KIT Signaling Promotes Growth of Colon Xenograft Tumors in Mice and Is Up-Regulated in a Subset of Human Colon Cancers.
2015; 149 (3): 705-17 e2
Receptor tyrosine kinase (RTK) inhibitors have advanced colon cancer treatment. We investigated the role of the RTK KIT in development of human colon cancer.An array of 137 patient-derived colon tumors and their associated xenografts were analyzed by immunohistochemistry to measure levels of KIT and its ligand KITLG. KIT and/or KITLG was stably knocked down by expression of small hairpin RNAs from lentiviral vectors in DLD1, HT29, LS174T, and COLO320 DM colon cancer cell lines, and in UM-COLON#8 and POP77 xenografts; cells transduced with only vector were used as controls. Cells were analyzed by real-time quantitative reverse transcription polymerase chain reaction, single-cell gene expression analysis, flow cytometry, and immunohistochemical, immunoblot, and functional assays. Xenograft tumors were grown from control and KIT-knockdown DLD1 and UM-COLON#8 cells in immunocompromised mice and compared. Some mice were given the RTK inhibitor imatinib after injection of cancer cells; tumor growth was measured based on bioluminescence. We assessed tumorigenicity using limiting dilution analysis.KIT and KITLG were expressed heterogeneously by a subset of human colon tumors. Knockdown of KIT decreased proliferation of colon cancer cell lines and growth of xenograft tumors in mice compared with control cells. KIT knockdown cells had increased expression of enterocyte markers, decreased expression of cycling genes, and, unexpectedly, increased expression of LGR5 associated genes. No activating mutations in KIT were detected in DLD1, POP77, or UM-COLON#8 cells. However, KITLG-knockdown DLD1 cells formed smaller xenograft tumors than control cells. Gene expression analysis of single CD44(+) cells indicated that KIT can promote growth via KITLG autocrine and/or paracrine signaling. Imatinib inhibited growth of KIT(+) colon cancer organoids in culture and growth of xenograft tumors in mice. Cancer cells with endogenous KIT expression were more tumorigenic in mice.KIT and KITLG are expressed by a subset of human colon tumors. KIT signaling promotes growth of colon cancer cells and organoids in culture and xenograft tumors in mice via its ligand, KITLG, in an autocrine or paracrine manner. Patients with KIT-expressing colon tumors can benefit from KIT RTK inhibitors.
View details for DOI 10.1053/j.gastro.2015.05.042
View details for PubMedID 26026391
- KIT Signaling Promotes Growth of Colon Xenograft Tumors in Mice and Is Up-Regulated in a Subset of Human Colon Cancers GASTROENTEROLOGY 2015; 149 (3): 705-?
Geometry of the Gene Expression Space of Individual Cells.
PLoS computational biology
2015; 11 (7)
There is a revolution in the ability to analyze gene expression of single cells in a tissue. To understand this data we must comprehend how cells are distributed in a high-dimensional gene expression space. One open question is whether cell types form discrete clusters or whether gene expression forms a continuum of states. If such a continuum exists, what is its geometry? Recent theory on evolutionary trade-offs suggests that cells that need to perform multiple tasks are arranged in a polygon or polyhedron (line, triangle, tetrahedron and so on, generally called polytopes) in gene expression space, whose vertices are the expression profiles optimal for each task. Here, we analyze single-cell data from human and mouse tissues profiled using a variety of single-cell technologies. We fit the data to shapes with different numbers of vertices, compute their statistical significance, and infer their tasks. We find cases in which single cells fill out a continuum of expression states within a polyhedron. This occurs in intestinal progenitor cells, which fill out a tetrahedron in gene expression space. The four vertices of this tetrahedron are each enriched with genes for a specific task related to stemness and early differentiation. A polyhedral continuum of states is also found in spleen dendritic cells, known to perform multiple immune tasks: cells fill out a tetrahedron whose vertices correspond to key tasks related to maturation, pathogen sensing and communication with lymphocytes. A mixture of continuum-like distributions and discrete clusters is found in other cell types, including bone marrow and differentiated intestinal crypt cells. This approach can be used to understand the geometry and biological tasks of a wide range of single-cell datasets. The present results suggest that the concept of cell type may be expanded. In addition to discreet clusters in gene-expression space, we suggest a new possibility: a continuum of states within a polyhedron, in which the vertices represent specialists at key tasks.
