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1.  Wnt Secretion from Epithelial Cells and Subepithelial Myofibroblasts Is Not Required in the Mouse Intestinal Stem Cell Niche In Vivo 
Stem Cell Reports  2014;2(2):127-134.
Summary
Wnt signaling is a crucial aspect of the intestinal stem cell niche required for crypt cell proliferation and differentiation. Paneth cells or subepithelial myofibroblasts are leading candidate sources of the required Wnt ligands, but this has not been tested in vivo. To abolish Wnt-ligand secretion, we used Porcupine (Porcn) conditional-null mice crossed to strains expressing inducible Cre recombinase in the epithelium, including Paneth cells (Villin-CreERT2); in smooth muscle, including subepithelial myofibroblasts (Myh11-CreERT2); and simultaneously in both compartments. Elimination of Wnt secretion from any of these compartments did not disrupt tissue morphology, cell proliferation, differentiation, or Wnt pathway activity. Thus, Wnt-ligand secretion from these cell populations is dispensable for intestinal homeostasis, revealing that a minor cell type or significant and unexpected redundancy is responsible for physiologic Wnt signaling in vivo.
Graphical Abstract
Highlights
•Porcn knockout in candidate intestinal niche cells eliminates Wnt secretion in vivo•Wnts from Paneth cells or intestinal subepithelial myofibroblasts are dispensable•Physiologic intestinal Wnt signaling is more complex than current views suggest
Wnt signaling is crucial in the intestinal stem cell niche, but the cell types producing essential Wnt ligands are unknown. Using Porcn conditional-null mice, Shivdasani and colleagues eliminated Wnt ligand secretion from intestinal epithelium, including Paneth cells; smooth muscle, including subepithelial myofibroblasts; and in both compartments. They found no defects in intestinal homeostasis or Wnt signaling, revealing unanticipated niche redundancy or complexity.
doi:10.1016/j.stemcr.2013.12.012
PMCID: PMC3923227  PMID: 24527386
2.  DOT1L-Mediated H3K79 Methylation in Chromatin Is Dispensable for Wnt Pathway-Specific and Other Intestinal Epithelial Functions 
Molecular and Cellular Biology  2013;33(9):1735-1745.
Methylation of H3K79 is associated with chromatin at expressed genes, though it is unclear if this histone modification is required for transcription of all genes. Recent studies suggest that Wnt-responsive genes depend particularly on H3K79 methylation, which is catalyzed by the methyltransferase DOT1L. Human leukemias carrying MLL gene rearrangements show DOT1L-mediated H3K79 methylation and aberrant expression of leukemogenic genes. DOT1L inhibitors reverse these effects, but their clinical use is potentially limited by toxicity in Wnt-dependent tissues such as intestinal epithelium. Genome-wide positioning of the H3K79me2 mark in Lgr5+ mouse intestinal stem cells and mature intestinal villus epithelium correlated with expression levels of all transcripts and not with Wnt-responsive genes per se. Selective Dot1l disruption in Lgr5+ stem cells or in whole intestinal epithelium eliminated H3K79me2 from the respective compartments, allowing genetic evaluation of DOT1L requirements. The absence of methylated H3K79 did not impair health, intestinal homeostasis, or expression of Wnt target genes in crypt epithelium for up to 4 months, despite increased crypt cell apoptosis. Global transcript profiles in Dot1l-null cells were barely altered. Thus, H3K79 methylation is not essential for transcription of Wnt-responsive or other intestinal genes, and intestinal toxicity is not imperative when DOT1L is rendered inactive in vivo.
doi:10.1128/MCB.01463-12
PMCID: PMC3624170  PMID: 23428873
3.  Gastrointestinal adenocarcinomas of the esophagus, stomach and colon exhibit distinct patterns of genome instability and oncogenesis 
Cancer research  2012;72(17):4383-4393.
