The pan-ErbB negative regulator Lrig1 is an intestinal stem cell marker that functions as a tumor suppressor.

Lineage mapping has identified both proliferative and quiescent intestinal stem cells, but the molecular circuitry controlling stem cell quiescence is incompletely understood. By lineage mapping, we show Lrig1, a pan-ErbB inhibitor, marks predominately noncycling, long-lived stem cells that are located at the crypt base and that, upon injury, proliferate and divide to replenish damaged crypts. Transcriptome profiling of Lrig1(+) colonic stem cells differs markedly from the profiling of highly proliferative, Lgr5(+) colonic stem cells; genes upregulated in the Lrig1(+) population include those involved in cell cycle repression and response to oxidative damage. Loss of Apc in Lrig1(+) cells leads to intestinal adenomas, and genetic ablation of Lrig1 results in heightened ErbB1-3 expression and duodenal adenomas. These results shed light on the relationship between proliferative and quiescent intestinal stem cells and support a model in which intestinal stem cell quiescence is maintained by calibrated ErbB signaling with loss of a negative regulator predisposing to neoplasia.

Lrig1+ gastric isthmal progenitor cells restore normal gastric lineage cells during damage recovery in adult mouse stomach.

OBJECTIVE: Lrig1 is a marker of proliferative and quiescent stem cells in the skin and intestine. We examined whether Lrig1-expressing cells are long-lived gastric progenitors in gastric glands in the mouse stomach. We also investigated how the Lrig1-expressing progenitor cells contribute to the regeneration of normal gastric mucosa by lineage commitment to parietal cells after acute gastric injury in mice. DESIGN: We performed lineage labelling using Lrig1-CreERT2/+;R26R-YFP/+ (Lrig1/YFP) or R26R-LacZ/+ (Lrig1/LacZ) mice to examine whether the Lrig1-YFP-marked cells are gastric progenitor cells. We studied whether Lrig1-YFP-marked cells give rise to normal gastric lineage cells in damaged mucosa using Lrig1/YFP mice after treatment with DMP-777 to induce acute injury. We also studied Lrig1-CreERT2/CreERT2 (Lrig1 knockout) mice to examine whether the Lrig1 protein is required for regeneration of gastric corpus mucosa after acute injury. RESULTS: Lrig1-YFP-marked cells give rise to gastric lineage epithelial cells both in the gastric corpus and antrum, in contrast to published results that Lgr5 only marks progenitor cells within the gastric antrum. Lrig1-YFP-marked cells contribute to replacement of damaged gastric oxyntic glands during the recovery phase after acute oxyntic atrophy in the gastric corpus. Lrig1 null mice recovered normally from acute gastric mucosal injury indicating that Lrig1 protein is not required for lineage differentiation. Lrig1+ isthmal progenitor cells did not contribute to transdifferentiating chief cell lineages after acute oxyntic atrophy. CONCLUSIONS: Lrig1 marks gastric corpus epithelial progenitor cells capable of repopulating the damaged oxyntic mucosa by differentiating into normal gastric lineage cells in mouse stomach.

LRIG1 Regulates Ontogeny of Smooth Muscle-Derived Subsets of Interstitial Cells of Cajal in Mice.

BACKGROUND & AIMS: Interstitial cells of Cajal (ICC) control intestinal smooth muscle contraction to regulate gut motility. ICC within the plane of the myenteric plexus (ICC-MY) arise from KIT-positive progenitor cells during mouse embryogenesis. However, little is known about the ontogeny of ICC associated with the deep muscular plexus (ICC-DMP) in the small intestine and ICC associated with the submucosal plexus (ICC-SMP) in the colon. Leucine-rich repeats and immunoglobulin-like domains protein 1 (LRIG1) marks intestinal epithelial stem cells, but the role of LRIG1 in nonepithelial intestinal cells has not been identified. We sought to determine the ontogeny of ICC-DMP and ICC-SMP, and whether LRIG1 has a role in their development. METHODS: Lrig1-null mice (homozygous Lrig1-CreERT2) and wild-type mice were analyzed by immunofluorescence and transit assays. Transit was evaluated by passage of orally administered rhodamine B-conjugated dextran. Lrig1-CreERT2 mice or mice with CreERT2 under control of an inducible smooth muscle promoter (Myh11-CreERT2) were crossed with Rosa26-LSL-YFP mice for lineage tracing analysis. RESULTS: In immunofluorescence assays, ICC-DMP and ICC-SMP were found to express LRIG1. Based on lineage tracing, ICC-DMP and ICC-SMP each arose from LRIG1-positive smooth muscle progenitors. In Lrig1-null mice, there was loss of staining for KIT in DMP and SMP regions, as well as for 2 additional ICC markers (anoctamin-1 and neurokinin 1 receptor). Lrig1-null mice had significant delays in small intestinal transit compared with control mice. CONCLUSIONS: LRIG1 regulates the postnatal development of ICC-DMP and ICC-SMP from smooth muscle progenitors in mice. Slowed small intestinal transit observed in Lrig1-null mice may be due, at least in part, to loss of the ICC-DMP population.

