register interest

Professor Simon Leedham

Research Area: Cell and Molecular Biology
Keywords: Stem cells and Morphogens
Web Links:

Simon Leedham is a Cancer Research UK Clinician Scientist and an Honorary Consultant Gastroenterologist

Outline of Research

Adult gastrointestinal stem cells are the targets of carcinogenic gene mutations and are believed to be the cells of origin of luminal gastrointestinal cancers. Our published work has examined the clonality and genetic mutation burden of pre-neoplastic gastrointestinal disease. Our current research focuses on the homeostatic cell-signaling pathways that control intestinal stem cells and the dysregulation of these pathways in carcinogenesis. The wnt pathway is the best characterised system and promotes the maintenance and proliferation of stem cells, however other signaling pathways such as the Bone Morphogenetic Protein (BMP) and Notch pathway cross-talk and interact with wnt signaling. Recent work from our laboratory on hereditary polyposis syndromes and genome-wide association studies in sporadic colorectal cancer patients has implicated the BMP pathway’s involvement in predisposition to colorectal cancer.

Projects in the lab

Clonality and clonal ordering.

We have developed techniques for examining the mutation burden of single intestinal crypts from archival paraffin embedded tissue. We use individual crypt mutation burden to analyse clonal evolution and construct tumor phylogeny by examining the spatial distribution of shared mutations in different phenotypic regions across single lesions; a technique that has been termed clonal ordering or genetic dependency analysis

‘Just-right’ wnt signal levels and regional intestinal tumour formation.

Mutations in the tumour suppressor gene APC result in increased canonical wnt signaling in intestinal stem cells and are found in up to 85% of colorectal tumours. The ‘just-right’ hypothesis predicts that an optimal but not excessive level of wnt signal in necessary for tumourigenesis and it is this that determines the APC mutation spectra. The optimal level of wnt signal varies throughout the intestinal tract. We are investigating the underlying basal wnt gradient in the human and mouse and have used transgenic mouse models to examine the effect of pan-intestinal wnt perturbation.

Mesenchymal control of intestinal stem cells.

There is increasing evidence to suggest that the epithelial stroma significantly influences tumour development and progression. BMP pathway constituents are mesenchymally expressed and act in a paracrine fashion upon the intestinal epithelium, antagonising wnt signaling and promoting differentiation in mature enterocytes. We are examining the epigenetic and transcriptional control of BMP pathway constituents and functionally characterizing the role of this pathway in intestinal stem cell control in vitro using colorectal cancer and myofibroblast cell lines and in vivo by developing transgenic animal models.

Name Department Institution Country
Professor Ian Tomlinson Wellcome Trust Centre for Human Genetics Oxford University, Henry Wellcome Building of Genomic Medicine United Kingdom
Dr Shazia Irshad Wellcome Trust Centre for Human Genetics Oxford University, Henry Wellcome Building of Genomic Medicine United Kingdom
Dr James East Experimental Medicine Division Oxford University, John Radcliffe Hospital United Kingdom
Leedham SJ, Chetty R. 2016. Wnt disruption in colorectal polyps - the traditional serrated adenoma enters the fray. J Pathol, 239 (4), pp. 387-390. | Show Abstract | Read more

The adenoma-carcinoma sequence describes the development of colorectal carcinoma (CRC) from benign colorectal precursor lesions. Molecular classification of established CRC has demonstrated considerable disease heterogeneity; however, as an emerging cancer frequently outgrows and destroys the initial precursor lesion, CRC molecular taxonomy can only be partially reconciled with histologically classified polyps. Thus, the molecular pathogenesis of some colorectal polyp types, including the traditional serrated adenoma (TSA), is still unclear. Now, candidate driver gene analysis of a cohort of different polyps reveals characteristic, but highly variable, mutations disrupting the Wnt signalling pathway across different histological polyp subtypes. How and when different precursor lesions acquire Wnt disruption reflects important distinctions in polyp biology, dependent on a combination of the dominant molecular pathway and the cell of origin of individual lesions. TSAs preferentially acquire ligand-dependent Wnt activating mutations, which means that the cancers that arise from these aggressive polyps may be sensitive to targeted Wnt inhibition. This paper demonstrates that applying next-generation sequencing technology to improve our understanding of colorectal precursor lesion molecular pathogenesis could also give important and translationally relevant insights into colorectal cancer biology. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Hellner K, Miranda F, Fotso Chedom D, Herrero-Gonzalez S, Hayden DM, Tearle R, Artibani M, KaramiNejadRanjbar M, Williams R, Gaitskell K et al. 2016. Premalignant SOX2 overexpression in the fallopian tubes of ovarian cancer patients: Discovery and validation studies. EBioMedicine, 10 pp. 137-149. | Show Abstract | Read more

Current screening methods for ovarian cancer can only detect advanced disease. Earlier detection has proved difficult because the molecular precursors involved in the natural history of the disease are unknown. To identify early driver mutations in ovarian cancer cells, we used dense whole genome sequencing of micrometastases and microscopic residual disease collected at three time points over three years from a single patient during treatment for high-grade serous ovarian cancer (HGSOC). The functional and clinical significance of the identified mutations was examined using a combination of population-based whole genome sequencing, targeted deep sequencing, multi-center analysis of protein expression, loss of function experiments in an in-vivo reporter assay and mammalian models, and gain of function experiments in primary cultured fallopian tube epithelial (FTE) cells. We identified frequent mutations involving a 40kb distal repressor region for the key stem cell differentiation gene SOX2. In the apparently normal FTE, the region was also mutated. This was associated with a profound increase in SOX2 expression (p<2(-16)), which was not found in patients without cancer (n=108). Importantly, we show that SOX2 overexpression in FTE is nearly ubiquitous in patients with HGSOCs (n=100), and common in BRCA1-BRCA2 mutation carriers (n=71) who underwent prophylactic salpingo-oophorectomy. We propose that the finding of SOX2 overexpression in FTE could be exploited to develop biomarkers for detecting disease at a premalignant stage, which would reduce mortality from this devastating disease.

East JE, Travis SPL, Leedham SJ. 2015. Causality and Chance in the Development of Cancer NEW ENGLAND JOURNAL OF MEDICINE, 373 (16), pp. 1578-1579. | Read more

East JE, Travis SP, Leedham SJ. 2015. Causality and Chance in the Development of Cancer. N Engl J Med, 373 (16), pp. 1578-1579. | Read more

Huels DJ, Ridgway RA, Radulescu S, Leushacke M, Campbell AD, Biswas S, Leedham S, Serra S, Chetty R, Moreaux G et al. 2015. E-cadherin can limit the transforming properties of activating β-catenin mutations. EMBO J, 34 (18), pp. 2321-2333. | Show Abstract | Read more

Wnt pathway deregulation is a common characteristic of many cancers. Only colorectal cancer predominantly harbours mutations in APC, whereas other cancer types (hepatocellular carcinoma, solid pseudopapillary tumours of the pancreas) have activating mutations in β-catenin (CTNNB1). We have compared the dynamics and the potency of β-catenin mutations in vivo. Within the murine small intestine (SI), an activating mutation of β-catenin took much longer to achieve Wnt deregulation and acquire a crypt-progenitor cell (CPC) phenotype than Apc or Gsk3 loss. Within the colon, a single activating mutation of β-catenin was unable to drive Wnt deregulation or induce the CPC phenotype. This ability of β-catenin mutation to differentially transform the SI versus the colon correlated with higher expression of E-cadherin and a higher number of E-cadherin:β-catenin complexes at the membrane. Reduction in E-cadherin synergised with an activating mutation of β-catenin resulting in a rapid CPC phenotype within the SI and colon. Thus, there is a threshold of β-catenin that is required to drive transformation, and E-cadherin can act as a buffer to sequester mutated β-catenin.

Biswas S, Davis H, Irshad S, Sandberg T, Worthley D, Leedham S. 2015. Microenvironmental control of stem cell fate in intestinal homeostasis and disease. J Pathol, 237 (2), pp. 135-145. | Show Abstract | Read more

The conventional model of intestinal epithelial architecture describes a unidirectional tissue organizational hierarchy with stem cells situated at the crypt base and daughter cells proliferating and terminally differentiating as they progress along the vertical (crypt-luminal) axis. In this model, the fate of a cell that has left the niche is determined and its lifespan limited. Evidence is accumulating to suggest that stem cell control and daughter cell fate determination is not solely an intrinsic, cell autonomous property but is heavily influenced by the microenvironment including paracrine, mesenchymal, and endogenous epithelial morphogen gradients. Recent research suggests that in intestinal homeostasis, stem cells transit reversibly between states of variable competence in the niche. Furthermore, selective pressures that disrupt the homeostatic balance, such as intestinal inflammation or morphogen dysregulation, can cause committed progenitor cells and even some differentiated cells to regain stem cell properties. Importantly, it has been recently shown that this disruption of cell fate determination can lead to somatic mutation and neoplastic transformation of cells situated outside the crypt base stem cell niche. This paper reviews the exciting developments in the study of stem cell dynamics in homeostasis, intestinal regeneration, and carcinogenesis, and explores the implications for human disease and cancer therapies.

Huels DJ, Ridgway RA, Radulescu S, Leushacke M, Campbell AD, Biswas S, Leedham S, Serra S, Chetty R, Moreaux G et al. 2015. E-cadherin can limit the transforming properties of activating β-catenin mutations EMBO Journal, 34 (18), pp. 2321-2333. | Show Abstract | Read more

© 2015 Cancer Research UK Beatson Institute. Published under the terms of the CC BY 4.0 license.Wnt pathway deregulation is a common characteristic of many cancers. Only colorectal cancer predominantly harbours mutations in APC, whereas other cancer types (hepatocellular carcinoma, solid pseudopapillary tumours of the pancreas) have activating mutations in β-catenin (CTNNB1). We have compared the dynamics and the potency of β-catenin mutations in vivo. Within the murine small intestine (SI), an activating mutation of β-catenin took much longer to achieve Wnt deregulation and acquire a crypt-progenitor cell (CPC) phenotype than Apc or Gsk3 loss. Within the colon, a single activating mutation of β-catenin was unable to drive Wnt deregulation or induce the CPC phenotype. This ability of β-catenin mutation to differentially transform the SI versus the colon correlated with higher expression of E-cadherin and a higher number of E-cadherin:β-catenin complexes at the membrane. Reduction in E-cadherin synergised with an activating mutation of β-catenin resulting in a rapid CPC phenotype within the SI and colon. Thus, there is a threshold of β-catenin that is required to drive transformation, and E-cadherin can act as a buffer to sequester mutated β-catenin. Synopsis In contrast to other Wnt-driven malignancies colorectal cancer is usually linked to APC loss, and very rarely to β-catenin activating mutations. Different E-cadherin levels can explain the variation in transforming potential of β-catenin mutations in different tumors. Activating β-catenin mutations in the mouse small intestine but not the colon lead to Wnt-activation. Increased E-cadherin levels can buffer mutated β-catenin in the colon epithelium. β-catenin activating mutations are linked to human cancers that show reduced levels of E-cadherin. In contrast to other Wnt-driven malignancies colorectal cancer is usually linked to APC loss, and very rarely to β-catenin activating mutations. Different E-cadherin levels can explain the variation in transforming potential of β-catenin mutations in different tumors.