A more detailed understanding of the somatic genetic events that drive gastrointestinal adenocarcinomas is necessary to improve diagnosis and therapy. Using data from high-density genomic profiling arrays, we conducted an analysis of somatic copy-number aberrations (SCNAs) in 486 gastrointestinal adenocarcinomas including 296 esophageal and gastric cancers. Focal amplifications were substantially more prevalent in gastric/esophageal adenocarcinomas than colorectal tumors. We identified 64 regions of significant recurrent amplification and deletion, some shared and others unique to the adenocarcinoma types examined. Amplified genes were noted in 37% of gastric/esophageal tumors, including in therapeutically targetable kinases such as ERBB2, FGFR1, FGFR2, EGFR, and MET, suggesting the potential utility of genomic amplifications as biomarkers to guide therapy of gastric and esophageal cancers where targeted therapeutics have been less developed compared to colorectal cancers. Amplified loci implicated genes with known involvement in carcinogenesis but also pointed to regions harboring potentially novel cancer genes, including a recurrent deletion found in 15% of esophageal tumors where the Runt transcription factor subunit RUNX1 was implicated, including by functional experiments in tissue culture. Together, our results defined genomic features that were common and distinct to various gut-derived adenocarcinomas, potentially informing novel opportunities for targeted therapeutic interventions.
doi:10.1158/0008-5472.CAN-11-3893
PMCID: PMC3432726  PMID: 22751462
Esophagus; stomach; colon; adenocarcinoma; copy-number
4.  Gastric Epithelial Stem Cells 
Gastroenterology  2010;140(2):412-424.
Advances in our understanding of stem cells in the gastrointestinal tract include the identification of molecular markers of stem and early progenitor cells in the small intestine. Although gastric epithelial stem cells have been localized, little is known about their molecular biology. Recent reports describe the use of inducible Cre recombinase activity to indelibly label candidate stem cells and their progeny in the distal stomach, (ie, the antrum and pylorus). No such lineage labeling of epithelial stem cells has been reported in the gastric body (corpus). Among stem cells in the alimentary canal, those of the adult corpus are unique in that they lie close to the lumen and increase proliferation following loss of a single mature progeny lineage, the acid-secreting parietal cell. They are also unique in that they neither depend on Wnt signaling nor express the surface marker Lgr5. Because pathogenesis of gastric adenocarcinoma has been associated with abnormal patterns of gastric differentiation and with chronic tissue injury, there has been much research on the response of stomach epithelial stem cells to inflammation. Chronic inflammation, as induced by infection with Helicobacter pylori, affects differentiation and promotes metaplasias. Several studies have identified cellular and molecular mechanisms in spasmolytic polypeptide–expressing (pseudopyloric) metaplasia. Researchers have also begun to identify signaling pathways and events that take place during embryonic development that eventually establish the adult stem cells to maintain the specific features and functions of the stomach mucosa. We review the cytologic, molecular, functional, and developmental properties of gastric epithelial stem cells.
doi:10.1053/j.gastro.2010.12.001
PMCID: PMC3708552  PMID: 21144849
Stomach Stem Cells; Epithelial Self-Renewal; Tissue Metaplasia
5.  Intestinal Master Transcription Factor CDX2 Controls Chromatin Access for Partner Transcription Factor Binding 
Molecular and Cellular Biology  2013;33(2):281-292.
Tissue-specific gene expression requires modulation of nucleosomes, allowing transcription factors to occupy cis elements that are accessible only in selected tissues. Master transcription factors control cell-specific genes and define cellular identities, but it is unclear if they possess special abilities to regulate cell-specific chromatin and if such abilities might underlie lineage determination and maintenance. One prevailing view is that several transcription factors enable chromatin access in combination. The homeodomain protein CDX2 specifies the embryonic intestinal epithelium, through unknown mechanisms, and partners with transcription factors such as HNF4A in the adult intestine. We examined enhancer chromatin and gene expression following Cdx2 or Hnf4a excision in mouse intestines. HNF4A loss did not affect CDX2 binding or chromatin, whereas CDX2 depletion modified chromatin significantly at CDX2-bound enhancers, disrupted HNF4A occupancy, and abrogated expression of neighboring genes. Thus, CDX2 maintains transcription-permissive chromatin, illustrating a powerful and dominant effect on enhancer configuration in an adult tissue. Similar, hierarchical control of cell-specific chromatin states is probably a general property of master transcription factors.
doi:10.1128/MCB.01185-12
PMCID: PMC3554120  PMID: 23129810
6.  Targeted Disruption of the BCL9/β-catenin Complex Inhibits Oncogenic Wnt Signaling 
Science translational medicine  2012;4(148):148ra117.
