The LIN28B-IMP1 post-transcriptional regulon has opposing effects on oncogenic signaling in the intestine.

RNA-binding proteins (RBPs) are expressed broadly during both development and malignant transformation, yet their mechanistic roles in epithelial homeostasis or as drivers of tumor initiation and progression are incompletely understood. Here we describe a novel interplay between RBPs LIN28B and IMP1 in intestinal epithelial cells. Ribosome profiling and RNA sequencing identified IMP1 as a principle node for gene expression regulation downstream from LIN28B In vitro and in vivo data demonstrate that epithelial IMP1 loss increases expression of WNT target genes and enhances LIN28B-mediated intestinal tumorigenesis, which was reversed when we overexpressed IMP1 independently in vivo. Furthermore, IMP1 loss in wild-type or LIN28B-overexpressing mice enhances the regenerative response to irradiation. Together, our data provide new evidence for the opposing effects of the LIN28B-IMP1 axis on post-transcriptional regulation of canonical WNT signaling, with implications in intestinal homeostasis, regeneration and tumorigenesis.

A single transcription factor is sufficient to induce and maintain secretory cell architecture.

We hypothesized that basic helix-loop-helix (bHLH) MIST1 (BHLHA15) is a "scaling factor" that universally establishes secretory morphology in cells that perform regulated secretion. Here, we show that targeted deletion of MIST1 caused dismantling of the secretory apparatus of diverse exocrine cells. Parietal cells (PCs), whose function is to pump acid into the stomach, normally lack MIST1 and do not perform regulated secretion. Forced expression of MIST1 in PCs caused them to expand their apical cytoplasm, rearrange mitochondrial/lysosome trafficking, and generate large secretory granules. Mist1 induced a cohort of genes regulated by MIST1 in multiple organs but did not affect PC function. MIST1 bound CATATG/CAGCTG E boxes in the first intron of genes that regulate autophagosome/lysosomal degradation, mitochondrial trafficking, and amino acid metabolism. Similar alterations in cell architecture and gene expression were also caused by ectopically inducing MIST1 in vivo in hepatocytes. Thus, MIST1 is a scaling factor necessary and sufficient by itself to induce and maintain secretory cell architecture. Our results indicate that, whereas mature cell types in each organ may have unique developmental origins, cells performing similar physiological functions throughout the body share similar transcription factor-mediated architectural "blueprints."

The oncogenic function of PLAGL2 is mediated via ASCL2 and IGF2 and a Wnt-independent mechanism in colorectal cancer.

Colorectal cancer (CRC) tumorigenesis and progression are linked to common oncogenic mutations, especially in the tumor suppressor APC, whose loss triggers the deregulation of TCF4/ß-Catenin activity. CRC tumorigenesis is also driven by multiple epimutational modifiers such as transcriptional regulators. We describe the common (and near-universal) activation of the zinc finger transcription factor and Let-7 target PLAGL2 in CRC and find that it is a key driver of intestinal epithelial transformation. PLAGL2 drives proliferation, cell cycle progression, and anchorage-independent growth in CRC cell lines and nontransformed intestinal cells. Investigating effects of PLAGL2 on downstream pathways revealed very modest effects on canonical Wnt signaling. Alternatively, we find pronounced effects on the direct PLAGL2 target genes IGF2, a fetal growth factor, and ASCL2, an intestinal stem cell-specific bHLH transcription factor. Inactivation of PLAGL2 in CRC cell lines has pronounced effects on ASCL2 reporter activity. Furthermore, ASCL2 expression can partially rescue deficits of proliferation and cell cycle progression caused by depletion of PLAGL2 in CRC cell lines. Thus, the oncogenic effects of PLAGL2 appear to be mediated via core stem cell and onco-fetal pathways, with minimal effects on downstream Wnt signaling.NEW & NOTEWORTHY A Let-7 target called PLAGL2 drives oncogenic transformation via Wnt-independent pathways. This work illustrates the robust effects of this zinc finger transcription factor in colorectal cancer (CRC) cell lines and nontransformed intestinal epithelium, with effects mediated, in part, via the direct target genes ASCL2 and IGF2. This has implications for the role of PLAGL2 in activation of onco-fetal and onco-stem cell pathways, contributing to immature and highly proliferative phenotypes in CRC.