Biswas S, Davis H, Irshad S, Sandberg T, Worthley D, Leedham S. 2015. Microenvironmental control of stem cell fate in intestinal homeostasis and disease Journal of Pathology, 237 (2), pp. 135-145. | Show Abstract | Read more

© 2015 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.The conventional model of intestinal epithelial architecture describes a unidirectional tissue organizational hierarchy with stem cells situated at the crypt base and daughter cells proliferating and terminally differentiating as they progress along the vertical (crypt-luminal) axis. In this model, the fate of a cell that has left the niche is determined and its lifespan limited. Evidence is accumulating to suggest that stem cell control and daughter cell fate determination is not solely an intrinsic, cell autonomous property but is heavily influenced by the microenvironment including paracrine, mesenchymal, and endogenous epithelial morphogen gradients. Recent research suggests that in intestinal homeostasis, stem cells transit reversibly between states of variable competence in the niche. Furthermore, selective pressures that disrupt the homeostatic balance, such as intestinal inflammation or morphogen dysregulation, can cause committed progenitor cells and even some differentiated cells to regain stem cell properties. Importantly, it has been recently shown that this disruption of cell fate determination can lead to somatic mutation and neoplastic transformation of cells situated outside the crypt base stem cell niche. This paper reviews the exciting developments in the study of stem cell dynamics in homeostasis, intestinal regeneration, and carcinogenesis, and explores the implications for human disease and cancer therapies.

Cited:

32

Scopus

Davis H, Irshad S, Bansal M, Rafferty H, Boitsova T, Bardella C, Jaeger E, Lewis A, Freeman-Mills L, Giner FC et al. 2015. Aberrant epithelial GREM1 expression initiates colonic tumorigenesis from cells outside the stem cell niche Nature Medicine, 21 (1), pp. 62-70. | Show Abstract | Read more

© 2014 Nature America, Inc. All rights reserved.Hereditary mixed polyposis syndrome (HMPS) is characterized by the development of mixed-morphology colorectal tumors and is caused by a 40-kb genetic duplication that results in aberrant epithelial expression of the gene encoding mesenchymal bone morphogenetic protein antagonist, GREM1. Here we use HMPS tissue and a mouse model of the disease to show that epithelial GREM1 disrupts homeostatic intestinal morphogen gradients, altering cell fate that is normally determined by position along the vertical epithelial axis. This promotes the persistence and/or reacquisition of stem cell properties in Lgr5-negative progenitor cells that have exited the stem cell niche. These cells form ectopic crypts, proliferate, accumulate somatic mutations and can initiate intestinal neoplasia, indicating that the crypt base stem cell is not the sole cell of origin of colorectal cancer. Furthermore, we show that epithelial expression of GREM1 also occurs in traditional serrated adenomas, sporadic premalignant lesions with a hitherto unknown pathogenesis, and these lesions can be considered the sporadic equivalents of HMPS polyps.

Davis H, Irshad S, Bansal M, Rafferty H, Boitsova T, Bardella C, Jaeger E, Lewis A, Freeman-Mills L, Giner FC et al. 2015. Aberrant epithelial GREM1 expression initiates colonic tumorigenesis from cells outside the stem cell niche. Nat Med, 21 (1), pp. 62-70. | Show Abstract | Read more

Hereditary mixed polyposis syndrome (HMPS) is characterized by the development of mixed-morphology colorectal tumors and is caused by a 40-kb genetic duplication that results in aberrant epithelial expression of the gene encoding mesenchymal bone morphogenetic protein antagonist, GREM1. Here we use HMPS tissue and a mouse model of the disease to show that epithelial GREM1 disrupts homeostatic intestinal morphogen gradients, altering cell fate that is normally determined by position along the vertical epithelial axis. This promotes the persistence and/or reacquisition of stem cell properties in Lgr5-negative progenitor cells that have exited the stem cell niche. These cells form ectopic crypts, proliferate, accumulate somatic mutations and can initiate intestinal neoplasia, indicating that the crypt base stem cell is not the sole cell of origin of colorectal cancer. Furthermore, we show that epithelial expression of GREM1 also occurs in traditional serrated adenomas, sporadic premalignant lesions with a hitherto unknown pathogenesis, and these lesions can be considered the sporadic equivalents of HMPS polyps.

Leedham SJ. 2014. Measuring stem cell dynamics in the human colon--where there's a wiggle, there's a way. J Pathol, 234 (3), pp. 292-295. | Show Abstract | Read more

The last decade has seen huge improvements in our understanding of intestinal stem cell biology, with major advances arising from the ability to transgenically label, and thus identify, murine stem cells and their progeny. In the human, transgenic labelling is not an available option and stem cell dynamic observations have been based on rare hereditary mutations and polymorphisms. Somatic mitochondrial DNA mutations cause a histochemically detectable, but neutrally selected, change in cytochrome c oxidase (CCO) enzyme activity and when this occurs in an intestinal stem cell, it can be used as an effective clonal marker in both health and disease. The intestinal crypt is the functional unit of the gut. Daughter cells are 'born' in the stem cell niche at the crypt base and proliferate, differentiate, and then apoptose as they migrate along the vertical crypt axis over 5-7 days. This stereotypical architecture provides a historical record of cell dynamics, as the distance travelled along the crypt axis is proportional to the time since the daughter cell was born. By staining, identifying, and carefully reconstructing crypt maps from serial en face sections of partially mutated mtDNA crypts, clonal ribbon images can be generated. 'Wiggles' in the width of the clonal ribbon reflect mtDNA mutated stem cell expansion or contraction events and these biological observations are applied in mathematical models. This clever approach is able to infer temporal evolutionary dynamics from a static, single time point measurement, in both normal and familial adenomatous polyposis tissue. As we have seen in the mouse, the simple ability to identify stem cell progeny can lead to a vast expansion in our understanding of stem cell evolution. The use of these techniques to trace recent stem cell dynamics in the human colon makes some headway into the knowledge gap in our understanding of murine and human intestinal stem cell biology. © 2014 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

East J, Leedham S. 2014. Colonoscopy, tumors Endoscopy, 46 (04), pp. 318-321. | Read more

Lewis A, Freeman-Mills L, de la Calle-Mustienes E, Giráldez-Pérez RM, Davis H, Jaeger E, Becker M, Hubner NC, Nguyen LN, Zeron-Medina J et al. 2014. A polymorphic enhancer near GREM1 influences bowel cancer risk through differential CDX2 and TCF7L2 binding. Cell Rep, 8 (4), pp. 983-990. | Show Abstract | Read more

A rare germline duplication upstream of the bone morphogenetic protein antagonist GREM1 causes a Mendelian-dominant predisposition to colorectal cancer (CRC). The underlying disease mechanism is strong, ectopic GREM1 overexpression in the intestinal epithelium. Here, we confirm that a common GREM1 polymorphism, rs16969681, is also associated with CRC susceptibility, conferring ∼20% differential risk in the general population. We hypothesized the underlying cause to be moderate differences in GREM1 expression. We showed that rs16969681 lies in a region of active chromatin with allele- and tissue-specific enhancer activity. The CRC high-risk allele was associated with stronger gene expression, and higher Grem1 mRNA levels increased the intestinal tumor burden in Apc(Min) mice. The intestine-specific transcription factor CDX2 and Wnt effector TCF7L2 bound near rs16969681, with significantly higher affinity for the risk allele, and CDX2 overexpression in CDX2/GREM1-negative cells caused re-expression of GREM1. rs16969681 influences CRC risk through effects on Wnt-driven GREM1 expression in colorectal tumors.

East JE, Leedham SJ. 2014. Colonoscopy, tumors. Endoscopy, 46 (4), pp. 318-321. | Read more

Gill P, Rafferty H, Munday D, Bailey A, Wang LM, East JE, Chetty R, Leedham SJ. 2013. Proximal colon cancer and serrated adenomas - hunting the missing 10%. Clin Med (Lond), 13 (6), pp. 557-561. | Show Abstract | Read more

There is a 10% shortfall in the number of proximal colorectal cancer cases detected by the UK Bowel Cancer Screening Programme and the actual number of UK-registered proximal colorectal cancers. Sessile serrated adenomas/polyps (SSA/P) are common premalignant lesions in the proximal colon and are notoriously difficult to spot endoscopically. Missed or dismissed SSA/Ps might contribute to this UK proximal colon cancer detection disparity. In Oxfordshire, a service evaluation audit and histological review has shown a linear increase in the detection rate of these lesions over the past 4 years. This is the result of increased endoscopist and pathologist awareness of these lesions and improved interdisciplinary communication. This is the result of increased endoscopist and pathologist awareness of these lesions, together with improved interdisciplinary communication, and we predict that this will lead to a comparable detection increase nationwide. Ongoing surveillance of an increasing number of these premalignant lesions could become a significant endoscopic resource requirement once UK guidelines on serrated lesion follow up are established.

Chetty R, Wang LM, Gill P, East JE, Leedham S. 2013. Left-sided sessile serrated polyps/adenomas. Hum Pathol, 44 (9), pp. 1959-1960. | Read more

Gill P, Wang LM, Bailey A, East JE, Leedham S, Chetty R. 2013. Reporting trends of right-sided hyperplastic and sessile serrated polyps in a large teaching hospital over a 4-year period (2009-2012) Journal of Clinical Pathology, 66 (8), pp. 655-658. | Show Abstract | Read more

Aim An audit of serrated polyps diagnosed over a 4-year period: 2009 to 2012 was undertaken to ascertain the reporting trends of sessile serrated polyps (SSP). Methods All right sided hyperplastic polyps (HP) proximal to the splenic flexure and all polyps designated SSP were retrieved from the study period. Three pathologists blinded to the original diagnosis re-examined the slides. Recent American College of Gastroenterology guidelines for the diagnosis of SSP was utilised. Results No cases of SSP were diagnosed in 2009. In 2010, 32 right-sided cases were encountered, 83 confirmed in 2011 and 134 confirmed in 2012. The vast majority of these were right-sided. With regards to right-sided HP that were re-classified as SSP the data is as follows: 20 of 66 in 2009 (30%); 58 of 91 in 2010 (64%); 42 of 106 (40%) in 2011 and 69 of 206 in 2012 (33%). Conclusions This study has demonstrated an almost exponential increase in the diagnosis of SSP over a 4-year period. In addition, 30 to 64% of right-sided HP were re-classified as SSP over the 4-year period suggesting that greater awareness of the diagnostic criteria for SSP is required. SSP is an important precursor lesion in the serrated pathway of colorectal cancer. Its recognition is important for surveillance and therapeutic strategies.