View details for DOI 10.1371/journal.pcbi.1004224
View details for PubMedID 26161936
- Geometry of the Gene Expression Space of Individual Cells PLOS COMPUTATIONAL BIOLOGY 2015; 11 (7)
Helicobacter pylori Activates and Expands Lgr5(+) Stem Cells Through Direct Colonization of the Gastric Glands.
2015; 148 (7): 1392-404 e21
Helicobacter pylori infection is the main risk factor for gastric cancer. We characterized the interactions of H pylori with gastric epithelial progenitor and stem cells in humans and mice and investigated how these interactions contribute to H pylori-induced pathology.We used quantitative confocal microscopy and 3-dimensional reconstruction of entire gastric glands to determine the localizations of H pylori in stomach tissues from humans and infected mice. Using lineage tracing to mark cells derived from leucine-rich repeat-containing G-protein coupled receptor 5-positive (Lgr5(+)) stem cells (Lgr5-eGFP-IRES-CreERT2/Rosa26-TdTomato mice) and in situ hybridization, we analyzed gastric stem cell responses to infection. Isogenic H pylori mutants were used to determine the role of specific virulence factors in stem cell activation and pathology.H pylori grow as distinct bacterial microcolonies deep in the stomach glands and interact directly with gastric progenitor and stem cells in tissues from mice and humans. These gland-associated bacteria activate stem cells, increasing the number of stem cells, accelerating Lgr5(+) stem cell proliferation, and up-regulating expression of stem cell-related genes. Mutant bacteria with defects in chemotaxis that are able to colonize the stomach surface but not the antral glands in mice do not activate stem cells. In addition, bacteria that are unable to inject the contact-dependent virulence factor CagA into the epithelium colonized stomach glands in mice, but did not activate stem cells or produce hyperplasia to the same extent as wild-type H pylori.H pylori colonize and manipulate the progenitor and stem cell compartments, which alters turnover kinetics and glandular hyperplasia. Bacterial ability to alter the stem cells has important implications for gastrointestinal stem cell biology and H pylori-induced gastric pathology.
View details for DOI 10.1053/j.gastro.2015.02.049
View details for PubMedID 25725293
- Helicobacter pylori Activates and Expands Lgr5(+) Stem Cells Through Direct Colonization of the Gastric Glands GASTROENTEROLOGY 2015; 148 (7): 1392-?
Lanthanum-Induced Gastrointestinal Histiocytosis.
ACG case reports journal
2015; 2 (3): 187-189
A patient with end-stage renal disease (ESRD) on hemodialysis presented with fever, anorexia, and nausea shortly after starting oral lanthanum carbonate for phosphate control. Gastric and duodenal biopsies demonstrated diffuse histiocytosis with intracellular aggregates of basophilic foreign material. Transmission electron microscopy, an underutilized diagnostic test, revealed the nature of the aggregates as heavy metal particles, consistent with lanthanum. Symptoms and histiocytosis improved after discontinuation of lanthanum. Lanthanum may be an underdiagnosed cause of gastrointestinal histiocytosis.
View details for DOI 10.14309/crj.2015.50
View details for PubMedID 26157959
A cell-intrinsic role for TLR2-MYD88 in intestinal and breast epithelia and oncogenesis.
Nature cell biology
2014; 16 (12): 1238-1248
It has been postulated that there is a link between inflammation and cancer. Here we describe a role for cell-intrinsic toll-like receptor-2 (TLR2; which is involved in inflammatory response) signalling in normal intestinal and mammary epithelial cells and oncogenesis. The downstream effectors of TLR2 are expressed by normal intestinal and mammary epithelia, including the stem/progenitor cells. Deletion of MYD88 or TLR2 in the intestinal epithelium markedly reduces DSS-induced colitis regeneration and spontaneous tumour development in mice. Limiting dilution transplantations of breast epithelial cells devoid of TLR2 or MYD88 revealed a significant decrease in mammary repopulating unit frequency compared with the control. Inhibition of TLR2, its co-receptor CD14, or its downstream targets MYD88 and IRAK1 inhibits growth of human breast cancers in vitro and in vivo. These results suggest that inhibitors of the TLR2 pathway merit investigation as possible therapeutic and chemoprevention agents.