Deregulated Wnt/β-catenin signaling underlies the pathogenesis of a broad range of human cancers, yet the development of targeted therapies to disrupt the aberrant transcription has proven difficult because the pathway incorporates large protein interaction surfaces and regulates many homeostatic functions. Therefore, we have directed our efforts toward blocking the interaction with BCL9, a co-activator for β-catenin-mediated transcription that is highly expressed in tumors but not in the cells of origin. BCL9 drives β-catenin signaling through direct binding mediated by its α-helical homology domain-2. We developed a Stabilized Alpha-Helix of BCL9 (SAH-BCL9), which we show targets β-catenin, dissociates native β-catenin/BCL9 complexes, selectively suppresses Wnt transcription, and exhibits mechanism-based anti-tumor effects. SAH-BCL9 also suppresses tumor growth, angiogenesis, invasion, and metastasis in mouse xenograft models of Colo-320 colorectal carcinoma and INA-6 multiple myeloma. By inhibiting the BCL9/β-catenin interaction and selectively suppressing oncogenic Wnt transcription, SAH-BCL9 may serve as a novel prototype therapy for cancers driven by deregulated Wnt signaling.
doi:10.1126/scitranslmed.3003808
PMCID: PMC3631420  PMID: 22914623
Wnt signaling; transcriptional activity; cancer; BCL9; B9L; stabilized alpha-helices; stapled peptides; metastasis; angiogenesis
7.  Differential WNT activity in colorectal cancer confers limited tumorigenic potential and is regulated by MAPK signaling 
Cancer research  2012;72(6):1547-1556.
Colorectal cancers (CRCs) express the WNT effector protein β-catenin in a heterogeneous subcellular pattern rather than uniformly in the nucleus. In this study, we investigated this important aspect of molecular heterogeneity in CRCs by analyzing its basis and relationship with tumor initiating capability. CRC cells expressing the highest WNT expression showed only a marginal increase in tumor initiation capacity. Notably, high WNT activity correlated with a coincident activation of robust MAPK signaling, which when upregulated by KRAS expression or downregulated by EGFR inhibition elicited parallel effects on WNT activity. These findings suggested that on its own high WNT activity may not be a reliable signifier of tumor-initiating potential or stem-like potential. Further, they suggest that MAPK signaling is a critical modifier of intratumoral heterogeneity that contributes signiificantly to determining the impact of WNT activity on stemness phenotypes inCRC cells.
doi:10.1158/0008-5472.CAN-11-3222
PMCID: PMC3571091  PMID: 22318865
Colon and colorectal cancer; beta-catenin; WNT signaling; KRAS and MAPK signaling; tumor-initiating cells
8.  Boundaries, Junctions and Transitions in the Gastrointestinal Tract 
Experimental cell research  2011;317(19):2711-2718.
Contiguous regions along the mammalian gastrointestinal tract, from the esophagus to the rectum, serve distinct digestive functions. Some organs, such as the esophagus and glandular stomach or the small bowel and colon, are separated by sharp boundaries. The duodenal, jejunal and ileal segments of the small intestine, by contrast, have imprecise borders. Because human esophageal and gastric cancers frequently arise in a background of tissue metaplasia and some intestinal disorders are confined to discrete regions, it is useful to appreciate the molecular and cellular basis of boundary formation and preservation. Here we review the anatomy and determinants of boundaries and transitions in the alimentary canal with respect to tissue morphology, gene expression, and, especially, transcriptional control of epithelial identity. We discuss the evidence for established and candidate molecular mechanisms of boundary formation, including the solitary and combinatorial actions of tissue-restricted transcription factors. Although the understanding remains sparse, genetic studies in mice do provide insights into dominant mechanisms and point the way for future investigation.
doi:10.1016/j.yexcr.2011.07.011
PMCID: PMC3210370  PMID: 21802415
digestive tract development; gastro-esophageal junction; gastro-duodenal boundary; transcriptional regulation of tissue identity; homeobox genes in gut development
9.  Activation of ERBB2 signaling causes resistance to the EGFR-directed therapeutic antibody cetuximab 
Science Translational Medicine  2011;3(99):99ra86.