Apobec1 complementation factor (A1CF) and RBM47 interact in tissue-specific regulation of C to U RNA editing in mouse intestine and liver.

Mammalian C to U RNA is mediated by APOBEC1, the catalytic deaminase, together with RNA binding cofactors (including A1CF and RBM47) whose relative physiological requirements are unresolved. Although A1CF complements APOBEC1 for in vitro RNA editing, A1cf-/- mice exhibited no change in apolipoproteinB (apoB) RNA editing, while Rbm47 mutant mice exhibited impaired intestinal RNA editing of apoB as well as other targets. Here we examined the role of A1CF and RBM47 in adult mouse liver and intestine, following deletion of either one or both gene products and also following forced (liver or intestinal) transgenic A1CF expression. There were minimal changes in hepatic and intestinal apoB RNA editing in A1cf-/- mice and no changes in either liver- or intestine-specific A1CF transgenic mice. Rbm47 liver-specific knockout (Rbm47LKO ) mice demonstrated reduced editing in a subset (11 of 20) of RNA targets, including apoB. By contrast, apoB RNA editing was virtually eliminated (<6% activity) in intestine-specific (Rbm47IKO ) mice with only five of 53 targets exhibiting C-to-U RNA editing. Double knockout of A1cf and Rbm47 in liver (ARLKO ) eliminated apoB RNA editing and reduced editing in the majority of other targets, with no changes following adenoviral APOBEC1 administration. Intestinal double knockout mice (ARIKO ) demonstrated further reduced editing (<10% activity) in four of five of the residual APOBEC1 targets identified in ARIKO mice. These data suggest that A1CF and RBM47 each function independently, yet interact in a tissue-specific manner, to regulate the activity and site selection of APOBEC1 dependent C-to-U RNA editing.

LIN28B promotes growth and tumorigenesis of the intestinal epithelium via Let-7.

The RNA-binding proteins LIN28A and LIN28B have diverse functions in embryonic stem cells, cellular reprogramming, growth, and oncogenesis. Many of these effects occur via direct inhibition of Let-7 microRNAs (miRNAs), although Let-7-independent effects have been surmised. We report that intestine targeted expression of LIN28B causes intestinal hypertrophy, crypt expansion, and Paneth cell loss. Furthermore, LIN28B fosters intestinal polyp and adenocarcinoma formation. To examine potential Let-7-independent functions of LIN28B, we pursued ribonucleoprotein cross-linking, immunoprecipitation, and high-throughput sequencing (CLIP-seq) to identify direct RNA targets. This revealed that LIN28B bound a substantial number of mRNAs and modestly augmented protein levels of these target mRNAs in vivo. Conversely, Let-7 had a profound effect; modulation of Let-7 levels via deletion of the mirLet7c2/mirLet7b genes recapitulated effects of Lin28b overexpression. Furthermore, intestine-specific Let-7 expression could reverse hypertrophy and Paneth cell depletion caused by Lin28b. This was independent of effects on insulin-PI3K-mTOR signaling. Our study reveals that Let-7 miRNAs are critical for repressing intestinal tissue growth and promoting Paneth cell differentiation. Let-7-dependent effects of LIN28B may supersede Let-7-independent effects on intestinal tissue growth. In summary, LIN28B can definitively act as an oncogene in the absence of canonical genetic alterations.

Interferon-γ directly induces gastric epithelial cell death and is required for progression to metaplasia.