Rafferty H, Gill P, Davis H, Bailey A, East J, Chetty R, Leedham S. 2013. SESSILE SERRATED ADENOMAS, UNDER-RECOGNISED ENDOSCOPICALLY AND UNDER DIAGNOSED PATHOLOGICALLY GUT, 62 (Suppl 1), pp. A196-A196. | Read more

Boitsova T, Rafferty H, Davis H, Bardella C, Gill P, East J, Tomlinson I, Chetty R, Silver A, Leedham S. 2013. BONE MORPHOGENETIC PROTEIN (BMP) PATHWAY DYSREGULATION SUBVERTS ONCOGENE INDUCED SENESCENCE MECHANISMS IN THE SERRATED PATHWAY OF TUMOURIGENESIS GUT, 62 (Suppl 1), pp. A200-A200. | Read more

Gill P, Wang LM, Bailey A, East JE, Leedham S, Chetty R. 2013. Reporting trends of right-sided hyperplastic and sessile serrated polyps in a large teaching hospital over a 4-year period (2009-2012). J Clin Pathol, 66 (8), pp. 655-658. | Show Abstract | Read more

AIM: An audit of serrated polyps diagnosed over a 4-year period: 2009 to 2012 was undertaken to ascertain the reporting trends of sessile serrated polyps (SSP). METHODS: All right sided hyperplastic polyps (HP) proximal to the splenic flexure and all polyps designated SSP were retrieved from the study period. Three pathologists blinded to the original diagnosis re-examined the slides. Recent American College of Gastroenterology guidelines for the diagnosis of SSP was utilised. RESULTS: No cases of SSP were diagnosed in 2009. In 2010, 32 right-sided cases were encountered, 83 confirmed in 2011 and 134 confirmed in 2012. The vast majority of these were right-sided. With regards to right-sided HP that were re-classified as SSP the data is as follows: 20 of 66 in 2009 (30%); 58 of 91 in 2010 (64%); 42 of 106 (40%) in 2011 and 69 of 206 in 2012 (33%). CONCLUSIONS: This study has demonstrated an almost exponential increase in the diagnosis of SSP over a 4-year period. In addition, 30 to 64% of right-sided HP were re-classified as SSP over the 4-year period suggesting that greater awareness of the diagnostic criteria for SSP is required. SSP is an important precursor lesion in the serrated pathway of colorectal cancer. Its recognition is important for surveillance and therapeutic strategies.

Leedham S, East JE, Chetty R. 2013. Diagnosis of sessile serrated polyps/adenomas: what does this mean for the pathologist, gastroenterologist and patient? J Clin Pathol, 66 (4), pp. 265-268. | Read more

Leedham S, East JE, Chetty R. 2013. Diagnosis of sessile serrated polyps/adenomas: What does this mean for the pathologist, gastroenterologist and patient Journal of Clinical Pathology, 66 (4), pp. 265-268. | Read more

Cited:

32

Scopus

Leedham SJ, Rodenas-Cuadrado P, Howarth K, Lewis A, Mallappa S, Segditsas S, Davis H, Jeffery R, Rodriguez-Justo M, Keshav S et al. 2013. A basal gradient of Wnt and stem-cell number influences regional tumour distribution in human and mouse intestinal tracts Gut, 62 (1), pp. 83-93. | Show Abstract | Read more

Objective: Wnt signalling is critical for normal intestinal development and homeostasis. Wnt dysregulation occurs in almost all human and murine intestinal tumours and an optimal but not excessive level of Wnt activation is considered favourable for tumourigenesis. The authors assessed effects of pan-intestinal Wnt activation on tissue homeostasis, taking into account underlying physiological Wnt activity and stem-cell number in each region of the bowel. Design: The authors generated mice that expressed temporally controlled, stabilised β-catenin along the crypt-villus axis throughout the intestines. Physiological Wnt target gene activity was assessed in different regions of normal mouse and human tissue. Human intestinal tumour mutation spectra were analysed. Results: In the mouse, β-catenin stabilisation resulted in a graduated neoplastic response, ranging from dysplastic transformation of the entire epithelium in the proximal small bowel to slightly enlarged crypts of non-dysplastic morphology in the colorectum. In contrast, stem and proliferating cell numbers were increased in all intestinal regions. In the normal mouse and human intestines, stem-cell and Wnt gradients were non-identical, but higher in the small bowel than large bowel in both species. There was also variation in the expression of some Wnt modulators. Human tumour analysis confirmed that different APC mutation spectra are selected in different regions of the bowel. Conclusions: There are variable gradients in stem-cell number, physiological Wnt activity and response to pathologically increased Wnt signalling along the cryptvillus axis and throughout the length of the intestinal tract. The authors propose that this variation influences regional mutation spectra, tumour susceptibility and lesion distribution in mice and humans.

Tomlinson I, Jaeger E, Leedham S, Thomas H. 2013. Reply to "The classification of intestinal polyposis". Nat Genet, 45 (1), pp. 2-3. | Read more

Khan S, Zeki S, Mitchell I, Harrison R, Dunn L, Leedham S, Barr H, Shepherd N, Graham T, Wright N, McDonald S. 2012. THE CLONAL PROGRESSION OF BARRETT'S OESOPHAGUS TO OESOPHAGEAL ADENOCARCINOMA GUT, 61 (Suppl 2), pp. A36-A37. | Read more

Jaeger E, Leedham S, Lewis A, Segditsas S, Becker M, Cuadrado PR, Davis H, Kaur K, Heinimann K, Howarth K et al. 2012. Hereditary mixed polyposis syndrome is caused by a 40-kb upstream duplication that leads to increased and ectopic expression of the BMP antagonist GREM1. Nat Genet, 44 (6), pp. 699-703. | Show Abstract | Read more

Hereditary mixed polyposis syndrome (HMPS) is characterized by apparent autosomal dominant inheritance of multiple types of colorectal polyp, with colorectal carcinoma occurring in a high proportion of affected individuals. Here, we use genetic mapping, copy-number analysis, exclusion of mutations by high-throughput sequencing, gene expression analysis and functional assays to show that HMPS is caused by a duplication spanning the 3' end of the SCG5 gene and a region upstream of the GREM1 locus. This unusual mutation is associated with increased allele-specific GREM1 expression. Whereas GREM1 is expressed in intestinal subepithelial myofibroblasts in controls, GREM1 is predominantly expressed in the epithelium of the large bowel in individuals with HMPS. The HMPS duplication contains predicted enhancer elements; some of these interact with the GREM1 promoter and can drive gene expression in vitro. Increased GREM1 expression is predicted to cause reduced bone morphogenetic protein (BMP) pathway activity, a mechanism that also underlies tumorigenesis in juvenile polyposis of the large bowel.

Leedham S, Tomlinson I. 2012. The continuum model of selection in human tumors: general paradigm or niche product? Cancer Res, 72 (13), pp. 3131-3134. | Show Abstract | Read more

Berger and colleagues recently proposed a continuum model of how somatic mutations cause tumors to grow, thus supplementing the established binary models, such as oncogene activation and "two hits" at tumor suppressor loci. In the basic continuum model, decreases or increases in gene function, short of full inactivation or activation, impact linearly on cancer development. An extension, called the fail-safe model, envisaged an optimum level of gene derangement for tumor growth, but proposed that the cell gained protection from tumorigenesis because additional mutations caused excessive derangement. Most of the evidence in support of the continuum model came from Pten mutant mice rather than humans. In this article, we assess the validity and applicability of the continuum and fail-safe models. We suggest that the latter is of limited use: In part, it restates the existing "just right" of optimum intermediate gene derangement in tumorigenesis, and in part it is inherently implausible that a cell should avoid becoming cancerous only when it is some way down the road to that state. In contrast, the basic continuum model is a very useful addition to the other genetic models of tumorigenesis, especially in certain scenarios. Fittingly for a quantitative model, we propose that the continuum model is most likely to apply where multiple, cancer-promoting mutations have relatively small, additive effects, either through the well-established case of additive germline predisposition alleles or in a largely hypothetical situation where cancers may have acquired several somatic "mini-driver" mutations, each with weaker effects than classical tumor suppressors or fully activated oncogenes.

Leedham SJ, Rodenas-Cuadrado P, Howarth K, Lewis A, Mallappa S, Segditsas S, Davis H, Jeffery R, Rodriguez-Justo M, Keshav S et al. 2013. A basal gradient of Wnt and stem-cell number influences regional tumour distribution in human and mouse intestinal tracts. Gut, 62 (1), pp. 83-93. | Show Abstract | Read more

OBJECTIVE: Wnt signalling is critical for normal intestinal development and homeostasis. Wnt dysregulation occurs in almost all human and murine intestinal tumours and an optimal but not excessive level of Wnt activation is considered favourable for tumourigenesis. The authors assessed effects of pan-intestinal Wnt activation on tissue homeostasis, taking into account underlying physiological Wnt activity and stem-cell number in each region of the bowel. DESIGN: The authors generated mice that expressed temporally controlled, stabilised β-catenin along the crypt-villus axis throughout the intestines. Physiological Wnt target gene activity was assessed in different regions of normal mouse and human tissue. Human intestinal tumour mutation spectra were analysed. RESULTS: In the mouse, β-catenin stabilisation resulted in a graduated neoplastic response, ranging from dysplastic transformation of the entire epithelium in the proximal small bowel to slightly enlarged crypts of non-dysplastic morphology in the colorectum. In contrast, stem and proliferating cell numbers were increased in all intestinal regions. In the normal mouse and human intestines, stem-cell and Wnt gradients were non-identical, but higher in the small bowel than large bowel in both species. There was also variation in the expression of some Wnt modulators. Human tumour analysis confirmed that different APC mutation spectra are selected in different regions of the bowel. CONCLUSIONS: There are variable gradients in stem-cell number, physiological Wnt activity and response to pathologically increased Wnt signalling along the crypt-villus axis and throughout the length of the intestinal tract. The authors propose that this variation influences regional mutation spectra, tumour susceptibility and lesion distribution in mice and humans.