The epidermal growth factor receptor directed antibody, cetuximab, is an effective clinical therapy for patients with colorectal, head and neck and non-small cell lung cancer patients particularly for those with KRAS and BRAF wild type cancers. Treatment in all patients is limited eventually by the development of acquired resistance but little is known about the underlying mechanism. Here we show, that activation of ERBB2 signaling, either through ERBB2 amplification or through heregulin upregulation, leads to persistent ERK 1/2 signaling and consequently cetuximab resistance. Inhibition of ERBB2 or disruption of ERBB2/ERBB3 heterodimerization restores cetuximab sensitivity in vitro and in vivo. A subset of colorectal cancer patients that exhibit either de novo or acquired resistance to cetuximab based therapy possess ERBB2 amplification or high levels of circulating heregulin. Collectively, these findings identify two distinct resistance mechanisms, both of which promote aberrant ERBB2 signaling, that mediate cetuximab resistance. Moreover, these results suggest that ERBB2 inhibitors, in combination with cetuximanb, represent a rational therapeutic strategy that should be assessed in cetuximab-resistant cancers.
doi:10.1126/scitranslmed.3002442
PMCID: PMC3268675  PMID: 21900593
Epidermal growth factor receptor; drug resistance; amplification; heregulin; cetuximab; colorectal cancer
10.  The Stem Cell Discovery Engine: an integrated repository and analysis system for cancer stem cell comparisons 
Nucleic Acids Research  2011;40(D1):D984-D991.
Mounting evidence suggests that malignant tumors are initiated and maintained by a subpopulation of cancerous cells with biological properties similar to those of normal stem cells. However, descriptions of stem-like gene and pathway signatures in cancers are inconsistent across experimental systems. Driven by a need to improve our understanding of molecular processes that are common and unique across cancer stem cells (CSCs), we have developed the Stem Cell Discovery Engine (SCDE)—an online database of curated CSC experiments coupled to the Galaxy analytical framework. The SCDE allows users to consistently describe, share and compare CSC data at the gene and pathway level. Our initial focus has been on carefully curating tissue and cancer stem cell-related experiments from blood, intestine and brain to create a high quality resource containing 53 public studies and 1098 assays. The experimental information is captured and stored in the multi-omics Investigation/Study/Assay (ISA-Tab) format and can be queried in the data repository. A linked Galaxy framework provides a comprehensive, flexible environment populated with novel tools for gene list comparisons against molecular signatures in GeneSigDB and MSigDB, curated experiments in the SCDE and pathways in WikiPathways. The SCDE is available at http://discovery.hsci.harvard.edu.
doi:10.1093/nar/gkr1051
PMCID: PMC3245064  PMID: 22121217
11.  Differentiation-specific histone modifications reveal dynamic chromatin interactions and alternative partners for the intestinal transcription factor CDX2 
Developmental cell  2010;19(5):713-726.
SUMMARY
Cell differentiation requires remodeling of tissue-specific gene loci and activities of key transcriptional regulators, which are recognized for their dominant control over cellular programs. Using epigenomic methods, we characterized enhancer elements specifically modified in differentiating intestinal epithelial cells and found enrichment of transcription factor-binding motifs corresponding to CDX2, a critical regulator of the intestine. Directed investigation revealed surprising lability in CDX2 occupancy of the genome, with redistribution from hundreds of sites occupied only in proliferating cells to thousands of new sites in differentiated cells. Knockout mice confirmed distinct Cdx2 requirements in dividing and mature adult intestinal cells, including responsibility for the active enhancer configuration associated with maturity. Dynamic CDX2 occupancy corresponds with condition-specific gene expression and, importantly, to differential co-occupancy with other tissue-restricted transcription factors such as GATA6 and HNF4A. These results reveal dynamic, context-specific functions and mechanisms of a prominent transcriptional regulator within a cell lineage.
doi:10.1016/j.devcel.2010.10.006
PMCID: PMC3001591  PMID: 21074721
12.  Essential and Redundant Functions of Caudal Family Proteins in Activating Adult Intestinal Genes ▿  
Molecular and Cellular Biology  2011;31(10):2026-2039.
Transcription factors that potently induce cell fate often remain expressed in the induced organ throughout life, but their requirements in adults are uncertain and varied. Mechanistically, it is unclear if they activate only tissue-specific genes or also directly repress heterologous genes. We conditionally inactivated mouse Cdx2, a dominant regulator of intestinal development, and mapped its genome occupancy in adult intestinal villi. Although homeotic transformation, observed in Cdx2-null embryos, was absent in mutant adults, gene expression and cell morphology were vitally compromised. Lethality was significantly accelerated in mice lacking both Cdx2 and its homolog Cdx1, with particular exaggeration of defects in villus enterocyte differentiation. Importantly, Cdx2 occupancy correlated with hundreds of transcripts that fell but not with equal numbers that rose with Cdx loss, indicating a predominantly activating role at intestinal cis-regulatory regions. Integrated consideration of a transcription factor's mutant phenotype and cistrome hence reveals the continued and distinct requirement in adults of a critical developmental regulator that activates tissue-specific genes.
doi:10.1128/MCB.01250-10
PMCID: PMC3133364  PMID: 21402776
13.  Notch signaling in stomach epithelial stem cell homeostasis 
In the mouse stomach epithelium, Notch signaling influences homeostasis and tumorigenesis in a cell type– and context-specific manner.