Chronic inflammation of the gastric mucosa, often caused by autoimmune gastritis and/or infection with Helicobacter pylori, can lead to atrophy of acid-secreting parietal cells with metaplasia of remaining cells. The histological pattern marks a critical step in the progression from chronic gastritis to gastric cancer, yet underlying mechanism(s) of inflammation-induced cell death of gastric epithelial cells are poorly understood. We investigated direct effects of a type 1 cytokine associated with autoimmunity and infection, interferon-γ (IFN-γ), on gastric epithelial cells. IFN-γ was applied to three-dimensional organoid cultures of gastric epithelial cells derived from gastric corpus gland (gastroids) of control and IFN-γ receptor-deficient mice. Gastroids were also treated with supernatants from activated immune cells isolated from a mouse model of autoimmune-mediated atrophic gastritis (TxA23) with and without IFN-γ expression. Finally, histopathological analysis of atrophy and metaplasia severity was performed in TxA23 mice and compared to TxA23 × Ifng-/- mice. Gastric epithelial cells in gastroid cultures expressed IFN-γ receptor in the basolateral membrane, and gastroids died when treated with IFN-γ in an IFN-γ receptor-dependent manner. Supernatants from immune cells containing high levels of IFN-γ were highly toxic to gastroids, and toxicity was tempered when IFN-γ was either neutralized using a monoclonal antibody or when supernatants from Ifng-/- mouse immune cells were used. Finally, TxA23 × Ifng-/- mice showed near-complete abrogation of pre-cancerous histopathological atrophy and metaplasia versus IFN-γ-sufficient controls. We identify IFN-γ as a critical promoter of parietal cell atrophy with metaplasia during the progression of gastritis to gastric atrophy and metaplasia. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

A stable but reversible integrated surrogate reporter for assaying CRISPR/Cas9-stimulated homology-directed repair.

The discovery and application of CRISPR/Cas9 technology for genome editing has greatly accelerated targeted mutagenesis in a variety of organisms. CRISPR/Cas9-mediated site-specific cleavage is typically exploited for the generation of insertions or deletions (indels) after aberrant dsDNA repair via the endogenous non-homology end-joining (NHEJ) pathway or, alternatively, for enhancing homology-directed repair to facilitate the generation of a specific mutation (or "knock-in"). However, there is a need for efficient cellular assays that can measure Cas9/guide RNA activity. Reliable methods for enriching and identifying desired mutants are also lacking. Here we describe a method using the Piggybac transposon for stable genomic integration of an H2B-GFP reporter or a hygromycin resistance gene for assaying Cas9 target cleavage and homology-directed repair. The H2B-GFP fusion protein provides increased stability and an obvious pattern of nuclear localization. This method, called SRIRACCHA (i.e. a stable, but reversible, integrated reporter for assaying CRISPR/Cas-stimulated HDR activity), enables the enrichment of mutants via selection of GFP-positive or hygromycin-resistant mammalian cells (immortalized or non-immortalized) as a surrogate for the modification of the endogenous target site. Currently available hyperactive Piggybac transposase mutants allow both delivery and removal of the surrogate reporters, with minimal risk of generating undesirable mutations. This assay permits rapid screening for efficient guide RNAs and the accelerated identification of mutant clones and is applicable to many cell types. We foresee the utility of this approach in contexts in which the maintenance of genomic integrity is essential, for example, when engineering cells for therapeutic purposes.

Let-7 Represses Carcinogenesis and a Stem Cell Phenotype in the Intestine via Regulation of Hmga2.

Let-7 miRNAs comprise one of the largest and most highly expressed family of miRNAs among vertebrates, and is critical for promoting differentiation, regulating metabolism, inhibiting cellular proliferation, and repressing carcinogenesis in a variety of tissues. The large size of the Let-7 family of miRNAs has complicated the development of mutant animal models. Here we describe the comprehensive repression of all Let-7 miRNAs in the intestinal epithelium via low-level tissue-specific expression of the Lin28b RNA-binding protein and a conditional knockout of the MirLet7c-2/Mirlet7b locus. This ablation of Let-7 triggers the development of intestinal adenocarcinomas concomitant with reduced survival. Analysis of both mouse and human intestinal cancer specimens reveals that stem cell markers were significantly associated with loss of Let-7 miRNA expression, and that a number of Let-7 targets were elevated, including Hmga1 and Hmga2. Functional studies in 3-D enteroids revealed that Hmga2 is necessary and sufficient to mediate many characteristics of Let-7 depletion, namely accelerating cell cycle progression and enhancing a stem cell phenotype. In addition, inactivation of a single Hmga2 allele in the mouse intestine epithelium significantly represses tumorigenesis driven by Lin28b. In aggregate, we conclude that Let-7 depletion drives a stem cell phenotype and the development of intestinal cancer, primarily via Hmga2.

