these activities appear insufficient by themselves to augment malignancy or metastasis in Apc1638N mice, they might explain the poor colon cancer prognosis associated with enhanced WNT5A expression.

Wnt signaling regulates the lineage differentiation potential of mouse embryonic stem cells through Tcf3 down-regulation.

Canonical Wnt signaling plays a rate-limiting role in regulating self-renewal and differentiation in mouse embryonic stem cells (ESCs). We have previously shown that mutation in the Apc (adenomatous polyposis coli) tumor suppressor gene constitutively activates Wnt signaling in ESCs and inhibits their capacity to differentiate towards ecto-, meso-, and endodermal lineages. However, the underlying molecular and cellular mechanisms through which Wnt regulates lineage differentiation in mouse ESCs remain to date largely unknown. To this aim, we have derived and studied the gene expression profiles of several Apc-mutant ESC lines encoding for different levels of Wnt signaling activation. We found that down-regulation of Tcf3, a member of the Tcf/Lef family and a key player in the control of self-renewal and pluripotency, represents a specific and primary response to Wnt activation in ESCs. Accordingly, rescuing Tcf3 expression partially restored the neural defects observed in Apc-mutant ESCs, suggesting that Tcf3 down-regulation is a necessary step towards Wnt-mediated suppression of neural differentiation. We found that Tcf3 down-regulation in the context of constitutively active Wnt signaling does not result from promoter DNA methylation but is likely to be caused by a plethora of mechanisms at both the RNA and protein level as shown by the observed decrease in activating histone marks (H3K4me3 and H3-acetylation) and the upregulation of miR-211, a novel Wnt-regulated microRNA that targets Tcf3 and attenuates early neural differentiation in mouse ESCs. Our data show for the first time that Wnt signaling down-regulates Tcf3 expression, possibly at both the transcriptional and post-transcriptional levels, and thus highlight a novel mechanism through which Wnt signaling inhibits neuro-ectodermal lineage differentiation in mouse embryonic stem cells.

Alterations in Wnt-ß-catenin and Pten signalling play distinct roles in endometrial cancer initiation and progression.

Endometrioid endometrial cancer arises through a gradual series of histological changes, each accompanied by specific alterations in gene expression and activity. Activation of the Wnt-ß-catenin pathway and loss of PTEN activity are frequently observed in endometrial cancers. However, the specific roles played by alterations in these pathways in the initiation and progression of endometrial cancer are currently unclear. Here, we investigated the effects of loss of Pten and Apc gene function in the mouse endometrium by employing tissue-specific and inducible mutant alleles, followed by immunohistochemical (IHC) and loss of heterozygosity (LOH) analysis of their corresponding cancerous lesions. Loss of the Apc function in the endometrium leads to cytoplasmic and nuclear ß-catenin accumulation in association with uterine hyperplasia and squamous cell metaplasia, but without malignant transformation. Loss of Pten function also resulted in squamous metaplasia but, in contrast to loss of Apc function, it initiates endometrial cancer. On the other hand, loss of Apc function in the endometrium accelerates Pten-driven endometrial tumourigenesis. Analysis of compound heterozygous mice confirmed that somatic loss of the wild-type Pten allele represents the rate-limiting initiation step in endometrial cancer. Simultaneous loss of Pten and Apc resulted in endometrial cancer characterized by earlier onset and a more aggressive malignant behaviour. These observations are indicative of the synergistic action between the Wnt-ß-catenin and Pten signalling pathways in endometrial cancer onset and progression.

Paneth cells in intestinal homeostasis and tissue injury.

Adult stem cell niches are often co-inhabited by cycling and quiescent stem cells. In the intestine, lineage tracing has identified Lgr5(+) cells as frequently cycling stem cells, whereas Bmi1(+), mTert(+), Hopx(+) and Lrig1(+) cells appear to be more quiescent. Here, we have applied a non-mutagenic and cell cycle independent approach to isolate and characterize small intestinal label-retaining cells (LRCs) persisting in the lower third of the crypt of Lieberkühn for up to 100 days. LRCs do not express markers of proliferation and of enterocyte, goblet or enteroendocrine differentiation, but are positive for Paneth cell markers. While during homeostasis, LR/Paneth cells appear to play a supportive role for Lgr5(+) stem cells as previously shown, upon tissue injury they switch to a proliferating state and in the process activate Bmi1 expression while silencing Paneth-specific genes. Hence, they are likely to contribute to the regenerative process following tissue insults such as chronic inflammation.

Generation and characterization of an inducible transgenic model for studying mouse esophageal biology.