The mammalian adult gastric epithelium self-renews continually through the activity of stem cells located in the isthmus of individual gland units. Mechanisms facilitating stomach stem and progenitor cell homeostasis are unknown. Here, we show that Notch signaling occurs in the mouse stomach epithelium during development and becomes restricted mainly to the isthmus in adult glands, akin to its known localization in the proliferative compartment of intestinal villi. Using genetic and chemical inhibition, we demonstrate that Notch signaling is required to maintain the gastric stem cell compartment. Activation of Notch signaling in lineage-committed stomach epithelial cells is sufficient to induce dedifferentiation into stem and/or multipotential progenitors that populate the mucosa with all major cell types. Prolonged Notch activation within dedifferentiated parietal cells eventually enhances cell proliferation and induces adenomas that show focal Wnt signaling. In contrast, Notch activation within native antral stomach stem cells does not affect cell proliferation. These results establish a role for Notch activity in the foregut and highlight the importance of cellular context in gastric tumorigenesis.
doi:10.1084/jem.20101737
PMCID: PMC3137787  PMID: 21402740
14.  Barx1-Mediated Inhibition of Wnt Signaling in the Mouse Thoracic Foregut Controls Tracheo-Esophageal Septation and Epithelial Differentiation 
PLoS ONE  2011;6(7):e22493.
Mesenchymal cells underlying the definitive endoderm in vertebrate animals play a vital role in digestive and respiratory organogenesis. Although several signaling pathways are implicated in foregut patterning and morphogenesis, and despite the clinical importance of congenital tracheal and esophageal malformations in humans, understanding of molecular mechanisms that allow a single tube to separate correctly into the trachea and esophagus is incomplete. The homoebox gene Barx1 is highly expressed in prospective stomach mesenchyme and required to specify this organ. We observed lower Barx1 expression extending contiguously from the proximal stomach domain, along the dorsal anterior foregut mesenchyme and in mesenchymal cells between the nascent esophagus and trachea. This expression pattern exactly mirrors the decline in Wnt signaling activity in late development of the adjacent dorsal foregut endoderm and medial mainstem bronchi. The hypopharynx in Barx1−/− mouse embryos is abnormally elongated and the point of esophago-tracheal separation shows marked caudal displacement, resulting in a common foregut tube that is similar to human congenital tracheo-esophageal fistula and explains neonatal lethality. Moreover, the Barx1−/− esophagus displays molecular and cytologic features of respiratory endoderm, phenocopying abnormalities observed in mouse embryos with activated ß-catenin. The zone of canonical Wnt signaling is abnormally prolonged and expanded in the proximal Barx1−/− foregut. Thus, as in the developing stomach, but distinct from the spleen, Barx1 control of thoracic foregut specification and tracheo-esophageal septation is tightly associated with down-regulation of adjacent Wnt pathway activity.
doi:10.1371/journal.pone.0022493
PMCID: PMC3142160  PMID: 21799872
15.  Transcription factor Foxq1 controls mucin gene expression and granule content in mouse stomach surface mucous cells 
Gastroenterology  2008;135(2):591-600.
Background and Aims
The gastric mucosa provides a stringent epithelial barrier and produces acid and enzymes that initiate digestion. In this regenerating tissue, progenitors differentiate continually into 4 principal specialized cell types, yet underlying mechanisms of differentiation are poorly understood. We identified stomach-restricted expression of the forkhead transcription factor FOXQ1.
Methods
We used a combination of genetic, histochemical, ultrastructural and molecular analysis to study gastric cell lineages with respect to FOXQ1.