A Novel CRISPR/Cas9-mediated Mouse Model of Colon Carcinogenesis.

BACKGROUND & AIMS: Human sporadic colorectal cancer (CRC) results from a multistep pathway with sequential acquisition of specific genetic mutations in the colorectal epithelium. Modeling CRC in vivo is critical for understanding the tumor microenvironment. To accurately recapitulate human CRC pathogenesis, mouse models must include these multi-step genetic abnormalities. The aim of this study was to generate a sporadic CRC model that more closely mimics this multi-step process and to use this model to study the role of a novel Let7 target PLAGL2 in CRC pathogenesis. METHODS: We generated a CRISPR/Cas9 somatic mutagenesis mouse model that is inducible and multiplexed for simultaneous inactivation of multiple genes involved in CRC pathogenesis. We used both a doxycycline-inducible transcriptional activator and a doxycycline-inactivated transcriptional repressor to achieve tight, non-leaky expression of the Cas9 nickase. This mouse has transgenic expression of multiple guide RNAs to induce sporadic inactivation in the gut epithelium of 4 tumor suppressor genes commonly mutated in CRC, Apc, Pten, Smad4, and Trp53. These were crossed to Vil-LCL-PLAGL2 mice, which have Cre-inducible overexpression of PLAGL2 in the gut epithelium. RESULTS: These mice exhibited random somatic mutations in all 4 targeted tumor suppressor genes, resulting in multiple adenomas and adenocarcinomas in the small bowel and colon. Crosses with Vil-LCL-PLAGL2 mice demonstrated that gut-specific PLAGL2 overexpression increased colon tumor growth. CONCLUSIONS: This conditional model represents a new CRISPR/Cas9-mediated mouse model of colorectal carcinogenesis. These mice can be used to investigate the role of novel, previously uncharacterized genes in CRC, in the context of multiple commonly mutated tumor suppressor genes and thus more closely mimic human CRC pathogenesis.

Hedgehog signaling controls homeostasis of adult intestinal smooth muscle.

The Hedgehog (Hh) pathway plays multiple patterning roles during development of the mammalian gastrointestinal tract, but its role in adult gut function has not been extensively examined. Here we show that chronic reduction in the combined epithelial Indian (Ihh) and Sonic (Shh) hedgehog signal leads to mislocalization of intestinal subepithelial myofibroblasts, loss of smooth muscle in villus cores and muscularis mucosa as well as crypt hyperplasia. In contrast, chronic over-expression of Ihh in the intestinal epithelium leads to progressive expansion of villus smooth muscle, but does not result in reduced epithelial proliferation. Together, these mouse models show that smooth muscle populations in the adult intestinal lamina propria are highly sensitive to the level of Hh ligand. We demonstrate further that Hh ligand drives smooth muscle differentiation in primary intestinal mesenchyme cultures and that cell-autonomous Hh signal transduction in C3H10T1/2 cells activates the smooth muscle master regulator Myocardin (Myocd) and induces smooth muscle differentiation. The rapid kinetics of Myocd activation by Hh ligands as well as the presence of an unusual concentration of Gli sties in this gene suggest that regulation of Myocd by Hh might be direct. Thus, these data indicate that Hh is a critical regulator of adult intestinal smooth muscle homeostasis and suggest an important link between Hh signaling and Myocd activation. Moreover, the data support the idea that lowered Hh signals promote crypt expansion and increased epithelial cell proliferation, but indicate that chronically increased Hh ligand levels do not dampen crypt proliferation as previously proposed.