Cited:

68

Scopus

Jaeger E, Leedham S, Lewis A, Segditsas S, Becker M, Cuadrado PR, Davis H, Kaur K, Heinimann K, Howarth K et al. 2012. Hereditary mixed polyposis syndrome is caused by a 40-kb upstream duplication that leads to increased and ectopic expression of the BMP antagonist GREM1 Nature Genetics, 44 (6), pp. 699-703. | Show Abstract | Read more

Hereditary mixed polyposis syndrome (HMPS) is characterized by apparent autosomal dominant inheritance of multiple types of colorectal polyp, with colorectal carcinoma occurring in a high proportion of affected individuals. Here, we use genetic mapping, copy-number analysis, exclusion of mutations by high-throughput sequencing, gene expression analysis and functional assays to show that HMPS is caused by a duplication spanning the 3′ end of the SCG5 gene and a region upstream of the GREM1 locus. This unusual mutation is associated with increased allele-specific GREM1 expression. Whereas GREM1 is expressed in intestinal subepithelial myofibroblasts in controls, GREM1 is predominantly expressed in the epithelium of the large bowel in individuals with HMPS. The HMPS duplication contains predicted enhancer elements; some of these interact with the GREM1 promoter and can drive gene expression in vitro. Increased GREM1 expression is predicted to cause reduced bone morphogenetic protein (BMP) pathway activity, a mechanism that also underlies tumorigenesis in juvenile polyposis of the large bowel. © 2012 Nature America, Inc. All rights reserved.

Galandiuk S, Rodriguez-Justo M, Jeffery R, Nicholson AM, Cheng Y, Oukrif D, Elia G, Leedham SJ, McDonald SA, Wright NA, Graham TA. 2012. Field cancerization in the intestinal epithelium of patients with Crohn's ileocolitis. Gastroenterology, 142 (4), pp. 855-864.e8. | Show Abstract | Read more

BACKGROUND & AIMS: Tumors that develop in patients with Crohn's disease tend be multifocal, so field cancerization (the replacement of normal cells with nondysplastic but tumorigenic clones) might contribute to intestinal carcinogenesis. We investigated patterns of tumor development from pretumor intestinal cell clones. METHODS: We performed genetic analyses of multiple areas of intestine from 10 patients with Crohn's disease and intestinal neoplasia. Two patients had multifocal neoplasia; longitudinal sections were collected from 3 patients. Individual crypts were microdissected and genotyped; clonal dependency analysis was used to determine the order and timing of mutations that led to tumor development. RESULTS: The same mutations in KRAS, CDKN2A(p16), and TP53 that were observed in neoplasias were also present in nontumor, nondysplastic, and dysplastic epithelium. In 2 patients, carcinogenic mutations were detected in nontumor epithelium 4 years before tumors developed. The same mutation (TP53 p.R248W) was detected at multiple sites along the entire length of the colon from 1 patient; it was the apparent founder mutation for synchronous tumors and multiple dysplastic areas. Disruption of TP53, CDKN2A, and KRAS were all seen as possible initial events in tumorigenesis; the sequence of mutations (the tumor development pathway) differed among lesions. CONCLUSIONS: Pretumor clones can grow extensively in the intestinal epithelium of patients with Crohn's disease. Segmental resections for neoplasia in patients with Crohn's disease might therefore leave residual pretumor disease, and dysplasia might be an unreliable biomarker for cancer risk. Characterization of the behavior of pretumor clones might be used to predict the development of intestinal neoplasia.

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Pearl RA, Leedham SJ, Pacifico MD. 2012. The safety of autologous fat transfer in breast cancer: Lessons from stem cell biology Journal of Plastic, Reconstructive and Aesthetic Surgery, 65 (3), pp. 283-288. | Show Abstract | Read more

Autologous fat grafting is versatile tool in plastic surgery and is increasing used for reconstruction following breast conserving surgery for breast cancer. Part of the reconstructive qualities of the transferred fat may be due to the presence of adipose derived mesenchymal stem cells (ADMSC) playing an angiogenic and an adipogenic role. In this context it must be considered if autologously engrafted fat tissue could contribute to carcinogenesis following breast conserving surgery. In this article we review the current stem cell biology evidence on engraftment, transdifferentiation and potential carcinogenic contribution in the breast and other solid organ stem cell niches in an attempt to highlight possible areas of concern. © 2011 British Association of Plastic, Reconstructive and Aesthetic Surgeons. Published by Elsevier Ltd. All rights reserved.

Jawad N, Direkze N, Leedham SJ. 2011. Inflammatory bowel disease and colon cancer. Recent Results Cancer Res, 185 pp. 99-115. | Show Abstract | Read more

The inflammatory bowel diseases (IBD); Crohn's and Ulcerative colitis, result from an altered host response to intestinal flora. Recurrent inflammation with ulceration and tissue restitution confers an increased risk of cancer in both UC and Crohns, and genome wide searches have identified a number of disease susceptibility alleles. The carcinogenesis pathway in colitis-associated colorectal cancer (CACRC) is less clearly understood than it's sporadic counterpart. Clonal ordering experiments have indicated the order and timing of chromosomal instability and common genetic mutations. Epigenetic changes such as DNA methylation and histone modification are thought to play an increasingly important role in inflammation induced carcinogenesis. Clonal expansion of procarcinogenic mutations can lead to large fields of mutant tissue from which colitis associated cancers can arise (field cancerisation). Endoscopic screening is the mainstay of surveillance in high-risk patients although the development of appropriate, clinically applicable biomarkers remains a research priority. Despite the expanding field of biological therapy in inflammatory bowel disease the ASA compounds remain the best-studied and most efficacious chemopreventive agents. Colitis associated CRC appears to have a different aetiology, carcinogenesis pathway and clinical course to its sporadic counterpart. Further research including long-term follow up of patient cohorts taking biological therapies will improve the detection and treatment of these important, inflammation-induced malignancies.

Pearl RA, Leedham SJ, Pacifico MD. 2012. The safety of autologous fat transfer in breast cancer: lessons from stem cell biology. J Plast Reconstr Aesthet Surg, 65 (3), pp. 283-288. | Show Abstract | Read more

Autologous fat grafting is versatile tool in plastic surgery and is increasing used for reconstruction following breast conserving surgery for breast cancer. Part of the reconstructive qualities of the transferred fat may be due to the presence of adipose derived mesenchymal stem cells (ADMSC) playing an angiogenic and an adipogenic role. In this context it must be considered if autologously engrafted fat tissue could contribute to carcinogenesis following breast conserving surgery. In this article we review the current stem cell biology evidence on engraftment, transdifferentiation and potential carcinogenic contribution in the breast and other solid organ stem cell niches in an attempt to highlight possible areas of concern.

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32

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Sakthianandeswaren A, Christie M, D'Andreti C, Tsui C, Jorissen RN, Li S, Fleming NI, Gibbs P, Lipton L, Malaterre J et al. 2011. PHLDA1 expression marks the putative epithelial stem cells and contributes to intestinal tumorigenesis Cancer Research, 71 (10), pp. 3709-3719. | Show Abstract | Read more

Studies employing mouse models have identified crypt base and position +4 cells as strong candidates for intestinal epithelial stem cells. Equivalent cell populations are thought to exist in the human intestine; however robust and specific protein markers are lacking. Here, we show that in the human small and large intestine, PHLDA1 is expressed in discrete crypt base and some position +4 cells. In small adenomas, PHLDA1 was expressed in a subset of undifferentiated and predominantly Ki-67-negative neoplastic cells, suggesting that a basic hierarchy of differentiation is retained in early tumorigenesis. In large adenomas, carcinomas, and metastases PHLDA1 expression became widespread, with increased expression and nuclear localization at invasive margins. siRNA-mediated suppression of PHLDA1 in colon cancer cells inhibited migration and anchorage-independent growth in vitro and tumor growth in vivo. The integrins ITGA2 and ITGA6 were downregulated in response to PHLDA1 suppression, and accordingly cell adhesion to laminin and collagen was significantly reduced. We conclude that PHLDA1 is a putative epithelial stem cell marker in the human small and large intestine and contributes to migration and proliferation in colon cancer cells. ©2011 AACR.

Gutierrez-Gonzalez L, Graham TA, Rodriguez-Justo M, Leedham SJ, Novelli MR, Gay LJ, Ventayol-Garcia T, Green A, Mitchell I, Stoker DL et al. 2011. The clonal origins of dysplasia from intestinal metaplasia in the human stomach. Gastroenterology, 140 (4), pp. 1251-1260.e1-6. | Show Abstract | Read more

BACKGROUND & AIMS: Studies of the clonal architecture of gastric glands with intestinal metaplasia are important in our understanding of the progression from metaplasia to dysplasia. It is not clear if dysplasias are derived from intestinal metaplasia or how dysplasias expand. We investigated whether cells within a metaplastic gland share a common origin, whether glands clonally expand by fission, and determine if such metaplastic glands are genetically related to the associated dysplasia. We also examined the clonal architecture of entire dysplastic lesions and the genetic changes associated with progression within dysplasia. METHODS: Cytochrome c oxidase-deficient (CCO⁻) metaplastic glands were identified using a dual enzyme histochemical assay. Clonality was assessed by laser capture of multiple cells throughout CCO⁻ glands and polymerase chain reaction sequencing of the entire mitochondrial DNA (mtDNA) genome. Nuclear DNA abnormalities in individual glands were identified by laser capture microdissection polymerase chain reaction sequencing for mutation hot spots and microsatellite loss of heterozygosity analysis. RESULTS: Metaplastic glands were derived from the same clone-all lineages shared a common mtDNA mutation. Mutated glands were found in patches that had developed through gland fission. Metaplastic and dysplastic glands can be genetically related, indicating the clonal origin of dysplasia from metaplasia. Entire dysplastic fields contained a founder mutation from which multiple, distinct subclones developed. CONCLUSIONS: There is evidence for a distinct clonal evolution from metaplasia to dysplasia in the human stomach. By field cancerization, a single clone can expand to form an entire dysplastic lesion. Over time, this field appears to become genetically diverse, indicating that gastric cancer can arise from a subclone of the founder mutation.