Results
Within the developing and adult gastrointestinal tract, Foxq1 mRNA is restricted to the stomach and expressed predominantly in foveolar (pit) cells, the abundant mucin-producing cells that line the mucosal surface. Mice carrying Foxq1 coding mutations show virtual absence of mRNA and protein for the backbone of the major stomach mucin, MUC5AC. These observations correspond to a paucity of foveolar-cell secretory vesicles and notable loss of stomach but not intestinal mucus. Transcriptional profiling identified a surprisingly restricted set of genes with altered expression in Foxq1 mutant stomachs. MUC5AC is a highly tissue-restricted product that similarly depends on FOXQ1 in its other major site of expression, conjunctival goblet cells.
Conclusions
Taken together, these observations imply that promotion of gastric MUC5AC synthesis is a primary, cell-autonomous function of FOXQ1. This study is the first to implicate a transcription factor in terminal differentiation of foveolar cells and begins to define the requirements to assemble highly specialized organelles and cells in the gastric mucosa.
doi:10.1053/j.gastro.2008.04.019
PMCID: PMC2955860  PMID: 18558092
mucin gene regulation; foveolar cell; stomach epithelial differentiation; forkhead; conjunctival goblet cell; surface mucous cell; MUC5AC; Satin; Foxq1
16.  Role of the homeodomain transcription factor Bapx1 in mouse distal stomach development 
Gastroenterology  2009;136(5):1701-1710.
Background & Aims
Expansion and patterning of the endoderm generate a highly ordered, multi-organ digestive system in vertebrate animals. Among distal foregut derivatives, the gastric corpus, antrum, pylorus and duodenum are distinct structures with sharp boundaries. Some homeodomain transcription factors expressed in gut mesenchyme convey positional information required for anterior-posterior patterning of the digestive tract. Barx1, in particular, controls stomach differentiation and morphogenesis. The NK homeobox gene Bapx1 (Nkx3-2) has an established role in skeletal development but its function in the mammalian gut is less clear.
Methods
We generated a Bapx1Cre knock-in allele to fate map Bapx1-expressing cells and evaluate its function in gastrointestinal development.
Results
Bapx1-expressing cells populate the gut mesenchyme with a rostral boundary in the hindstomach, near the junction of the gastric corpus and antrum. Smooth muscle differentiation and distribution of early regional markers are ostensibly normal in Bapx1Cre/Cre gut, but there are distinctive morphologic abnormalities near this rostral Bapx1 domain: the antral segment of the stomach is markedly shortened and the pyloric constriction is lost. Comparison of expression domains and examination of stomach phenotypes in single and compound Barx1 and Bapx1 mutant mice suggest a hierarchy between these two factors; Bapx1 expression is lost in the absence of Barx1.
Conclusions
This study reveals the non-redundant requirement for Bapx1 in distal stomach development, places it within a Barx1-dependent pathway, and illustrates the pervasive influence of gut mesenchyme homeobox genes on endoderm differentiation and digestive organogenesis.
doi:10.1053/j.gastro.2009.01.009
PMCID: PMC2955323  PMID: 19208343
Gastrointestinal development; stomach; mesoderm; endoderm; Nkx3-2; Bapx1; pyloric sphincter; Barx1; antrum
18.  Relationship of CDX2 Loss with Molecular Features and Prognosis in Colorectal Cancer: Implications for Clinical and Pathology Practice 
Purpose
The homeodomain transcription factor CDX2 is a relatively specific immunohistochemical marker for gastrointestinal carcinoma. However, no study has comprehensively examined the relationship between CDX2 expression in colon cancer and clinical, pathologic, prognostic and molecular features, including microsatellite instability (MSI) and CpG island methylator phenotype (CIMP).
Experimental Design
Utilizing 621 colorectal cancers with clinical outcome and molecular data, CDX2 loss was detected in 183 (29%) tumors by immunohistochemistry.
Results
In multivariate logistic regression analysis, CDX2 loss was associated with female gender [odds ratio (OR)=3.32; p<0.0001], CIMP-high (OR=4.42; p=0.0003), high tumor grade (OR=2.69; p=0.0085), stage IV disease (OR=2.03; p=0.019), and inversely with LINE-1 hypomethylation (for a 30% decline; OR=0.33; p=0.0031), p53 expression (OR=0.55; p=0.011), β-catenin activation (OR=0.60; p=0.037), but not with body mass index, tumor location, MSI, BRAF, KRAS, PIK3CA, p21 or COX-2. CDX2 loss was not independently associated with patient survival. However, the prognostic effect of CDX2 loss appeared to differ according to family history of colorectal cancer (Pinteraction=0.0094). CDX2 loss was associated with high overall mortality [multivariate hazard ratio (HR)=2.40; 95% CI, 1.28–4.51] among patients with a family history of colorectal cancer; no such association was present (multivariate HR=0.97; 95% CI, 0.66–1.41) among patients without a family history of colorectal cancer.