Inducible loss of one Apc allele in Lrig1-expressing progenitor cells results in multiple distal colonic tumors with features of familial adenomatous polyposis.

Individuals with familial adenomatous polyposis (FAP) harbor a germline mutation in adenomatous polyposis coli (APC). The major clinical manifestation is development of multiple colonic tumors at a young age due to stochastic loss of the remaining APC allele. Extracolonic features, including periampullary tumors, gastric abnormalities, and congenital hypertrophy of the retinal pigment epithelium, may occur. The objective of this study was to develop a mouse model that simulates these features of FAP. We combined our Lrig1-CreERT2/+ mice with Apcfl/+ mice, eliminated one copy of Apc in leucine-rich repeats and immunoglobulin-like domains protein 1 (Lrig1)-positive (Lrig1(+)) progenitor cells with tamoxifen injection, and monitored tumor formation in the colon by colonoscopy and PET. Initial loss of one Apc allele in Lrig1(+) cells results in a predictable pattern of preneoplastic changes, culminating in multiple distal colonic tumors within 50 days of induction, as well as the extracolonic manifestations of FAP mentioned above. We show that tumor formation can be monitored by noninvasive PET imaging. This inducible stem cell-driven model recapitulates features of FAP and offers a tractable platform on which therapeutic interventions can be monitored over time by colonoscopy and noninvasive imaging.

Pathology of rodent models of intestinal cancer: progress report and recommendations.

In October 2010, a pathology review of rodent models of intestinal neoplasia was held at The Jackson Laboratory. This review complemented 2 other concurrent events: a workshop on methods of modeling colon cancer in rodents and a conference on current issues in murine and human colon cancer. We summarize the results of the pathology review and the committee's recommendations for tumor nomenclature. A virtual high-resolution image slide box of these models has been developed. This report discusses significant recent developments in rodent modeling of intestinal neoplasia, including the role of stem cells in cancer and the creation of models of metastatic intestinal cancer.

Characterization of the intestinal cancer stem cell marker CD166 in the human and mouse gastrointestinal tract.

BACKGROUND & AIMS: CD166 (also called activated leukocyte cell adhesion molecule [ALCAM]) is a marker of colorectal cancer (CRC) stem cells; it is expressed by aggressive tumors. Although the presence of CD166 at the tumor cell surface has been correlated with shortened survival, little is known about its function and expression in normal intestinal epithelia. METHODS: We characterized the expression pattern of CD166 in normal intestinal tissue samples from humans and mice using immunohistochemisty, flow cytometry, and quantitative reverse-transcriptase polymerase chain reaction. Human and mouse intestinal tumors were also analyzed. RESULTS: CD166 was expressed on the surface of epithelial cells within the stem cell niche and along the length of the intestine; expression was conserved across species. In the small intestine, CD166 was observed on crypt-based Paneth cells and intervening crypt-based columnar cells (putative stem cells). A subset of CD166-positive, crypt-based columnar cells coexpressed the stem cell markers Lgr5, Musashi-1, or Dcamkl-1. CD166 was located in the cytoplasm and at the surface of cells within human CRC tumors. CD166-positive cells were also detected in benign adenomas in mice; rare cells coexpressed CD166 and CD44 or epithelial-specific antigen. CONCLUSIONS: CD166 is highly expressed within the endogenous intestinal stem cell niche. CD166-positive cells appear at multiple stages of intestinal carcinoma progression, including benign and metastatic tumors. Further studies should investigate the function of CD166 in stem cells and the stem cell niche, which might have implications for normal intestinal homeostasis. CD166 has potential as a therapeutic target for CRC.