Erratum: Cas-CLOVER is a novel high-fidelity nuclease for safe and robust generation of TSCM-enriched allogeneic CAR-T cells.

[This corrects the article DOI: 10.1016/j.omtn.2022.06.003.].

Cas-CLOVER is a novel high-fidelity nuclease for safe and robust generation of TSCM-enriched allogeneic CAR-T cells.

The use of T cells from healthy donors for allogeneic chimeric antigen receptor T (CAR-T) cell cancer therapy is attractive because healthy donor T cells can produce versatile off-the-shelf CAR-T treatments. To maximize safety and durability of allogeneic products, the endogenous T cell receptor and major histocompatibility complex class I molecules are often removed via knockout of T cell receptor beta constant (TRBC) (or T cell receptor alpha constant [TRAC]) and B2M, respectively. However, gene editing tools (e.g., CRISPR-Cas9) can display poor fidelity, which may result in dangerous off-target mutations. Additionally, many gene editing technologies require T cell activation, resulting in a low percentage of desirable stem cell memory T cells (TSCM). We characterize an RNA-guided endonuclease, called Cas-CLOVER, consisting of the Clo051 nuclease domain fused with catalytically dead Cas9. In primary T cells from multiple donors, we find that Cas-CLOVER is a high-fidelity site-specific nuclease, with low off-target activity. Notably, Cas-CLOVER yields efficient multiplexed gene editing in resting T cells. In conjunction with the piggyBac transposon for delivery of a CAR transgene against the B cell maturation antigen (BCMA), we produce allogeneic CAR-T cells composed of high percentages of TSCM cells and possessing potent in vivo anti-tumor cytotoxicity.

Hedgehog is an anti-inflammatory epithelial signal for the intestinal lamina propria.

BACKGROUND & AIMS: Epithelial Hedgehog (Hh) ligands regulate several aspects of fetal intestinal organogenesis, and emerging data implicate the Hh pathway in inflammatory signaling in the adult colon. Here, we investigated the effects of chronic Hh inhibition in vivo and profiled molecular pathways acutely modulated by Hh signaling in the intestinal mesenchyme. METHODS: The progression of inflammatory disease was characterized in a bi-transgenic mouse model of chronic Hh inhibition (VFHhip). In parallel, microarray and bioinformatic analyses (Gene Ontology terms overrepresentation analysis, hierarchical clustering, and MeSH term filtration) were performed on isolated cultured intestinal mesenchyme acutely exposed to Hh ligand. RESULTS: Six- to 10-month-old VFHhip animals exhibited villus smooth muscle loss and subsequent villus atrophy. Areas of villus loss became complicated by spontaneous inflammation and VFHhip animals succumbed to wasting and death. Phenotypic similarities were noted between the VFHhip phenotype and human inflammatory disorders, especially human celiac disease. Microarray analysis revealed that inflammatory pathways were acutely activated in intestinal mesenchyme cultured in the absence of epithelium, and the addition of Hh ligand alone was sufficient to largely reverse this inflammatory response within 24 hours. CONCLUSIONS: Hh ligand is a previously unrecognized anti-inflammatory epithelial modulator of the mesenchymal inflammatory milieu. Acute modulation of Hh signals results in changes in inflammatory pathways in intestinal mesenchyme, while chronic inhibition of Hh signaling in adult animals leads to spontaneous intestinal inflammation and death. Regulation of epithelial Hh signaling may be an important mechanism to modulate tolerogenic versus proinflammatory signaling in the small intestine.

Conditional deletion of beta1 integrins in the intestinal epithelium causes a loss of Hedgehog expression, intestinal hyperplasia, and early postnatal lethality.