Conclusions
CDX2 loss in colorectal cancer is independently associated with female gender, CIMP-high, high-level LINE-1 methylation, high tumor grade, and advanced stage. CDX2 loss may be associated with poor prognosis among patients with a family history of colorectal cancer.
doi:10.1158/1078-0432.CCR-09-0401
PMCID: PMC2777758  PMID: 19584150
colon cancer; CDX2; CIMP; family history; prognosis
19.  SOX2 Is an Amplified Lineage Survival Oncogene in Lung and Esophageal Squamous Cell Carcinomas 
Nature genetics  2009;41(11):1238-1242.
Lineage survival oncogenes are activated by somatic DNA alterations in cancers arising from the cell lineages in which these genes play a role in normal development.1,2 Here we show that a peak of genomic amplification on chromosome 3q26.33, found in squamous cell carcinomas (SCCs) of the lung and esophagus, contains the transcription factor gene SOX2—which is mutated in hereditary human esophageal malformations3 and necessary for normal esophageal squamous development4, promotes differentiation and proliferation of basal tracheal cells5 and co-operates in induction of pluripotent stem cells.6,7,8 SOX2 expression is required for proliferation and anchorage-independent growth of lung and esophageal cell lines, as shown by RNA interference experiments. Furthermore, ectopic expression of SOX2 cooperated with FOXE1 or FGFR2 to transform immortalized tracheobronchial epithelial cells. SOX2-driven tumors show expression of markers of both squamous differentiation and pluripotency. These observations identify SOX2 as a novel lineage survival oncogene in lung and esophageal SCC.
doi:10.1038/ng.465
PMCID: PMC2783775  PMID: 19801978
20.  The 8q24 cancer risk variant rs6983267 demonstrates long-range interaction with MYC in colorectal cancer 
Nature genetics  2009;41(8):882-884.
An inherited variant on chromosome 8q24, rs6983267, is significantly associated with cancer pathogenesis. We present evidence that this region is a transcriptional enhancer, that the risk region physically interacts with the MYC proto-oncogene, and that the alleles of rs6983267 differentially bind transcription factor 7-like 2 (TCF7L2). These data provide strong support for a biological mechanism underlying this non-protein coding risk variant.
doi:10.1038/ng.403
PMCID: PMC2763485  PMID: 19561607
22.  Profiling Critical Cancer Gene Mutations in Clinical Tumor Samples 
PLoS ONE  2009;4(11):e7887.
Background
Detection of critical cancer gene mutations in clinical tumor specimens may predict patient outcomes and inform treatment options; however, high-throughput mutation profiling remains underdeveloped as a diagnostic approach. We report the implementation of a genotyping and validation algorithm that enables robust tumor mutation profiling in the clinical setting.
Methodology
We developed and implemented an optimized mutation profiling platform (“OncoMap”) to interrogate ∼400 mutations in 33 known oncogenes and tumor suppressors, many of which are known to predict response or resistance to targeted therapies. The performance of OncoMap was analyzed using DNA derived from both frozen and FFPE clinical material in a diverse set of cancer types. A subsequent in-depth analysis was conducted on histologically and clinically annotated pediatric gliomas. The sensitivity and specificity of OncoMap were 93.8% and 100% in fresh frozen tissue; and 89.3% and 99.4% in FFPE-derived DNA. We detected known mutations at the expected frequencies in common cancers, as well as novel mutations in adult and pediatric cancers that are likely to predict heightened response or resistance to existing or developmental cancer therapies. OncoMap profiles also support a new molecular stratification of pediatric low-grade gliomas based on BRAF mutations that may have immediate clinical impact.
Conclusions
Our results demonstrate the clinical feasibility of high-throughput mutation profiling to query a large panel of “actionable” cancer gene mutations. In the future, this type of approach may be incorporated into both cancer epidemiologic studies and clinical decision making to specify the use of many targeted anticancer agents.
doi:10.1371/journal.pone.0007887
PMCID: PMC2774511  PMID: 19924296
23.  CDK8 is a colorectal cancer oncogene that regulates β-catenin activity 
Nature  2008;455(7212):547-551.