Wnt-reporter expression pattern in the mouse intestine during homeostasis.

BACKGROUND: The canonical Wnt signaling pathway is a known regulator of cell proliferation during development and maintenance of the intestinal epithelium. Perturbations in this pathway lead to aberrant epithelial proliferation and intestinal cancer. In the mature intestine, proliferation is confined to the relatively quiescent stem cells and the rapidly cycling transient-amplifying cells in the intestinal crypts. Although the Wnt signal is believed to regulate all proliferating intestinal cells, surprisingly, this has not been thoroughly demonstrated. This important determination has implications on intestinal function, especially during epithelial expansion and regeneration, and warrants an extensive characterization of Wnt-activated cells. METHODS: To identify intestinal epithelial cells that actively receive a Wnt signal, we analyzed intestinal Wnt-reporter expression patterns in two different mouse lines using immunohistochemistry, enzymatic activity, in situ hybridization and qRT-PCR, then corroborated results with reporter-independent analyses. Wnt-receiving cells were further characterized for co-expression of proliferation markers, putative stem cell markers and cellular differentiation markers using an immunohistochemical approach. Finally, to demonstrate that Wnt-reporter mice have utility in detecting perturbations in intestinal Wnt signaling, the reporter response to gamma-irradiation was examined. RESULTS: Wnt-activated cells were primarily restricted to the base of the small intestinal and colonic crypts, and were highest in numbers in the proximal small intestine, decreasing in frequency in a gradient toward the large intestine. Interestingly, the majority of the Wnt-reporter-expressing cells did not overlap with the transient-amplifying cell population. Further, while Wnt-activated cells expressed the putative stem cell marker Musashi-1, they did not co-express DCAMKL-1 or cell differentiation markers. Finally, gamma-irradiation stimulated an increase in Wnt-activated intestinal crypt cells. CONCLUSION: We show, for the first time, detailed characterization of the intestine from Wnt-reporter mice. Further, our data show that the majority of Wnt-receiving cells reside in the stem cell niche of the crypt base and do not extend into the proliferative transient-amplifying cell population. We also show that the Wnt-reporter mice can be used to detect changes in intestinal epithelial Wnt signaling upon physiologic injury. Our findings have an important impact on understanding the regulation of the intestinal stem cell hierarchy during homeostasis and in disease states.

p120-Catenin is an obligate haploinsufficient tumor suppressor in intestinal neoplasia.

p120-Catenin (p120) functions as a tumor suppressor in intestinal cancer, but the mechanism is unclear. Here, using conditional p120 knockout in Apc-sensitized mouse models of intestinal cancer, we have identified p120 as an "obligatory" haploinsufficient tumor suppressor. Whereas monoallelic loss of p120 was associated with a significant increase in tumor multiplicity, loss of both alleles was never observed in tumors from these mice. Moreover, forced ablation of the second allele did not further enhance tumorigenesis, but instead induced synthetic lethality in combination with Apc loss of heterozygosity. In tumor-derived organoid cultures, elimination of both p120 alleles resulted in caspase-3-dependent apoptosis that was blocked by inhibition of Rho kinase (ROCK). With ROCK inhibition, however, p120-ablated organoids exhibited a branching phenotype and a substantial increase in cell proliferation. Access to data from Sleeping Beauty mutagenesis screens afforded an opportunity to directly assess the tumorigenic impact of p120 haploinsufficiency relative to other candidate drivers. Remarkably, p120 ranked third among the 919 drivers identified. Cofactors α-catenin and epithelial cadherin (E-cadherin) were also among the highest scoring candidates, indicating a mechanism at the level of the intact complex that may play an important role at very early stages of of intestinal tumorigenesis while simultaneously restricting outright loss via synthetic lethality.

Using a new Lrig1 reporter mouse to assess differences between two Lrig1 antibodies in the intestine.