Graham TA, Humphries A, Sanders T, Rodriguez-Justo M, Tadrous PJ, Preston SL, Novelli MR, Leedham SJ, McDonald SA, Wright NA. 2011. Use of methylation patterns to determine expansion of stem cell clones in human colon tissue. Gastroenterology, 140 (4), pp. 1241-1250.e1-9. | Show Abstract | Read more

BACKGROUND & AIMS: It is a challenge to determine the dynamics of stem cells within human epithelial tissues such as colonic crypts. By tracking methylation patterns of nonexpressed genes, we have been able to determine how rapidly individual stem cells became dominant within a human colonic crypt. We also analyzed methylation patterns to study clonal expansion of entire crypts via crypt fission. METHODS: Colonic mucosa was obtained from 9 patients who received surgery for colorectal cancer. The methylation patterns of Cardiac-specific homeobox, Myoblast determination protein 1, and Biglycan were examined within clonal cell populations, comprising either part of, or multiple adjacent, normal human colonic crypts. Clonality was demonstrated by following cytochrome c oxidase-deficient (CCO⁻) cells that shared an identical somatic point mutation in mitochondrial DNA. RESULTS: Methylation pattern diversity among CCO⁻ clones that occupied only part of a crypt was proportional to clone size; this allowed us to determine rates of clonal expansion. Analysis indicated a slow rate of niche succession within the crypt. The 2 arms of bifurcating crypts had distinct methylation patterns, indicating that fission can disrupt epigenetic records of crypt ancestry. Adjacent clonal CCO⁻ crypts usually had methylation patterns as dissimilar to one another as methylation patterns of 2 unrelated crypts. Mathematical models indicated that stem cell dynamics and epigenetic drift could account for observed dissimilarities in methylation patterns. CONCLUSIONS: Methylation patterns can be analyzed to determine the rates of recent clonal expansion of stem cells, but determination of clonality over many decades is restricted by epigenetic drift. We developed a technique to follow changes in intestinal stem cell dynamics in human epithelial tissues that might be used to study premalignant disease.

Will OC, Leedham SJ, Elia G, Phillips RK, Clark SK, Tomlinson IP. 2010. Location in the large bowel influences the APC mutations observed in FAP adenomas. Fam Cancer, 9 (3), pp. 389-393. | Show Abstract | Read more

The right colon differs from the left, in embryological origin, luminal environment, and function. In both sporadic colorectal cancer and Familial Adenomatous Polyposis (FAP), polyp density and cancer susceptibility vary markedly by colonic site. Adenomas in FAP have a different mutational spectrum in small intestine versus colon. This study aimed to investigate whether colonic location also influences the APC mutation spectrum in FAP. 127 1-2 mm mildly dysplastic adenomas from 5 patients with a codon 1309 germline mutation, and 41 from 3 patients with mutations proximal to codon 1265, were analysed to assess the frequency of loss of heterozygosity (LOH). We chose polyps from different locations in the colon. Immunohistochemistry for beta-catenin, caspase-3 and Ki-67 was performed to assess Wnt pathway activation, apoptosis and proliferation. In polyps from patients with a 1309 mutation, the frequency of LOH showed a gradient from rectum (highest) to caecum/ascending colon (lowest), but this was not present in patients with proximal germline APC mutations. Crypt-by-crypt analysis confirmed the LOH findings from whole polyps. Beta-catenin and caspase-3 expression showed no significant variation by colonic region, but Ki-67 expression decreased from ascending colon to rectum in tumours and normal tissue. Colonic site alters the mutational spectrum of APC, and crypt cell proliferation. The higher frequency of LOH in rectal polyps from patients with codon 1309 mutations may help to explain their increased polyp burden at this site compared with patients who have other germline APC mutations.

Thirlwell C, Will OC, Domingo E, Graham TA, McDonald SA, Oukrif D, Jeffrey R, Gorman M, Rodriguez-Justo M, Chin-Aleong J et al. 2010. Clonality assessment and clonal ordering of individual neoplastic crypts shows polyclonality of colorectal adenomas. Gastroenterology, 138 (4), pp. 1441-1454.e7. | Show Abstract | Read more

BACKGROUND & AIMS: According to the somatic mutation theory, monoclonal colorectal lesions arise from sequential mutations in the progeny of a single stem cell. However, studies in a sex chromosome mixoploid mosaic (XO/XY) patient indicated that colorectal adenomas were polyclonal. We assessed adenoma clonality on an individual crypt basis and completed a genetic dependency analysis in carcinomas-in-adenomas to assess mutation order and timing. METHODS: Polyp samples were analyzed from the XO/XY individual, patients with familial adenomatous polyposis and attenuated familial adenomatous polyposis, patients with small sporadic adenomas, and patients with sporadic carcinoma-in-adenomas. Clonality was analyzed using X/Y chromosome fluorescence in situ hybridization, analysis of 5q loss of heterozygosity in XO/XY tissue, and sequencing of adenomatous polyposis coli. Individual crypts and different phenotypic areas of carcinoma-in-adenoma lesions were analyzed for mutations in adenomatous polyposis coli, p53, and K-RAS; loss of heterozygosity at 5q, 17p, and 18q; and aneuploidy. Phylogenetic trees were constructed. RESULTS: All familial adenomatous polyposis-associated adenomas and some sporadic lesions had polyclonal genetic defects. Some independent clones appeared to be maintained in advanced adenomas. No clear obligate order of genetic events was established. Top-down growth of dysplastic tissue into neighboring crypts was a possible mechanism of clonal competition. CONCLUSIONS: Human colorectal microadenomas are polyclonal and may arise from a combination of host genetic features, mucosal exposures, and active crypt interactions. Analyses of tumor phylogenies show that most lesions undergo intermittent genetic homogenization, but heterotypic mutation patterns indicate that independent clonal evolution can occur throughout adenoma development. Based on observations of clonal ordering the requirement and timing of genetic events during neoplastic progression may be more variable than previously thought.

Graham TA, Leedham SJ. 2010. Field defects in DNA repair: is loss of MGMT an initial event in colorectal carcinogenesis? Gut, 59 (11), pp. 1452-1453. | Read more

Gutierrez-Gonzalez L, Deheragoda M, Leedham SJ, Elia G, Shankar A, Imber C, Turnbull DM, Novelli M, Jankowski JA, Wright NW, McDonald SAC. 2009. HUMAN SMALL INTESTINAL CRYPTS ARE CLONAL, CONTAIN MULTIPLE STEM CELLS AND MUTATED CRYPTS DIVIDE BY FISSION GUT, 58 pp. A2-A2.

Segditsas S, Rowan AJ, Howarth K, Jones A, Leedham S, Wright NA, Gorman P, Chambers W, Domingo E, Roylance RR et al. 2009. APC and the three-hit hypothesis. Oncogene, 28 (1), pp. 146-155. | Show Abstract | Read more

The seminal 'two-hit hypothesis' implicitly assumes that bi-allelic tumour suppressor gene (TSG) mutations cause loss of protein function. All subsequent events in that tumour therefore take place on an essentially null background for that TSG protein. We have shown that the two-hit model requires modification for the APC TSG, because mutant APC proteins probably retain some function and the two hits are co-selected to produce an optimal level of Wnt activation. We wondered whether the optimal Wnt level might change during tumour progression, leading to selection for more than two hits at the APC locus. Comprehensive screening of a panel of colorectal cancer (CRC) cell lines and primary CRCs showed that some had indeed acquired third hits at APC. These third hits were mostly copy number gains or deletions, but could be protein-truncating mutations. Third hits were significantly less common when the second hit at APC had arisen by copy-neutral loss of heterozygosity. Both polyploid and near-diploid CRCs had third hits, and the third hits did not simply arise as a result of acquiring a polyploid karyotype. The third hits affected mRNA and protein levels, with potential functional consequences for Wnt signalling and tumour growth. Although some third hits were probably secondary to genomic instability, others did appear specifically to target APC. Whilst it is generally believed that tumours develop and progress through stepwise accumulation of mutations in different functional pathways, it also seems that repeated targeting of the same pathway and/or gene is selected in some cancers.

Leedham SJ, Graham TA, Oukrif D, McDonald SA, Rodriguez-Justo M, Harrison RF, Shepherd NA, Novelli MR, Jankowski JA, Wright NA. 2009. Clonality, founder mutations, and field cancerization in human ulcerative colitis-associated neoplasia. Gastroenterology, 136 (2), pp. 542-50.e6. | Show Abstract | Read more

BACKGROUND & AIMS: The clonality of colitis-associated neoplasia has not been fully determined. One previous report showed polyclonal origins with subsequent monoclonal outgrowth. We aimed to assess the clonality and mutation burden of individual crypts in colitis-associated neoplasias to try to identify gatekeeping founder mutations, and explore the clonality of synchronous lesions to look for field effects. METHODS: Individual crypts (range, 8-21 crypts) were microdissected from across 17 lesions from 10 patients. Individual crypt adenomatous polyposis coli (APC), p53, K-RAS, and 17p loss of heterozygosity mutation burden was established using polymerase chain reaction and sequencing analysis. Serial sections underwent immunostaining for p53, beta-catenin, and image cytometry to detect aneuploidy. RESULTS: In most lesions an oncogenic mutation could be identified in all crypts across the lesion showing monoclonality. This founder mutation was a p53 lesion in the majority of neoplasms but 4 tumors had an initiating K-RAS mutation. Some nondysplastic crypts surrounding areas of dysplasia were found to contain clonal p53 mutations and in one case 3 clonal tumors arose from a patch of nondysplastic crypts containing a K-RAS mutation. CONCLUSIONS: This study used mutation burden analysis of individual crypts across colitis-associated neoplasms to show lesion monoclonality. This study confirmed p53 mutation as initiating mutation in the majority of lesions, but also identified K-RAS activation as an alternative gatekeeping mutation. Local and segmental field cancerization was found by showing pro-oncogenic mutations in nondysplastic crypts surrounding neoplasms, although field changes are unlikely to involve the entire colon because widely separated tumors were genetically distinct.

Gutierrez-Gonzalez L, Deheragoda M, Elia G, Leedham SJ, Shankar A, Imber C, Jankowski JA, Turnbull DM, Novelli M, Wright NA, McDonald SA. 2009. Analysis of the clonal architecture of the human small intestinal epithelium establishes a common stem cell for all lineages and reveals a mechanism for the fixation and spread of mutations. J Pathol, 217 (4), pp. 489-496. | Show Abstract | Read more

Little is known about the clonal structure or stem cell architecture of the human small intestinal crypt/villus unit, or how mutations spread and become fixed. Using mitochondrial DNA (mtDNA) mutations as a marker of clonal expansion of stem cell progeny, we aimed to provide answers to these questions. Enzyme histochemistry (for cytochrome c oxidase and succinate dehydrogenase) was performed on frozen sections of normal human duodenum. Laser-capture microdissected cells were taken from crypts/villi. The entire mitochondrial genome was amplified using a nested PCR protocol; sequencing identified mutations and immunohistochemistry demonstrated specific cell lineages. Cytochrome c oxidase-deficient small bowel crypts were observed within all sections: negative crypts contained the same clonal mutation and all differentiated epithelial lineages were present, indicating a common stem cell origin. Mixed crypts were also detected, confirming the existence of multiple stem cells. We observed crypts where Paneth cells were positive but the rest of the crypt was deficient. We have demonstrated patches of deficient crypts that shared a common mutation, suggesting that they have divided by fission. We have shown that all cells within a small intestinal crypt are derived from one common stem cell. Partially-mutated crypts revealed some novel features of Paneth cell biology, suggesting that either they are long-lived or a committed Paneth cell-specific long-lived progenitor was present. We have demonstrated that mutations are fixed in the small bowel by fission and this has important implications for adenoma development.