BACKGROUND: To facilitate the in vivo study of esophageal (stem) cell biology in homeostasis and cancer, novel mouse models are necessary to elicit expression of candidate genes in a tissue-specific and inducible fashion. To this aim, we developed and studied a mouse model to allow labeling of esophageal cells with the histone 2B-GFP (H2B-GFP) fusion protein. RESULTS: First, we generated a transgenic mouse model expressing the reverse tetracycline transactivator rtTA2-M2 under control of the promoter (ED-L2) of the Epstein-Barr virus (EBV) gene encoding the latent membrane protein-1 (LMP-1). The newly generated ED-L2-rtTA2-M2 (ED-L2-rtTA) mice were then bred with the previously developed tetO-HIST1H2BJ/GFP (tetO-H2B-GFP) model to assess inducibility and tissue-specificity. Expression of the H2B-GFP fusion protein was observed upon doxycycline induction but was restricted to the terminally differentiated cells above the basal cell layer. To achieve expression in the basal compartment of the esophagus, we subsequently employed a different transgenic model expressing the reverse transactivator rtTA2S-M2 under the control of the ubiquitous, methylation-free CpG island of the human hnRNPA2B1-CBX3 gene (hnRNP-rtTA). Upon doxycycline administration to the compound hnRNP-rtTA/tetO-H2B-GFP mice, near-complete labeling of all esophageal cells was achieved. Pulse-chase experiments confirmed that complete turnover of the esophageal epithelium in the adult mouse is achieved within 7-10 days. CONCLUSIONS: We show that the esophagus-specific promoter ED-L2 is expressed only in the differentiated cells above the basal layer. Moreover, we confirmed that esophageal turn-over in the adult mouse does not exceed 7-10 days.

Induced Wnt5a expression perturbs embryonic outgrowth and intestinal elongation, but is well-tolerated in adult mice.

Wnt5a is essential during embryonic development, as indicated by mouse Wnt5a knockout embryos displaying outgrowth defects of multiple structures including the gut. The dynamics of Wnt5a involvement in these processes is unclear, and perinatal lethality of Wnt5a knockout embryos has hampered investigation of Wnt5a during postnatal stages in vivo. Although in vitro studies have suggested a relevant role for Wnt5a postnatally, solid evidence for a significant impact of Wnt5a within the complexity of an adult organism is lacking. We generated a tightly-regulated inducible Wnt5a transgenic mouse model and investigated the effects of Wnt5a induction during different time-frames of embryonic development and in adult mice, focusing on the gastrointestinal tract. When induced in embryos from 10.5 dpc onwards, Wnt5a expression led to severe outgrowth defects affecting the gastrointestinal tracts, limbs, facial structures and tails, closely resembling the defects observed in Wnt5a knockout mice. However, Wnt5a induction from 13.5 dpc onwards did not cause this phenotype, indicating that the most critical period for Wnt5a in embryonic development is prior to 13.5 dpc. In adult mice, induced Wnt5a expression did not reveal abnormalities, providing the first in vivo evidence that Wnt5a has no major impact on mouse intestinal homeostasis postnatally. Protein expression of Wnt5a receptor Ror2 was strongly reduced in adult intestine compared to embryonic stages. Moreover, we uncovered a regulatory process where induction of Wnt5a causes downregulation of its receptor Ror2. Taken together, our results indicate a role for Wnt5a during a restricted time-frame of embryonic development, but suggest no impact during homeostatic postnatal stages.

Loss of APC function in mesenchymal cells surrounding the Müllerian duct leads to myometrial defects in adult mice.

The WNT signal transduction pathway plays a rate limiting role in early development of many different organs. To study the functional consequences of constitutive activation of the canonical WNT pathway in the developing uterus, we generated a novel mouse model where loss of the tumor suppressor gene Apc was induced. A mouse model was generated and evaluated where Amhr2(Cre/+) driven loss of Apc exon 15 was induced. The Apc recombination was detected mainly in the myometrial layer of the adult uterus. A significant loss of muscle fibers in myometrium was apparent, though with very few muscle cells earmarked by nuclear ß-catenin. The finding was confirmed in the Pgr(Cre/+);Apc(15lox/15lox) mouse model. Loss of APC function in mesenchymal cells surrounding the fetal Müllerian ducts results in severe defects in the myometrial layers of the uterus in adult mice, suggesting that the WNT signaling pathway plays important roles in maintaining myometrial integrity.

ß-catenin tyrosine 654 phosphorylation increases Wnt signalling and intestinal tumorigenesis.