Lrig1 is an intestinal stem cell marker important for epithelial homeostasis. However, the position of the Lrig1(+) population in the intestinal crypt has been debated, largely due to discrepant staining patterns using two Lrig1 antibodies. Here, we set out to decipher the differences between these Lrig1 antibodies to clarify their use for Lrig1-related studies. We confirmed that the commercially available Lrig1-R&D antibody stained the bottom third of the colonic crypt, whereas an independently generated Lrig1-VU antibody recognized a subset of anti-Lrig1-R&D(+) cells. Biochemically, we found that anti-Lrig1-VU recognized a non-glycosylated form of Lrig1; in contrast, anti-Lrig1-R&D recognized both glycosylated and non-glycosylated forms of Lrig1. In addition, we generated a reporter mouse (Lrig1-Apple) as an independent readout of Lrig1 transcriptional activity. Flow cytometry of isolated colonic epithelial cells from Lrig1-Apple mice demonstrated anti-Lrig1-R&D recognized mostly RFP-hi cells, while anti-Lrig1-VU recognized cells that were largely RFP-mid. Of note, by qRT-PCR, Lgr5 was expressed in the RFP-hi population, but not in the RFP-mid population. We conclude that anti-Lrig1-R&D appears to recognize all Lrig1(+) cells, while anti-Lrig1-VU recognizes a subpopulation of Lrig1(+) cells.

Excess PLAC8 promotes an unconventional ERK2-dependent EMT in colon cancer.

The epithelial-to-mesenchymal transition (EMT) transcriptional program is characterized by repression of E-cadherin (CDH1) and induction of N-cadherin (CDH2), and mesenchymal genes like vimentin (VIM). Placenta-specific 8 (PLAC8) has been implicated in colon cancer; however, how PLAC8 contributes to disease is unknown, and endogenous PLAC8 protein has not been studied. We analyzed zebrafish and human tissues and found that endogenous PLAC8 localizes to the apical domain of differentiated intestinal epithelium. Colon cancer cells with elevated PLAC8 levels exhibited EMT features, including increased expression of VIM and zinc finger E-box binding homeobox 1 (ZEB1), aberrant cell motility, and increased invasiveness. In contrast to classical EMT, PLAC8 overexpression reduced cell surface CDH1 and upregulated P-cadherin (CDH3) without affecting CDH2 expression. PLAC8-induced EMT was linked to increased phosphorylated ERK2 (p-ERK2), and ERK2 knockdown restored cell surface CDH1 and suppressed CDH3, VIM, and ZEB1 upregulation. In vitro, PLAC8 directly bound and inactivated the ERK2 phosphatase DUSP6, thereby increasing p-ERK2. In a murine xenograft model, knockdown of endogenous PLAC8 in colon cancer cells resulted in smaller tumors, reduced local invasion, and decreased p-ERK2. Using MultiOmyx, a multiplex immunofluorescence-based methodology, we observed coexpression of cytosolic PLAC8, CDH3, and VIM at the leading edge of a human colorectal tumor, supporting a role for PLAC8 in cancer invasion in vivo.

Helicobacter pylori promotes the expression of Krüppel-like factor 5, a mediator of carcinogenesis, in vitro and in vivo.

Helicobacter pylori is the strongest known risk factor for the development of gastric adenocarcinoma. H. pylori expresses a repertoire of virulence factors that increase gastric cancer risk, including the cag pathogenicity island and the vacuolating cytotoxin (VacA). One host element that promotes carcinogenesis within the gastrointestinal tract is Krüppel-like factor 5 (KLF5), a transcription factor that mediates key cellular functions. To define the role of KLF5 within the context of H. pylori-induced inflammation and injury, human gastric epithelial cells were co-cultured with the wild-type cag(+) H. pylori strain 60190. KLF5 expression was significantly upregulated following co-culture with H. pylori, but increased expression was independent of the cag island or VacA. To translate these findings into an in vivo model, C57BL/6 mice were challenged with the wild-type rodent-adapted cag(+) H. pylori strain PMSS1 or a PMSS1 cagE(-) isogenic mutant. Similar to findings in vitro, KLF5 staining was significantly enhanced in gastric epithelium of H. pylori-infected compared to uninfected mice and this was independent of the cag island. Flow cytometry revealed that the majority of KLF5(+) cells also stained positively for the stem cell marker, Lrig1, and KLF5(+)/Lrig1(+) cells were significantly increased in H. pylori-infected versus uninfected tissue. To extend these results into the natural niche of this pathogen, levels of KLF5 expression were assessed in human gastric biopsies isolated from patients with or without premalignant lesions. Levels of KLF5 expression increased in parallel with advancing stages of neoplastic progression, being significantly elevated in gastritis, intestinal metaplasia, and dysplasia compared to normal gastric tissue. These results indicate that H. pylori induces expression of KLF5 in gastric epithelial cells in vitro and in vivo, and that the degree of KLF5 expression parallels the severity of premalignant lesions in human gastric carcinogenesis.