View details for DOI 10.1038/ncb3058
View details for PubMedID 25362351
- A cell-intrinsic role for TLR2 MYD88 in intestinal and breast epithelia and oncogenesis NATURE CELL BIOLOGY 2014; 16 (12): 1238-U245
Quantitative assessment of single-cell RNA-sequencing methods.
2014; 11 (1): 41-46
Interest in single-cell whole-transcriptome analysis is growing rapidly, especially for profiling rare or heterogeneous populations of cells. We compared commercially available single-cell RNA amplification methods with both microliter and nanoliter volumes, using sequence from bulk total RNA and multiplexed quantitative PCR as benchmarks to systematically evaluate the sensitivity and accuracy of various single-cell RNA-seq approaches. We show that single-cell RNA-seq can be used to perform accurate quantitative transcriptome measurement in individual cells with a relatively small number of sequencing reads and that sequencing large numbers of single cells can recapitulate bulk transcriptome complexity.
View details for DOI 10.1038/nmeth.2694
View details for PubMedID 24141493
- Gastrointestinal stromal tumor: an unusual cause of gastrointestinal bleeding. Digestive diseases and sciences 2013; 58 (11): 3112-3116
- 5-ASA Induced Recurrent Myopericarditis and Cardiac Tamponade in a Patient with Ulcerative Colitis DIGESTIVE DISEASES AND SCIENCES 2013; 58 (8): 2148-2150
Innate immune response to homologous rotavirus infection in the small intestinal villous epithelium at single-cell resolution
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2012; 109 (50): 20667-20672
"Bulk" measurements of antiviral innate immune responses from pooled cells yield averaged signals and do not reveal underlying signaling heterogeneity in infected and bystander single cells. We examined such heterogeneity in the small intestine during rotavirus (RV) infection. Murine RV EW robustly activated type I IFNs and several antiviral genes (IFN-stimulated genes) in the intestine by bulk analysis, the source of induced IFNs primarily being hematopoietic cells. Flow cytometry and microfluidics-based single-cell multiplex RT-PCR allowed dissection of IFN responses in single RV-infected and bystander intestinal epithelial cells (IECs). EW replicates in IEC subsets differing in their basal type I IFN transcription and induces IRF3-dependent and IRF3-augmented transcription, but not NF-?B-dependent or type I IFN transcripts. Bystander cells did not display enhanced type I IFN transcription but had elevated levels of certain IFN-stimulated genes, presumably in response to exogenous IFNs secreted from immune cells. Comparison of IRF3 and NF-?B induction in STAT1(-/-) mice revealed that murine but not simian RRV mediated accumulation of IkB-? protein and decreased transcription of NF-?B-dependent genes. RRV replication was significantly rescued in IFN types I and II, as well as STAT1 (IFN types I, II, and III) deficient mice in contrast to EW, which was only modestly sensitive to IFNs I and II. Resolution of "averaged" innate immune responses in single IECs thus revealed unexpected heterogeneity in both the induction and subversion of early host antiviral immunity, which modulated host range.
View details for DOI 10.1073/pnas.1212188109
View details for Web of Science ID 000312605600104
View details for PubMedID 23188796
Identification of a cKit(+) Colonic Crypt Base Secretory Cell That Supports Lgr5(+) Stem Cells in Mice
2012; 142 (5): 1195-?