Objective Deregulation of the Wnt signalling pathway by mutations in the Apc or ß-catenin genes underlies colorectal carcinogenesis. As a result, ß-catenin stabilises, translocates to the nucleus, and activates gene transcription. Intestinal tumours show a heterogeneous pattern of nuclear ß-catenin, with the highest levels observed at the invasion front. Activation of receptor tyrosine kinases in these tumour areas by growth factors expressed by surrounding stromal cells phosphorylate ß-catenin at tyrosine residues, which is thought to increase ß-catenin nuclear translocation and tumour invasiveness. This study investigates the relevance of ß-catenin tyrosine phosphorylation for Wnt signalling and intestinal tumorigenesis in vivo. Design A conditional knock-in mouse model was generated into which the phospho-mimicking Y654E modification in the endogenous ß-catenin gene was introduced. Results This study provided in vivo evidence that ß-catenin(E654) is characterised by reduced affinity for cadherins, increased signalling and strongly increased phosphorylation at serine 675 by protein kinase A (PKA). In addition, homozygosity for the ß-catenin(E654) targeted allele caused embryonic lethality, whereas heterozygosity predisposed to intestinal tumour development, and strongly enhanced Apc-driven intestinal tumour initiation associated with increased nuclear accumulation of ßcatenin. Surprisingly, the expression of ß-catenin(E654) did not affect histological grade or induce tumour invasiveness. Conclusions A thus far unknown mechanism was uncovered in which Y654 phosphorylation of ß-catenin facilitates additional phosphorylation at serine 675 by PKA. In addition, in contrast to the current belief that ß-catenin Y654 phosphorylation increases tumour progression to a more invasive phenotype, these results show that it rather increases tumour initiation by enhancing Wnt signalling.

Leiden Open Variation Database of the MUTYH gene.

The MUTYH gene encodes a DNA glycosylase involved in base excision repair (BER). Biallelic pathogenic MUTYH variants have been associated with colorectal polyposis and cancer. The pathogenicity of a few variants is beyond doubt, including c.536A4G/p.Tyr179Cys and c.1187G4A/p.Gly396Asp (previously c.494A4G/p.Tyr165Cys and c.1145G4A/p.Gly382Asp).However, for a substantial fraction of the detected variants, the clinical significance remains uncertain,compromising molecular diagnostics and thereby genetic counseling. We have established an interactive MUTYH gene sequence variant database (www.lovd.nl/MUTYH) with the aim of collecting and sharing MUTYH genotype and phenotype data worldwide. To support standard variant description, we chose NM_001128425.1 as the reference sequence. The database includes records with variants per individual, linked to available phenotype and geographic origin data as well as records with in vitro functional and in silico test data. As of April 2010, the database contains 1968 published and 423 unpublished submitted entries, and 230 and 61 unique variants,respectively. This open-access repository allows all involved to quickly share all variants encountered and communicate potential consequences, which will be especially useful to classify variants of uncertain significance.

Barrett's oesophageal adenocarcinoma encompasses tumour-initiating cells that do not express common cancer stem cell markers.

Accumulating evidence has suggested that tumours have a hierarchical organization in which only the cancer stem cells (CSCs) have tumour-initiating properties. Several surface antigens have been employed to isolate CSCs from various malignancies, although not from oesophageal adenocarcinoma (EA). We tested whether Barrett's oesophagus (BE) and EA might serve as a model for the CSC concept. In vivo assays were performed by transplantation of serially diluted bulk EA cells into NOD-SCID mice to establish the presence and frequency of tumour-initiating cells. These were found to be present as ca. 1 in 64 000 cells. The transplanted tumours fully recapitulated the primary lesions. Subsequently, a panel of previously established CSC markers was employed for immunohistochemistry. CD24, CD29 and CD44 showed heterogeneous staining in EA. Nuclear beta-catenin accumulation increased during progression from metaplasia to dysplasia and was often observed in the basal compartment with CD24 and CD29 staining. However, the overall staining patterns were not such to clearly point out specific candidate markers. Accordingly, all markers were employed to sort the corresponding subpopulations of cancer cells and transplant them at low multiplicities in NOD-SCID mice. No increased tumour-initiating capacity of sorted EA cells was observed upon transplantation. These results indicate that tumour-initiating cells are present in EA, thus reflecting a hierarchical organization. However, antibodies directed against novel surface antigens are needed to detect subpopulations enriched for CSCs in EA by transplantation assays.

Progesterone inhibition of Wnt/beta-catenin signaling in normal endometrium and endometrial cancer.