Fusion between Intestinal epithelial cells and macrophages in a cancer context results in nuclear reprogramming.

The most deadly phase in cancer progression is attributed to the inappropriate acquisition of molecular machinery leading to metastatic transformation and spread of disease to distant organs. Although it is appreciated that metastasis involves epithelial-mesenchymal interplay, the underlying mechanism defining this process is poorly understood. Specifically, how cancer cells evade immune surveillance and gain the ability to navigate the circulatory system remains a focus. One possible mechanism underlying metastatic conversion is fusion between blood-derived immune cells and cancer cells. While this notion is a century old, in vivo evidence that cell fusion occurs within tumors and imparts genetic or physiologic changes remains controversial. We have previously demonstrated in vivo cell fusion between blood cells and intestinal epithelial cells in an injury setting. Here, we hypothesize that immune cells, such as macrophages, fuse with tumor cells imparting metastatic capabilities by transferring their cellular identity. We used parabiosis to introduce fluorescent-labeled bone marrow-derived cells to mice with intestinal tumors, finding that fusion between circulating blood-derived cells and tumor epithelium occurs during the natural course of tumorigenesis. Moreover, we identify the macrophage as a key cellular partner for this process. Interestingly, cell fusion hybrids retain a transcriptome identity characteristic of both parental derivatives, while also expressing a unique subset of transcripts. Our data supports the novel possibility that tumorigenic cell fusion may impart physical behavior attributed to migratory macrophages, including navigation of circulation and immune evasion. As such, cell fusion may represent a promising novel mechanism underlying the metastatic conversion of cancer cells.

Lessons from development: A role for asymmetric stem cell division in cancer.

Asymmetric stem cell division has emerged as a major regulatory mechanism for physiologic control of stem cell numbers. Reinvigoration of the cancer stem cell theory suggests that tumorigenesis may be regulated by maintaining the balance between asymmetric and symmetric cell division. Therefore, mutations affecting this balance could result in aberrant expansion of stem cells. Although a number of molecules have been implicated in regulation of asymmetric stem cell division, here, we highlight known tumor suppressors with established roles in this process. While a subset of these tumor suppressors were originally defined in developmental contexts, recent investigations reveal they are also lost or mutated in human cancers. Mutations in tumor suppressors involved in asymmetric stem cell division provide mechanisms by which cancer stem cells can hyperproliferate and offer an intriguing new focus for understanding cancer biology. Our discussion of this emerging research area derives insight from a frontier area of basic science and links these discoveries to human tumorigenesis. This highlights an important new focus for understanding the mechanism underlying expansion of cancer stem cells in driving tumorigenesis.

Inflammation and proliferation act together to mediate intestinal cell fusion.

Cell fusion between circulating bone marrow-derived cells (BMDCs) and non-hematopoietic cells is well documented in various tissues and has recently been suggested to occur in response to injury. Here we illustrate that inflammation within the intestine enhanced the level of BMDC fusion with intestinal progenitors. To identify important microenvironmental factors mediating intestinal epithelial cell fusion, we performed bone marrow transplantation into mouse models of inflammation and stimulated epithelial proliferation. Interestingly, in a non-injury model or in instances where inflammation was suppressed, an appreciable baseline level of fusion persisted. This suggests that additional mediators of cell fusion exist. A rigorous temporal analysis of early post-transplantation cellular dynamics revealed that GFP-expressing donor cells first trafficked to the intestine coincident with a striking increase in epithelial proliferation, advocating for a required fusogenic state of the host partner. Directly supporting this hypothesis, induction of augmented epithelial proliferation resulted in a significant increase in intestinal cell fusion. Here we report that intestinal inflammation and epithelial proliferation act together to promote cell fusion. While the physiologic impact of cell fusion is not yet known, the increased incidence in an inflammatory and proliferative microenvironment suggests a potential role for cell fusion in mediating the progression of intestinal inflammatory diseases and cancer.