Paneth cells contribute to the small intestinal niche of Lgr5(+) stem cells. Although the colon also contains Lgr5(+) stem cells, it does not contain Paneth cells. We investigated the existence of colonic Paneth-like cells that have a distinct transcriptional signature and support Lgr5(+) stem cells.We used multicolor fluorescence-activated cell sorting to isolate different subregions of colon crypts, based on known markers, from dissociated colonic epithelium of mice. We performed multiplexed single-cell gene expression analysis with quantitative reverse transcriptase polymerase chain reaction followed by hierarchical clustering analysis to characterize distinct cell types. We used immunostaining and fluorescence-activated cell sorting analyses with in vivo administration of a Notch inhibitor and in vitro organoid cultures to characterize different cell types.Multicolor fluorescence-activated cell sorting could isolate distinct regions of colonic crypts. Four major epithelial subtypes or transcriptional states were revealed by gene expression analysis of selected populations of single cells. One of these, the goblet cells, contained a distinct cKit/CD117(+) crypt base subpopulation that expressed Dll1, Dll4, and epidermal growth factor, similar to Paneth cells, which were also marked by cKit. In the colon, cKit(+) goblet cells were interdigitated with Lgr5(+) stem cells. In vivo, this colonic cKit(+) population was regulated by Notch signaling; administration of a ?-secretase inhibitor to mice increased the number of cKit(+) cells. When isolated from mouse colon, cKit(+) cells promoted formation of organoids from Lgr5(+) stem cells, which expressed Kitl/stem cell factor, the ligand for cKit. When organoids were depleted of cKit(+) cells using a toxin-conjugated antibody, organoid formation decreased.cKit marks small intestinal Paneth cells and a subset of colonic goblet cells that are regulated by Notch signaling and support Lgr5(+) stem cells.
View details for DOI 10.1053/j.gastro.2012.02.006
View details for Web of Science ID 000303113600038
View details for PubMedID 22333952
View details for PubMedCentralID PMC3911891
Single-cell dissection of transcriptional heterogeneity in human colon tumors
2011; 29 (12): 1120-U11
Cancer is often viewed as a caricature of normal developmental processes, but the extent to which its cellular heterogeneity truly recapitulates multilineage differentiation processes of normal tissues remains unknown. Here we implement single-cell PCR gene-expression analysis to dissect the cellular composition of primary human normal colon and colon cancer epithelia. We show that human colon cancer tissues contain distinct cell populations whose transcriptional identities mirror those of the different cellular lineages of normal colon. By creating monoclonal tumor xenografts from injection of a single (n = 1) cell, we demonstrate that the transcriptional diversity of cancer tissues is largely explained by in vivo multilineage differentiation and not only by clonal genetic heterogeneity. Finally, we show that the different gene-expression programs linked to multilineage differentiation are strongly associated with patient survival. We develop two-gene classifier systems (KRT20 versus CA1, MS4A12, CD177, SLC26A3) that predict clinical outcomes with hazard ratios superior to those of pathological grade and comparable to those of microarray-derived multigene expression signatures.
View details for DOI 10.1038/nbt.2038
View details for Web of Science ID 000298038700023
View details for PubMedID 22081019
View details for PubMedCentralID PMC3237928
The Myc Connection: ES Cells and Cancer
2010; 143 (2): 184-186
Gene profiling experiments have revealed similarities between cancer and embryonic stem (ES) cells. Kim et al. (2010) dissect the gene expression signature of ES cells into three functional modules and find that the Myc module, including genes targeted by Myc-interacting proteins, accounts for most of the similarity between ES and cancer cells.
View details for DOI 10.1016/j.cell.2010.09.046
View details for Web of Science ID 000283052200007
View details for PubMedID 20946977
- Evidence for an Evolutionarily-Conserved Mechanism Regulating Stem and Progenitor Cell Numbers in the Regenerative Response to Ulcerative Colitis Gastroenterology 2010; 138 (5): S111
- Successful Use of Octreotide to Treat Menetrier's Disease: A Rare Cause of Abdominal Pain, Weight Loss, Edema, and Hypoalbuminemia DIGESTIVE DISEASES AND SCIENCES 2009; 54 (7): 1403-1407
- Adenovirus-Induced Acute Liver Failure DIGESTIVE DISEASES AND SCIENCES 2009; 54 (2): 218-221
- Cancer Stem Cells (Chapter 53) Essentials of Stem Cell Biology 2nd ed. (ed. Robert Lanza) 2009
Antibiotics in the management of hepatic encephalopathy: an evidence-based review.