Conditional deletion of beta1 integrins in the intestinal epithelium, unlike in epidermal and mammary epithelia, of mice does not result in decreased cell adhesion and proliferation, but instead causes a profound increase in epithelial proliferation with dysplasia and polypoid structures. The increased epithelial proliferation inhibited epithelial differentiation that caused severe malnutrition and early postnatal lethality. The striking similarities between beta1 integrin-deleted mice and neonatal mice with defective Hedgehog signaling led to the discovery that Hedgehog expression was markedly reduced in the former mice. beta1 integrins were found to drive the expression of Hedgehogs in intestinal epithelial cells in an HNF-3beta (Foxa2)-dependent fashion. The expression of Tcf-4, a transcription factor known to be required for intestinal epithelial stem cell proliferation, was increased and mislocalized in the intestinal epithelia of the beta1 integrin-deleted mice and in newborn mice treated with the Hedgehog signaling inhibitor cyclopamine. This study shows that beta1 integrins are key regulators of proliferation and homeostasis in the intestine and achieve this not through anchorage-dependent effects but by generating Hh expression and signaling.

Apobec1 complementation factor overexpression promotes hepatic steatosis, fibrosis, and hepatocellular cancer.

The RNA-binding protein Apobec1 complementation factor (A1CF) regulates posttranscriptional ApoB mRNA editing, but the range of RNA targets and the long-term effect of altered A1CF expression on liver function are unknown. Here we studied hepatocyte-specific A1cf-transgenic (A1cf+/Tg), A1cf+/Tg Apobec1-/-, and A1cf-/- mice fed chow or high-fat/high-fructose diets using RNA-Seq, RNA CLIP-Seq, and tissue microarrays from human hepatocellular cancer (HCC). A1cf+/Tg mice exhibited increased hepatic proliferation and steatosis, with increased lipogenic gene expression (Mogat1, Mogat2, Cidea, Cd36) associated with shifts in polysomal RNA distribution. Aged A1cf+/Tg mice developed spontaneous fibrosis, dysplasia, and HCC, and this development was accelerated on a high-fat/high-fructose diet and was independent of Apobec1. RNA-Seq revealed increased expression of mRNAs involved in oxidative stress (Gstm3, Gpx3, Cbr3), inflammatory response (Il19, Cxcl14, Tnfα, Ly6c), extracellular matrix organization (Mmp2, Col1a1, Col4a1), and proliferation (Kif20a, Mcm2, Mcm4, Mcm6), and a subset of mRNAs (including Sox4, Sox9, Cdh1) were identified in RNA CLIP-Seq. Increased A1CF expression in human HCC correlated with advanced fibrosis and with reduced survival in a subset with nonalcoholic fatty liver disease. In conclusion, we show that hepatic A1CF overexpression selectively alters polysomal distribution and mRNA expression, promoting lipogenic, proliferative, and inflammatory pathways leading to HCC.

Paracrine Hedgehog signaling in stomach and intestine: new roles for hedgehog in gastrointestinal patterning.

BACKGROUND & AIMS: Hedgehog signaling is critical in gastrointestinal patterning. Mice deficient in Hedgehog signaling exhibit abnormalities that mirror deformities seen in the human VACTERL (vertebral, anal, cardiac, tracheal, esophageal, renal, limb) association. However, the direction of Hedgehog signal flow is controversial and the cellular targets of Hedgehog signaling change with time during development. We profiled cellular Hedgehog response patterns from embryonic day 10.5 (E10.5) to adult in murine antrum, pyloric region, small intestine, and colon. METHODS: Hedgehog signaling was profiled using Hedgehog pathway reporter mice and in situ hybridization. Cellular targets were identified by immunostaining. Ihh-overexpressing transgenic animals were generated and analyzed. RESULTS: Hedgehog signaling is strictly paracrine from antrum to colon throughout embryonic and adult life. Novel findings include the following: mesothelial cells of the serosa transduce Hedgehog signals in fetal life; the hindgut epithelium expresses Ptch but not Gli1 at E10.5; the 2 layers of the muscularis externa respond differently to Hedgehog signals; organogenesis of the pyloric sphincter is associated with robust Hedgehog signaling; dramatically different Hedgehog responses characterize stomach and intestine at E16; and after birth, the muscularis mucosa and villus smooth muscle consist primarily of Hedgehog-responsive cells and Hh levels actively modulate villus core smooth muscle. CONCLUSIONS: These studies reveal a previously unrecognized association of paracrine Hedgehog signaling with several gastrointestinal patterning events involving the serosa, pylorus, and villus smooth muscle. The results may have implications for several human anomalies and could potentially expand the spectrum of the human VACTERL association.