PURPOSE: Wnt signaling regulates the fine balance between stemness and differentiation. Here, the role of Wnt signaling to maintain the balance between estrogen-induced proliferation and progesterone-induced differentiation during the menstrual cycle, as well as during the induction of hyperplasia and carcinogenesis of the endometrium, was investigated. EXPERIMENTAL DESIGN: Endometrial gene expression profiles from estradiol (E(2)) and E(2) + medroxyprogesterone acetate-treated postmenopausal patients were combined with profiles obtained during the menstrual cycle (PubMed; GEO DataSets). Ishikawa cells were transfected with progesterone receptors and Wnt inhibitors dickkopf homologue 1 (DKK1) and forkhead box O1 (FOXO1), measuring Wnt activation. Expression of DKK1 and FOXO1 was inhibited by use of sequence-specific short hairpins. Furthermore, patient samples (hormone-treated endometria, hyperplasia, and endometrial cancer) were stained for Wnt activation using nuclear beta-catenin and CD44. RESULTS: In vivo, targets and components of the Wnt signaling pathway (among them DKK1 and FOXO1) are regulated by E(2) and progesterone. In Wnt-activated Ishikawa cells, progesterone inhibits Wnt signaling by induction of DKK1 and FOXO1. Furthermore, using siRNA-mediated knockdown of both DKK1 and FOXO1, progesterone inhibition of Wnt signaling was partly circumvented. Subsequently, immunohistochemical analysis of the Wnt target gene CD44 showed that progesterone acted as an inhibitor of Wnt signaling in hyperplasia and in well-differentiated endometrial cancer. CONCLUSION: Progesterone induction of DKK1 and FOXO1 results in inhibition of Wnt signaling in the human endometrium. This Wnt inhibitory effect of progesterone is likely to play a rate-limiting role in the maintenance of endometrial homeostasis and, on its loss, in tumor onset and progression toward malignancy.

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/beta-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/beta-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/beta-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/beta-catenin signaling activity differentially affect tissue homeostasis and initiate tumorigenesis in an organ-specific fashion.

Generation of a tightly regulated doxycycline-inducible model for studying mouse intestinal biology.

To develop a sensitive and inducible system to study intestinal biology, we generated a transgenic mouse model expressing the reverse tetracycline transactivator rtTA2-M2 under control of the 12.4 kb murine Villin promoter. The newly generated Villin-rtTA2-M2 mice were then bred with the previously developed tetO-HIST1H2BJ/GFP model to assess inducibility and tissue-specificity. Expression of the histone H2B-GFP fusion protein was observed exclusively upon doxycycline induction and was uniformly distributed throughout the intestinal epithelium. The Villin-rtTA2-M2 was also found to drive transgene expression in the developing mouse intestine. Furthermore, we could detect transgene expression in the proximal tubules of the kidney and in a population of alleged gastric progenitor cells. By administering different concentrations of doxycycline, we show that the Villin-rtTA2-M2 system drives transgene expression in a dosage-dependent fashion. Thus, we have generated a novel doxycycline-inducible mouse model, providing a valuable tool to study the effect of different gene dosages on intestinal physiology and pathology.

Smad4 haploinsufficiency: a matter of dosage.

BACKGROUND: The inactivation of tumor suppressor genes follows Alfred Knudson's 'two-hit' model: both alleles need to be inactivated by independent mutation events to trigger tumor formation. However, in a minority of tumor suppressor genes a single hit is sufficient to initiate tumorigenesis notwithstanding the presence of the wild-type allele, a condition known as haploinsufficiency. The SMAD4 gene is an intracellular mediator of the TGF-beta and BMP signal transduction pathways and a tumor suppressor involved in pancreatic and colorectal tumorigenesis. In Smad4-mutant mouse models, haploinsufficiency characterizes the development of gastrointestinal polyps with initial retention of the wild-type allele and protein expression within the nascent tumors and in their direct microenvironment. Similarly, germline SMAD4 mutations are responsible for a subset of patients affected by juvenile polyposis syndrome, an autosomal dominant intestinal cancer syndrome. To date, the molecular and cellular consequences of SMAD4 haploinsufficiency on TGF-beta and BMP signaling and on genome-wide gene expression have not been investigated. RESULTS: Here we show that, similar to previous observations in Smad4-mutant mouse models, haploinsufficiency characterizes a substantial fraction of the juvenile polyps arising in patients with germline SMAD4 mutations. Also, mouse embryonic and intestinal cells heterozygous for a targeted Smad4 null mutation are characterized by a corresponding 50% reduction of the Smad4 protein levels. Reporter assays revealed that mouse Smad4+/- cells exert intermediate inhibitory effects on both TGF-beta and BMP signaling. Genome-wide expression profiling analysis of Smad4+/- and Smad4-/- cells pinpointed a subset of dosage-dependent transcriptional target genes encompassing, among others, members of the TGF-beta and Wnt signaling pathways. These SMAD4 dosage-dependent transcriptional changes were confirmed and validated in a subset of target genes in intestinal tissues from juvenile polyposis syndrome patients. CONCLUSION: Smad4 haploinsufficiency is sufficient to significantly inhibit both TGF-beta and BMP signal transduction and results in the differential expression of a broad subset of target genes likely to underlie tumor formation both from the mesenchymal and epithelial compartments. The results of our study, performed in normal rather than tumor cells where additional (epi-) genetic alterations may confound the analysis, are relevant for our understanding and elucidation of the initial steps underlying SMAD4-driven intestinal tumorigenesis.