Reviews in gastroenterological disorders
2005; 5: 26-35
Hepatic encephalopathy (HE) is an increasingly prevalent and debilitating condition that occurs in functional hepatic insufficiency. It is marked by fluctuating neuropsychiatric and cognitive impairment, which can be severe and life threatening. Hepatic encephalopathy is a diagnosis of exclusion; thus, it is challenging to diagnose definitively and to investigate in clinical trials. High response rates in the placebo arms of well-conducted studies demonstrate that the most effective treatment for HE is the correction of known precipitating triggers. However, pharmacological therapies may also be helpful. Although the precise pathogenesis remains unknown, bacterially derived neurotoxins from enteric flora likely play an important role. Based on this hypothesis and on accumulating clinical experience documented in randomized trials, oral antibiotics have emerged as an important treatment adjunct. This article addresses the qualities of an ideal antibiotic and reviews the literature on 4 antibiotics used to treat HE: neomycin, metronidazole, vancomycin, and rifaximin, with the most promising of these drugs appearing to be rifaximin. Unfortunately, most studies of the treatment of HE are difficult to interpret due to small sample sizes, methodological flaws, vulnerability to bias, and the intrinsic challenges of studying HE. Many studies have erroneously concluded that treatments are equivalent simply because no significant difference between treatment arms was detected. Consequently, the literature generally lacks definitive data from large, randomized, placebo-controlled trials. Nevertheless, the data suggest that minimally absorbed antibiotics are emerging as a safe and effective approach for the treatment of HE.
View details for PubMedID 17713457
- Cell biology - The hippo hypothesis NATURE 2003; 425 (6957): 469-470
Drosophila pod-1 crosslinks both actin and microtubules and controls the targeting of axons
2003; 39 (5): 779-791
Actin and microtubules (MTs) are tightly coordinated during neuronal growth cone navigation and are dynamically regulated in response to guidance cues; however, little is known about the underlying molecular mechanisms. Here, we characterize Drosophila pod-1 (dpod1) and show that purified Dpod1 can crosslink both actin and MTs. In cultured S2 cells, Dpod1 colocalizes with lamellar actin and MTs, and overexpression remodels the cytoskeleton to promote dynamic neurite-like actin-dependent projections. Consistent with these observations, Dpod1 localizes to the tips of growing axons, regions where actin and MTs interact, and is especially abundant at navigational choice points. In either the absence or overabundance of Dpod1, growth cone targeting but not outgrowth is disrupted. Taken together, these results reveal novel activities for pod-1 and show that proper levels of Dpod1, an actin/MT crosslinker, must be maintained in the growth cone for correct axon guidance.
View details for Web of Science ID 000185079300008
View details for PubMedID 12948445
salvador - The persistence of proliferation
2002; 2 (3): 171-173
Despite years of extensive studies on genes that regulate proliferation and cell death, two processes that must be tightly coordinated throughout development to regulate cell number, remarkably few genes have been shown to affect both processes. Using an elegant genetic screen in the fly eye, have identified a gene, salvador, which is especially significant, because it not only regulates and coordinates both exit from the cell cycle and apoptosis, but also has a human homolog that may play a key role in tumorigenesis.
View details for Web of Science ID 000178353000004
View details for PubMedID 12242148
casanova plays an early and essential role in endoderm formation in zebrafish
1999; 215 (2): 343-357
The cellular and molecular mechanisms that regulate endoderm development in vertebrates have only recently begun to be explored. Here we show that the zebrafish locus casanova plays an early and essential role in this process. casanova mutants lack a gut tube and do not express any molecular markers of endoderm differentiation. The early endodermal expression of genes such as axial, gata5, and fkd2 does not initiate in casanova mutants, indicating that the endoderm is defective from the onset of gastrulation. Mosaic analysis demonstrates that casanova functions cell autonomously within the endodermal progenitors. We also report the isolation of a zebrafish homologue of Mixer, a gene important for early endoderm formation in Xenopus. casanova does not encode zebrafish Mixer, and mixer expression is normal in casanova mutants, indicating that casanova acts downstream of, or parallel to, mixer to promote endoderm formation. We further find that the forerunner cells, a specialized group of noninvoluting dorsal mesendodermal cells, do not form in casanova mutants. Studies of casanova mutants do not support an important role for the forerunner cells in either dorsal axis or tail development, as has been previously proposed. In addition, although different populations of mesodermal precursors are generated normally in casanova mutants, morphogenetic defects in the heart, vasculature, blood, and kidney are apparent, suggesting a possible role for the endoderm in morphogenesis of these organs.