Preston SL, Leedham SJ, Oukrif D, Deheregoda M, Goodlad RA, Poulsom R, Alison MR, Wright NA, Novelli M. 2008. The development of duodenal microadenomas in FAP patients: the human correlate of the Min mouse. J Pathol, 214 (3), pp. 294-301. | Show Abstract | Read more

UNLABELLED: The morphological changes associated with the adenoma-carcinoma sequence are well documented in the colorectum. Small intestinal carcinogenesis is thought to progress through a similar adenoma-to-carcinoma pathway, but there is a relative dearth of studies examining the associated morphological changes. The best-known mouse model of intestinal neoplasia, the multiple intestinal neoplasia (Min) mouse, has been criticized as a genetic model of intestinal neoplasia, as the majority of its tumours occur in the small intestine. We examined pancreatico-duodenal resection specimens from seven familial adenomatous polyposis (FAP) patients. Serial sections of these were stained with haematoxylin and eosin for beta-catenin and its downstream target CD44, for BMPR1a, lysozyme, carbonic anhydrase II, and with MIB-1. Individual dysplastic crypts were isolated and mutations in the FAP (APC) gene compared between the top and bottom of the crypt. We found that: (a) duodenal microadenomas are extremely common in FAP patients; (b) these grow in the core of duodenal villi, forming lesions similar to those described in the Min mouse; (c) many lesions arise as monocryptal adenomas and grow by a process of crypt fission and branching; (d) migrating adenomatous cells lose their dysplastic phenotype as they migrate up the crypt villous axis; and (e) Paneth cells lose positional information. IN CONCLUSION: (a) the morphological similarity of adenomas in the Min mouse and human suggest the Min mouse is a good model of FAP; (b) duodenal adenomas in FAP originate in monocryptal adenomas and follow the 'bottom-up' rather than the 'top-down' model of morphogenesis; (c) early microadenomas show evidence of cellular differentiation; (d) defects in the positioning of Paneth cells suggests disruption of the EphB2:EphB3 receptor system.

Leedham SJ, Wright NA. 2008. Expansion of a mutated clone: from stem cell to tumour. J Clin Pathol, 61 (2), pp. 164-171. | Show Abstract | Read more

Intestinal stem cells are adult, tissue-based stem cells located at the base of the intestinal crypt and are capable of regenerating all intestinal cell types. The progeny of mutated stem cells can expand to fill an entire crypt as a consequence of genetic drift, selective advantage or hitchhiking-eventually forming a clonal crypt population by a process called "niche succession". Cancer is believed to be a disease of stem cells. The digestive tract has a very high cancer prevalence partly due to rapid epithelial cell turnover and exposure to dietary toxins. Work on the hereditary cancer syndromes, including familial adenomatous polyposis (FAP), has led to significant advances, including the adenoma-carcinoma sequence. The initial mutation involved in this stepwise progression is in the "gatekeeper" tumour suppressor gene adenomatous polyposis coli (APC). In FAP somatic, second hits in this gene are non-random events, selected for by the position of the germline mutation. The early growth of adenomas is contentious, with two main theories, the "top-down" and "bottom-up" hypotheses, attempting to explain the spread of dysplastic tissue in the bowel. Initial X chromosome inactivation studies suggested that colorectal tumours were monoclonal; however, work on a rare XO/XY human patient with FAP and chimeric Min mice showed that approximately 76% of adenomas were polyclonal. A reduction in tumour multiplicity in the chimeric mouse model has been achieved by the introduction of a homozygous tumour resistance allele. This model has been used to suggest that short-range interaction between adjacent initiated crypts, not random polyp collision, is responsible for tumour polyclonality.

McDonald SA, Greaves LC, Gutierrez-Gonzalez L, Rodriguez-Justo M, Deheragoda M, Leedham SJ, Taylor RW, Lee CY, Preston SL, Lovell M et al. 2008. Mechanisms of field cancerization in the human stomach: the expansion and spread of mutated gastric stem cells. Gastroenterology, 134 (2), pp. 500-510. | Show Abstract | Read more

BACKGROUND & AIMS: How mutations are established and spread through the human stomach is unclear because the clonal structure of gastric mucosal units is unknown. Here we investigate, using mitochondrial DNA (mtDNA) mutations as a marker of clonal expansion, the clonality of the gastric unit and show how mutations expand in normal mucosa and gastric mucosa showing intestinal metaplasia. This has important implications in gastric carcinogenesis. METHODS: Mutated units were identified by a histochemical method to detect activity of cytochrome c oxidase. Negative units were laser-capture microdissected, and mutations were identified by polymerase chain reaction sequencing. Differentiated epithelial cells were identified by immunohistochemistry for lineage markers. RESULTS: We show that mtDNA mutations establish themselves in stem cells within normal human gastric body units, and are passed on to all their differentiated progeny, thereby providing evidence for clonal conversion to a new stem cell-derived unit-monoclonal conversion, encompassing all gastric epithelial lineages. The presence of partially mutated units indicates that more than one stem cell is present in each unit. Mutated units can divide by fission to form patches, with each unit sharing an indentical, mutant mtDNA genotype. Furthermore, we show that intestinal metaplastic crypts are clonal, possess multiple stem cells, and that fission is a mechanism by which intestinal metaplasia spreads. CONCLUSIONS: These data show that human gastric body units are clonal, contain multiple multipotential stem cells, and provide definitive evidence for how mutations spread within the human stomach, and show how field cancerization develops.

Leedham SJ, Wright NA. 2008. Human tumour clonality assessment--flawed but necessary. J Pathol, 215 (4), pp. 351-354. | Show Abstract | Read more

One of the premises of the somatic mutation theory of carcinogenesis is that tumours are clonal lesions derived from a single mutated stem cell progenitor. This theory spawned a proliferation of clonality studies, using a variety of different molecular markers to try to determine tumour clonality in multiple organs. In order to establish true clonality, it is necessary to identify the original founding mutation that occurred at the initiation of the progenitor clone. Use of other lesions may only serve to identify sub-clones. As founding mutations have not been properly established in many organ systems, human clonality assessments carry this caveat. However it is only through clonality and mutation burden assessments that phylogenetic tress become established. Here, we review the advantages, disadvantages and use of different clonality markers.

Leedham SJ, Preston SL, McDonald SA, Elia G, Bhandari P, Poller D, Harrison R, Novelli MR, Jankowski JA, Wright NA. 2008. Individual crypt genetic heterogeneity and the origin of metaplastic glandular epithelium in human Barrett's oesophagus. Gut, 57 (8), pp. 1041-1048. | Show Abstract | Read more

OBJECTIVES: Current models of clonal expansion in human Barrett's oesophagus are based upon heterogenous, flow-purified biopsy analysis taken at multiple segment levels. Detection of identical mutation fingerprints from these biopsy samples led to the proposal that a mutated clone with a selective advantage can clonally expand to fill an entire Barrett's segment at the expense of competing clones (selective sweep to fixation model). We aimed to assess clonality at a much higher resolution by microdissecting and genetically analysing individual crypts. The histogenesis of Barrett's metaplasia and neo-squamous islands has never been demonstrated. We investigated the oesophageal gland squamous ducts as the source of both epithelial sub-types. METHODS: Individual crypts across Barrett's biopsy and oesophagectomy blocks were dissected. Determination of tumour suppressor gene loss of heterozygosity patterns, p16 and p53 point mutations were carried out on a crypt-by-crypt basis. Cases of contiguous neo-squamous islands and columnar metaplasia with oesophageal squamous ducts were identified. Tissues were isolated by laser capture microdissection and genetically analysed. RESULTS: Individual crypt dissection revealed mutation patterns that were masked in whole biopsy analysis. Dissection across oesophagectomy specimens demonstrated marked clonal heterogeneity, with multiple independent clones present. We identified a p16 point mutation arising in the squamous epithelium of the oesophageal gland duct, which was also present in a contiguous metaplastic crypt, whereas neo-squamous islands arising from squamous ducts were wild-type with respect to surrounding Barrett's dysplasia. CONCLUSIONS: By studying clonality at the crypt level we demonstrate that Barrett's heterogeneity arises from multiple independent clones, in contrast to the selective sweep to fixation model of clonal expansion previously described. We suggest that the squamous gland ducts situated throughout the oesophagus are the source of a progenitor cell that may be susceptible to gene mutation resulting in conversion to Barrett's metaplastic epithelium. Additionally, these data suggest that wild-type ducts may be the source of neo-squamous islands.

Alison MR, Murphy G, Leedham S. 2008. Stem cells and cancer: a deadly mix. Cell Tissue Res, 331 (1), pp. 109-124. | Show Abstract | Read more

Stem cells and cancer are inextricably linked; the process of carcinogenesis initially affects normal stem cells or their closely related progenitors and then, at some point, neoplastic stem cells are generated that propagate and ultimately maintain the process. Many, if not all, cancers contain a minority population of self-renewing stem cells, "cancer stem cells", that are entirely responsible for sustaining the tumour and for giving rise to proliferating but progressively differentiating cells that contribute to the cellular heterogeneity typical of many solid tumours. Thus, the bulk of the tumour is often not the clinical problem, and so the identification of cancer stem cells and the factors that regulate their behaviour are likely to have an enormous bearing on the way that we treat neoplastic disease in the future. This review summarises (1) our knowledge of the origins of some cancers from normal stem cells and (2) the evidence for the existence of cancer stem cells; it also illustrates some of the stem cell renewal pathways that are frequently aberrant in cancer and that may represent druggable targets.

Leedham S, Jankowski J. 2007. The evidence base of proton pump inhibitor chemopreventative agents in Barrett's esophagus--the good, the bad, and the flawed! Am J Gastroenterol, 102 (1), pp. 21-23. | Show Abstract | Read more

Gastric acid is believed to be an important etiological factor in the pathogenesis of Barrett's esophagus. Pulsatile acid exposure increases cell proliferation in ex vivo Barrrett's tissue and normalization of esophageal pH reverses this. Proton pump inhibitors (PPIs) are the mainstay of therapy in Barrett's esophagus, and have numerous beneficial effects including symptom control, reduction of inflammation, and promotion of the development of squamous islands. However, PPI therapy causes hypergastrinemia and has not prevented recent increase in the incidences of esophageal cancer. Additionally, evidence presented here by Feagins et al. suggests that acid exposure has a p53-mediated, antiproliferative effect on a nondysplastic Barrett's epithelial cell line, an effect that acid suppression might abrogate. These complex pH, inflammation, and growth factor biological interactions can be most reliably tested in large clinical trials with hard end points like cancer conversion or all causes of mortality. Combining the anti-inflammatory effects of acid suppression with aspirin, a nonsteroidal anti-inflammatory agent, is the subject of the AspECT clinical trial, and this may be the future of chemoprevention in Barrett's.