Prospective identification of a multilineage progenitor in murine stomach epithelium.

BACKGROUND & AIMS: Epithelial stem cells in the stomach are responsible for constant renewal of the epithelium through generation of multiple gastric cell lineages that populate the gastric glands. However, gastric stem or progenitor cells have not been well-characterized because of the lack of specific markers that permit their prospective recognition. We identified an intestinal promoter that is active in a rare subpopulation of gastric epithelial cells and investigated whether these cells possess multilineage potential. METHODS: A marked allele of the endogenous mouse villin locus was used to visualize single beta-galactosidase-positive cells located in the lower third of antral glands. A 12.4-kb villin promoter/enhancer fragment drives several transgenes (EGFP, beta-galactosidase, and Cre recombinase) in these cells in a pattern similar to that of the marked villin allele. Reporter gene activity was used to track these cells during development and to examine cell number in the context of inflammatory challenge while Cre activity allowed lineage tracing in vivo. RESULTS: We show that these rare epithelial cells are normally quiescent, but multiply in response to interferon gamma. Lineage tracing studies confirm that these cells give rise to all gastric lineages of the antral glands. In the embryo, these cells are located basally in the stomach epithelium before completion of gastric gland morphogenesis. CONCLUSIONS: We have identified a rare subpopulation of gastric progenitors with multilineage potential. The ability to prospectively identify and manipulate such progenitors in situ represents a major step forward in gastric stem cell biology and has potential implications for gastric cancer.

Transforming growth factor beta receptor type II inactivation induces the malignant transformation of intestinal neoplasms initiated by Apc mutation.

The transforming growth factor-beta (TGF-beta) signaling pathway is a tumor-suppressor pathway that is commonly inactivated in colon cancer. TGF-beta is a secreted ligand that mediates its effects through a transmembrane heteromeric receptor complex, which consists of type I (TGFBR1) and type II subunits (TGFBR2). Approximately 30% of colon cancers carry TGFBR2 mutations, demonstrating that it is a common target for mutational inactivation in this cancer. To assess the functional role of TGFBR2 inactivation in the multistep progression sequence of colon cancer, we generated a mouse model that recapitulates two common genetic events observed in human colon cancer by mating Apc(1638N/wt) mice with mice that are null for Tgfbr2 in the intestinal epithelium, Villin-Cre;Tgfbr2(E2flx/E2flx) mice. In this model, we observed a dramatic increase in the number of intestinal adenocarcinomas in the Apc(1638N/wt);Villin-Cre;Tgfbr2(E2flx/E2flx) mice (called Apc(1638N/wt);Tgfbr2(IEKO)) compared with those mice with intact Tgfbr2 (Apc(1638N/wt);Tgfbr2(E2flx/E2flx)). Additionally, in vitro analyses of epithelial tumor cells derived from the Apc(1638N/wt);Tgfbr2(IEKO) mice showed enhanced expression and activity of matrix metalloproteinase MMP-2 and MMP-9, as well as increased TGF-beta1 secretion in the conditioned medium. Similarly, primary tumor tissues from the Apc(1638N/wt);Tgfbr2(IEKO) mice also showed elevated amounts of TGF-beta1 as well as higher MMP-2 activity in comparison with Apc(1638N/wt);Tgfbr2(E2flx/E2flx)-derived tumors. Thus, loss of TGFBR2 in intestinal epithelial cells promotes the invasion and malignant transformation of tumors initiated by Apc mutation, providing evidence that Wnt signaling deregulation and TGF-beta signaling inactivation cooperate to drive the initiation and progression, respectively, of intestinal cancers in vivo.