View details for Web of Science ID 000083726600017
View details for PubMedID 10545242
Partner of numb colocalizes with numb during mitosis and directs numb asymmetric localization in Drosophila neural and muscle progenitors
1998; 95 (2): 225-235
During mitosis of multiple types of precursor cells in Drosophila, Numb is asymmetrically distributed between the two daughter cells and confers distinct daughter cell fates. Here we report the identification of a novel gene product, Partner of Numb (PON), based on its physical interaction with Numb. PON is asymmetrically localized during mitosis and colocalizes with Numb. Loss of pon function disrupts Numb localization in muscle progenitors and delays Numb crescent formation in neural precursors. Moreover, ectopically expressed PON responds to the apical-basal polarity of epithelial cells and is sufficient to localize Numb basally. We propose that PON is one component of a multimolecular machinery that localizes Numb by responding to polarity cues conserved in neural precursors and epithelial cells.
View details for Web of Science ID 000076538300010
View details for PubMedID 9790529
Mot(3) a Zn finger transcription factor that modulates gene expression and attenuates mating pheromone signaling in Saccharomyces cerevisiae
1998; 149 (2): 879-892
In the yeast Saccharomyces cerevisiae, mating pheromone response is initiated by activation of a G protein- and mitogen-activated protein (MAP) kinase-dependent signaling pathway and attenuated by several mechanisms that promote adaptation or desensitization. To identify genes whose products negatively regulate pheromone signaling, we screened for mutations that suppress the hyperadaptive phenotype of wild-type cells overexpressing signaling-defective G protein beta subunits. This identified recessive mutations in MOT3, which encodes a nuclear protein with two Cys2-His2 Zn fingers. MOT3 was found to be a dosage-dependent inhibitor of pheromone response and pheromone-induced gene expression and to require an intact signaling pathway to exert its effects. Several results suggested that Mot3 attenuates expression of pheromone-responsive genes by mechanisms distinct from those used by the negative transcriptional regulators Cdc36, Cdc39, and Mot2. First, a Mot3-lexA fusion functions as a transcriptional activator. Second, Mot3 is a dose-dependent activator of several genes unrelated to pheromone response, including CYC1, SUC2, and LEU2. Third, insertion of consensus Mot3 binding sites (C/A/T)AGG(T/C)A activates a promoter in a MOT3-dependent manner. These findings, and the fact that consensus binding sites are found in the 5' flanking regions of many yeast genes, suggest that Mot3 is a globally acting transcriptional regulator. We hypothesize that Mot3 regulates expression of factors that attenuate signaling by the pheromone response pathway.
View details for Web of Science ID 000074028400035
View details for PubMedID 9611199
Numb-associated kinase interacts with the phosphotyrosine binding domain of numb and antagonizes the function of numb in vivo
MOLECULAR AND CELLULAR BIOLOGY
1998; 18 (1): 598-607
During asymmetric cell division, the membrane-associated Numb protein localizes to a crescent in the mitotic progenitor and is segregated predominantly to one of the two daughter cells. We have identified a putative serine/threonine kinase, Numb-associated kinase (Nak), which interacts physically with the phosphotyrosine binding (PTB) domain of Numb. The PTB domains of Shc and insulin receptor substrate bind to an NPXY motif which is not present in the region of Nak that interacts with Numb PTB domain. We found that the Numb PTB domain but not the Shc PTB domain interacts with Nak through a peptide of 11 amino acids, implicating a novel and specific protein-protein interaction. Overexpression of Nak in the sensory organs causes both daughters of a normally asymmetric cell division to adopt the same cell fate, a transformation similar to the loss of numb function phenotype and opposite the cell fate transformation caused by overexpression of Numb. The frequency of cell fate transformation is sensitive to the numb gene dosage, as expected from the physical interaction between Nak and Numb. These findings indicate that Nak may play a role in cell fate determination during asymmetric cell divisions.
View details for Web of Science ID 000071195700060
View details for PubMedID 9418906