Leedham SJ, Jankowski JA, Wright NA, Tomlinson IPM. 2006. Genetics of inflammatory bowel disease and associated cancers Current Colorectal Cancer Reports, 2 (4), pp. 191-199. | Show Abstract | Read more

The inflammatory bowel diseases (IBD), Crohn's disease and ulcerative colitis, result from an altered host response to intestinal flora, and genome-wide searches have identified a number of disease susceptibility alleles, such as NOD2/CARD15. IBD confers a high risk of development of a number of malignancies, especially colorectal cancer, and this risk is related to chronic inflammation. Genomic instability in the form of gross chromosomal changes is common, with microsatellite instability occurring in a small subset of colitis-associated lesions. The carcinogenesis pathway in colitis-associated cancers is less clearly understood than its sporadic counterpart. Mutations in the APC gene appear to be less frequent and occur later, whereas inflammation-induced p53 mutations are found early in nondysplastic tissue. Selection and clonal expansion of inflammation-induced mutations is likely to account for the high mutational load seen in carcinoma tissue. Development of an effective biomarker to predict development of malignancy in colitis has so far been unsuccessful. Copyright © 2006 by Current Science Inc.

Greaves LC, Preston SL, Tadrous PJ, Taylor RW, Barron MJ, Oukrif D, Leedham SJ, Deheragoda M, Sasieni P, Novelli MR et al. 2006. Mitochondrial DNA mutations are established in human colonic stem cells, and mutated clones expand by crypt fission. Proc Natl Acad Sci U S A, 103 (3), pp. 714-719. | Show Abstract | Read more

The understanding of the fixation of mutations within human tissues and their subsequent clonal expansion is a considerable problem, of which little is known. We have previously shown that nononcogenic mutations in the mitochondrial genome occur in one of a number of morphologically normal colonic crypt stem cells, the progeny of which later occupy the whole crypt. We propose that these wholly mutated crypts then clonally expand by crypt fission, where each crypt divides into two mutated daughter crypts. Here we show that (i) mutated crypts in the process of fission share the same mutated mitochondrial genotype not present in neighboring cytochrome c oxidase-positive crypts (the odds of this being a random event are >or=2.48 x 10(9):1); (ii) neighboring mutated crypts have the same genotype, which is different from adjacent cytochrome c oxidase-positive crypts; (iii) mutated crypts are clustered together throughout the colon; and (iv) patches of cytochrome c oxidase-deficient crypts increase in size with age. We thus demonstrate definitively that crypt fission is the mechanism by which mutations spread in the normal human colon. This has important implications for the biology of the normal adult human colon and possibly for the growth and spread of colorectal neoplasms.

McDonald SA, Preston SL, Greaves LC, Leedham SJ, Lovell MA, Jankowski JA, Turnbull DM, Wright NA. 2006. Clonal expansion in the human gut: mitochondrial DNA mutations show us the way. Cell Cycle, 5 (8), pp. 808-811. | Show Abstract | Read more

The mechanisms of how DNA mutations are fixed within the human gastrointestinal tract and how they spread are poorly understood and are hotly debated. It has been well documented that human colonic crypts are clonal units; one epithelial stem cell within the crypt becoming dominant and taking over the crypts' entire stem cell population--so called monoclonal conversion. Studies have revealed that crypts can exist as families and develop into patches. The questions have been how do such patches in the human colon develop? Does this have implications on how DNA mutations spread? We have previously shown that mitochondrial DNA (mtDNA) mutations, which result in the deficiency of cytochrome c oxidase, are established within a single colonic crypt stem cell, resulting in a crypt with a mixed phenotype. Over time that mutated stem cell can take over the entire stem cell population resulting in a wholly-mutated crypt. We have furthered this research by showing that entirely cytochrome c oxidase-deficient crypts are able to divide by a process called crypt fission, to form two cytochrome c oxidase-deficient daughter crypts, each sharing the exact parental mtDNA mutation. Furthermore, patches of these crypts also possess a founder mtDNA mutation suggesting that fission repeats itself to form patches, which increase in size with age. Here, we hypothesize that this can be expanded into other areas of the gastrointestinal tract, especially the stomach, where there is a paucity of data regarding clonality and the spread of DNA mutations. We ask if these mutated crypts expand at a different rate to wild type ones. We also discuss the implications for the spread of potential carcinogenic mutations within the gut.

Leedham SJ, Brittan M, Preston SL, McDonald SA, Wright NA. 2006. The stomach periglandular fibroblast sheath: all present and correct. Gut, 55 (2), pp. 295-296.

Leedham SJ, Thliveris AT, Halberg RB, Newton MA, Wright NA. 2005. Gastrointestinal stem cells and cancer: bridging the molecular gap. Stem Cell Rev, 1 (3), pp. 233-241. | Show Abstract | Read more

Cancer is believed to be a disease involving stem cells. The digestive tract has a very high cancer prevalence partly owing to rapid epithelial cell turnover and exposure to dietary toxins. Work on the hereditary cancer syndromes including familial adenomatous polyposis (FAP) has led to significant advances, including the adenoma-carcinoma sequence. The initial mutation involved in this stepwise progression is in the "gatekeeper" tumor suppressor gene adenomatous polyposis coli (APC). In FAP somatic, second hits in this gene are nonrandom events, selected for by the position of the germ-line mutation. Extensive work in both the mouse and human has shown that crypts are clonal units and mutated stem cells may develop a selective advantage, eventually forming a clonal crypt population by a process called "niche succession." Aberrant crypt foci are then formed by the longitudinal division of crypts into two daughter units--crypt fission. The early growth of adenomas is contentious with two main theories, the "top-down" and "bottom-up" hypotheses, attempting to explain the spread of dysplastic tissue in the bowel. Initial X chromosome inactivation studies suggested that colorectal tumors were monoclonal; however, work on a rare XO/XY human patient with FAP and chimeric Min mice showed that 76% of adenomas were polyclonal. A reduction in tumor multiplicity in the chimeric mouse model has been achieved by the introduction of a homozygous tumor resistance allele. This model has been used to suggest that short-range interaction between adjacent initiated crypts, not random polyp collision, is responsible for tumor polyclonality.

Leedham SJ, Brittan M, McDonald SA, Wright NA. 2005. Intestinal stem cells. J Cell Mol Med, 9 (1), pp. 11-24. | Show Abstract | Read more

The intestinal tract has a rapid epithelial cell turnover, which continues throughout life. The process is regulated and maintained by a population of stem cells, which give rise to all the intestinal epithelial cell lineages. Studies in both the mouse and the human show that these cells are capable of forming clonal crypt populations. Stem cells remain hard to identify, however it is thought that they reside in a 'niche' towards the base of the crypt and their activity is regulated by the paracrine secretion of growth factors and cytokines from surrounding mesenchymal cells. Stem cell division is usually asymmetric with the formation of an identical daughter stem cell and committed progenitor cells. Progenitor cells retain the ability to divide until they terminally differentiate. Occasional symmetric division produces either 2 daughter cells with stem cell loss, or 2 stem cells and eventual clone dominance. This stochastic extinction of stem cell lines with eventual dominance of one cell line is called 'niche succession'. The discovery of plasticity, the ability of stem cells to engraft into, and in some cases replace the function of damaged host tissues has generated a large amount of scientific and clinical interest: however the concept remains controversial and is still a subject of hot debate. Studies are beginning to identify the complex molecular, genetic and cellular pathways underlying stem cell function such as Wnt signalling, bone morphogenetic protein (BMP) and Notch/Delta pathways. The derangement of these pathways within stem cells plays an integral part in the development of malignancy within the intestinal tract.

Leedham SJ, Schier S, Thliveris AT, Halberg RB, Newton MA, Wright NA. 2005. From gene mutations to tumours--stem cells in gastrointestinal carcinogenesis. Cell Prolif, 38 (6), pp. 387-405. | Show Abstract | Read more

Stem cells share many properties with malignant cells, such as the ability to self-renew and proliferate. Cancer is believed to be a disease of stem cells. The gastrointestinal tract has high cancer prevalence partly because of rapid epithelial cell turnover and exposure to dietary toxins. The molecular pathways of carcinogenesis differ according to the tissue. Work on hereditary cancer syndromes including familial adenomatous polyposis (FAP) has led to advances in our understanding of the events that occur in tumour development from a gastrointestinal stem cell. The initial mutation involved in the adenoma-carcinoma sequence is in the 'gatekeeper' tumour-suppressor gene adenomatous polyposis coli (APC). Somatic hits in this gene are non-random in FAP, with the type of mutation selected for by the position of the germline mutation. In the stomach, a metaplasia-dysplasia sequence occurs and is often related to Helicobacter pylori infection. Clonal expansion of mutated cells occurs by niche succession. Further expansion of the aberrant clone then occurs by the longitudinal division of crypts into two daughter units--crypt fission. Two theories seek to explain the early development of adenomas--the 'top down' and 'bottom up' hypotheses. Initial studies suggested that colorectal tumours were monoclonal; however, later work on chimeric mice and a sex chromosome mixoploid patient with FAP suggested that up to 76% of early adenomas were polyclonal. Introduction of a homozygous resistance allele has reduced tumour multiplicity in the mouse and has been used to rule out random collision of polyps as the cause of these observations. It is likely that short-range interaction between adjacent initiated crypts is responsible for polyclonality.