Aberrant activation of the canonical Wnt/β-catenin pathway occurs in almost all colorectal cancers and contributes to their growth, invasion and survival. Although dysregulated β-catenin activity drives colon tumorigenesis, additional genetic perturbations are required to elaborate fully malignant disease. To identify genes that both modulate β-catenin activity and are essential for colon cancer cell proliferation, we conducted two loss-of-function screens in human colon cancer cells and compared genes identified in these screens with an analysis of copy-number alterations in colon cancer specimens. One of these genes, CDK8, which encodes a member of the mediator complex, is located at 13q12.13, a region of recurrent copy number gain in a substantial fraction of colon cancers. Suppression of CDK8 expression inhibited proliferation in colon cancer cells characterized by high levels of CDK8 and β-catenin hyperactivity. CDK8 kinase activity was necessary for β-catenin driven transformation and expression of several β-catenin transcriptional targets. Together these observations suggest that therapeutic interventions targeting CDK8 may confer clinical benefit in β-catenin-driven malignancies.
doi:10.1038/nature07179
PMCID: PMC2587138  PMID: 18794900
24.  Expression and function of Nkx6.3 in vertebrate hindbrain 
Brain research  2008;1222:42-50.
Homeodomain transcription factors serve important functions in organogenesis and tissue differentiation, particularly with respect to the positional identity of individual cells. The Nkx6 subfamily controls tissue differentiation in the developing central nervous system where they function as transcriptional repressor proteins. Recent work indicates that Nkx6.3 is expressed in hindbrain V2 interneurons that co-express Nkx6.1, suggesting the possibility of functional redundancy. Here, we report that Nkx6.3 expression is specific to Chx10+ V2a interneurons but not Gata3+ V2b interneurons of the hindbrain, and that Nkx6.3 expression appears to mark cells of the prospective medullary reticular formation. Molecular analysis of Nkx6.3 null embryonic mouse hindbrain did not reveal detectable defects in progenitor markers, motor neuron or V2 interneuron sub-types. Forced expression of Nkx6.3 and Nkx6.1 promote V2 interneuron differentiation in the developing chick hindbrain. These findings indicate Nkx6.3 function is dispensable for CNS development and lead to the proposal that absence of overt defects is due to functional compensation from a related homeodomain transcription factor.
doi:10.1016/j.brainres.2008.04.072
PMCID: PMC2555971  PMID: 18586225
Nervous System Development; Regeneration and Aging; Homeobox transcription factor; CNS development; V2 interneuron; Nkx6.3; Nkx6.1; rhombomere; medullary reticular formation
25.  UNCOUPLING OF CHONDROCYTE DIFFERENTIATION AND PERICHONDRIAL MINERALIZATION UNDERLIES THE SKELETAL DYSPLASIA IN TRICHO-RHINO-PHALANGEAL SYNDROME 
Human molecular genetics  2008;17(14):2244-2254.
Tricho-rhino-phalangeal syndrome (TRPS) is an autosomal dominant craniofacial and skeletal dysplasia that is caused by mutations involving the TRPS1 gene. Patients with TRPS have short stature, hip abnormalities, cone-shaped epiphyses, and premature closure of growth plates reflecting defects in endochondral ossification. The TRPS1 gene encodes for the transcription factor TRPS1 that has been demonstrated to repress transcription in vitro. To elucidate molecular mechanisms underlying skeletal abnormalities in TRPS, we analyzed Trps1 mutant mice (Trps1ΔGT mice). Analyses of growth plates demonstrated delayed chondrocyte differentiation and accelerated mineralization of perichondrium in Trps1 mutant mice. These abnormalities were accompanied by increased Runx2 and Ihh expression and increased Ihh signaling. We demonstrated that Trps1 physically interacts with Runx2 and represses Runx2-mediated transactivation. Importantly, generation of Trps1ΔGT/+; Runx2+/− double heterozygous mice rescued the opposite growth plate phenotypes of single mutants demonstrating the genetic interaction between Trps1 and Runx2 transcription factors. Collectively, these data suggest that skeletal dysplasia in TRPS is caused by dysregulation of chondrocyte and perichondrium development partially due to loss of Trps1 repression of Runx2.
doi:10.1093/hmg/ddn125
PMCID: PMC2710999  PMID: 18424451

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