Leedham SJ, Chetty R. 2016. Wnt disruption in colorectal polyps - the traditional serrated adenoma enters the fray. J Pathol, 239 (4), pp. 387-390. | Show Abstract | Read more

The adenoma-carcinoma sequence describes the development of colorectal carcinoma (CRC) from benign colorectal precursor lesions. Molecular classification of established CRC has demonstrated considerable disease heterogeneity; however, as an emerging cancer frequently outgrows and destroys the initial precursor lesion, CRC molecular taxonomy can only be partially reconciled with histologically classified polyps. Thus, the molecular pathogenesis of some colorectal polyp types, including the traditional serrated adenoma (TSA), is still unclear. Now, candidate driver gene analysis of a cohort of different polyps reveals characteristic, but highly variable, mutations disrupting the Wnt signalling pathway across different histological polyp subtypes. How and when different precursor lesions acquire Wnt disruption reflects important distinctions in polyp biology, dependent on a combination of the dominant molecular pathway and the cell of origin of individual lesions. TSAs preferentially acquire ligand-dependent Wnt activating mutations, which means that the cancers that arise from these aggressive polyps may be sensitive to targeted Wnt inhibition. This paper demonstrates that applying next-generation sequencing technology to improve our understanding of colorectal precursor lesion molecular pathogenesis could also give important and translationally relevant insights into colorectal cancer biology. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Huels DJ, Ridgway RA, Radulescu S, Leushacke M, Campbell AD, Biswas S, Leedham S, Serra S, Chetty R, Moreaux G et al. 2015. E-cadherin can limit the transforming properties of activating β-catenin mutations. EMBO J, 34 (18), pp. 2321-2333. | Show Abstract | Read more

Wnt pathway deregulation is a common characteristic of many cancers. Only colorectal cancer predominantly harbours mutations in APC, whereas other cancer types (hepatocellular carcinoma, solid pseudopapillary tumours of the pancreas) have activating mutations in β-catenin (CTNNB1). We have compared the dynamics and the potency of β-catenin mutations in vivo. Within the murine small intestine (SI), an activating mutation of β-catenin took much longer to achieve Wnt deregulation and acquire a crypt-progenitor cell (CPC) phenotype than Apc or Gsk3 loss. Within the colon, a single activating mutation of β-catenin was unable to drive Wnt deregulation or induce the CPC phenotype. This ability of β-catenin mutation to differentially transform the SI versus the colon correlated with higher expression of E-cadherin and a higher number of E-cadherin:β-catenin complexes at the membrane. Reduction in E-cadherin synergised with an activating mutation of β-catenin resulting in a rapid CPC phenotype within the SI and colon. Thus, there is a threshold of β-catenin that is required to drive transformation, and E-cadherin can act as a buffer to sequester mutated β-catenin.

Biswas S, Davis H, Irshad S, Sandberg T, Worthley D, Leedham S. 2015. Microenvironmental control of stem cell fate in intestinal homeostasis and disease. J Pathol, 237 (2), pp. 135-145. | Show Abstract | Read more

The conventional model of intestinal epithelial architecture describes a unidirectional tissue organizational hierarchy with stem cells situated at the crypt base and daughter cells proliferating and terminally differentiating as they progress along the vertical (crypt-luminal) axis. In this model, the fate of a cell that has left the niche is determined and its lifespan limited. Evidence is accumulating to suggest that stem cell control and daughter cell fate determination is not solely an intrinsic, cell autonomous property but is heavily influenced by the microenvironment including paracrine, mesenchymal, and endogenous epithelial morphogen gradients. Recent research suggests that in intestinal homeostasis, stem cells transit reversibly between states of variable competence in the niche. Furthermore, selective pressures that disrupt the homeostatic balance, such as intestinal inflammation or morphogen dysregulation, can cause committed progenitor cells and even some differentiated cells to regain stem cell properties. Importantly, it has been recently shown that this disruption of cell fate determination can lead to somatic mutation and neoplastic transformation of cells situated outside the crypt base stem cell niche. This paper reviews the exciting developments in the study of stem cell dynamics in homeostasis, intestinal regeneration, and carcinogenesis, and explores the implications for human disease and cancer therapies.

Davis H, Irshad S, Bansal M, Rafferty H, Boitsova T, Bardella C, Jaeger E, Lewis A, Freeman-Mills L, Giner FC et al. 2015. Aberrant epithelial GREM1 expression initiates colonic tumorigenesis from cells outside the stem cell niche. Nat Med, 21 (1), pp. 62-70. | Show Abstract | Read more

Hereditary mixed polyposis syndrome (HMPS) is characterized by the development of mixed-morphology colorectal tumors and is caused by a 40-kb genetic duplication that results in aberrant epithelial expression of the gene encoding mesenchymal bone morphogenetic protein antagonist, GREM1. Here we use HMPS tissue and a mouse model of the disease to show that epithelial GREM1 disrupts homeostatic intestinal morphogen gradients, altering cell fate that is normally determined by position along the vertical epithelial axis. This promotes the persistence and/or reacquisition of stem cell properties in Lgr5-negative progenitor cells that have exited the stem cell niche. These cells form ectopic crypts, proliferate, accumulate somatic mutations and can initiate intestinal neoplasia, indicating that the crypt base stem cell is not the sole cell of origin of colorectal cancer. Furthermore, we show that epithelial expression of GREM1 also occurs in traditional serrated adenomas, sporadic premalignant lesions with a hitherto unknown pathogenesis, and these lesions can be considered the sporadic equivalents of HMPS polyps.

Lewis A, Freeman-Mills L, de la Calle-Mustienes E, Giráldez-Pérez RM, Davis H, Jaeger E, Becker M, Hubner NC, Nguyen LN, Zeron-Medina J et al. 2014. A polymorphic enhancer near GREM1 influences bowel cancer risk through differential CDX2 and TCF7L2 binding. Cell Rep, 8 (4), pp. 983-990. | Show Abstract | Read more

A rare germline duplication upstream of the bone morphogenetic protein antagonist GREM1 causes a Mendelian-dominant predisposition to colorectal cancer (CRC). The underlying disease mechanism is strong, ectopic GREM1 overexpression in the intestinal epithelium. Here, we confirm that a common GREM1 polymorphism, rs16969681, is also associated with CRC susceptibility, conferring ∼20% differential risk in the general population. We hypothesized the underlying cause to be moderate differences in GREM1 expression. We showed that rs16969681 lies in a region of active chromatin with allele- and tissue-specific enhancer activity. The CRC high-risk allele was associated with stronger gene expression, and higher Grem1 mRNA levels increased the intestinal tumor burden in Apc(Min) mice. The intestine-specific transcription factor CDX2 and Wnt effector TCF7L2 bound near rs16969681, with significantly higher affinity for the risk allele, and CDX2 overexpression in CDX2/GREM1-negative cells caused re-expression of GREM1. rs16969681 influences CRC risk through effects on Wnt-driven GREM1 expression in colorectal tumors.

Gill P, Rafferty H, Munday D, Bailey A, Wang LM, East JE, Chetty R, Leedham SJ. 2013. Proximal colon cancer and serrated adenomas - hunting the missing 10%. Clin Med (Lond), 13 (6), pp. 557-561. | Show Abstract | Read more

There is a 10% shortfall in the number of proximal colorectal cancer cases detected by the UK Bowel Cancer Screening Programme and the actual number of UK-registered proximal colorectal cancers. Sessile serrated adenomas/polyps (SSA/P) are common premalignant lesions in the proximal colon and are notoriously difficult to spot endoscopically. Missed or dismissed SSA/Ps might contribute to this UK proximal colon cancer detection disparity. In Oxfordshire, a service evaluation audit and histological review has shown a linear increase in the detection rate of these lesions over the past 4 years. This is the result of increased endoscopist and pathologist awareness of these lesions and improved interdisciplinary communication. This is the result of increased endoscopist and pathologist awareness of these lesions, together with improved interdisciplinary communication, and we predict that this will lead to a comparable detection increase nationwide. Ongoing surveillance of an increasing number of these premalignant lesions could become a significant endoscopic resource requirement once UK guidelines on serrated lesion follow up are established.

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Leedham SJ, Rodenas-Cuadrado P, Howarth K, Lewis A, Mallappa S, Segditsas S, Davis H, Jeffery R, Rodriguez-Justo M, Keshav S et al. 2013. A basal gradient of Wnt and stem-cell number influences regional tumour distribution in human and mouse intestinal tracts Gut, 62 (1), pp. 83-93. | Show Abstract | Read more

Objective: Wnt signalling is critical for normal intestinal development and homeostasis. Wnt dysregulation occurs in almost all human and murine intestinal tumours and an optimal but not excessive level of Wnt activation is considered favourable for tumourigenesis. The authors assessed effects of pan-intestinal Wnt activation on tissue homeostasis, taking into account underlying physiological Wnt activity and stem-cell number in each region of the bowel. Design: The authors generated mice that expressed temporally controlled, stabilised β-catenin along the crypt-villus axis throughout the intestines. Physiological Wnt target gene activity was assessed in different regions of normal mouse and human tissue. Human intestinal tumour mutation spectra were analysed. Results: In the mouse, β-catenin stabilisation resulted in a graduated neoplastic response, ranging from dysplastic transformation of the entire epithelium in the proximal small bowel to slightly enlarged crypts of non-dysplastic morphology in the colorectum. In contrast, stem and proliferating cell numbers were increased in all intestinal regions. In the normal mouse and human intestines, stem-cell and Wnt gradients were non-identical, but higher in the small bowel than large bowel in both species. There was also variation in the expression of some Wnt modulators. Human tumour analysis confirmed that different APC mutation spectra are selected in different regions of the bowel. Conclusions: There are variable gradients in stem-cell number, physiological Wnt activity and response to pathologically increased Wnt signalling along the cryptvillus axis and throughout the length of the intestinal tract. The authors propose that this variation influences regional mutation spectra, tumour susceptibility and lesion distribution in mice and humans.

Jaeger E, Leedham S, Lewis A, Segditsas S, Becker M, Cuadrado PR, Davis H, Kaur K, Heinimann K, Howarth K et al. 2012. Hereditary mixed polyposis syndrome is caused by a 40-kb upstream duplication that leads to increased and ectopic expression of the BMP antagonist GREM1. Nat Genet, 44 (6), pp. 699-703. | Show Abstract | Read more

Hereditary mixed polyposis syndrome (HMPS) is characterized by apparent autosomal dominant inheritance of multiple types of colorectal polyp, with colorectal carcinoma occurring in a high proportion of affected individuals. Here, we use genetic mapping, copy-number analysis, exclusion of mutations by high-throughput sequencing, gene expression analysis and functional assays to show that HMPS is caused by a duplication spanning the 3' end of the SCG5 gene and a region upstream of the GREM1 locus. This unusual mutation is associated with increased allele-specific GREM1 expression. Whereas GREM1 is expressed in intestinal subepithelial myofibroblasts in controls, GREM1 is predominantly expressed in the epithelium of the large bowel in individuals with HMPS. The HMPS duplication contains predicted enhancer elements; some of these interact with the GREM1 promoter and can drive gene expression in vitro. Increased GREM1 expression is predicted to cause reduced bone morphogenetic protein (BMP) pathway activity, a mechanism that also underlies tumorigenesis in juvenile polyposis of the large bowel.

Cited:

68

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Jaeger E, Leedham S, Lewis A, Segditsas S, Becker M, Cuadrado PR, Davis H, Kaur K, Heinimann K, Howarth K et al. 2012. Hereditary mixed polyposis syndrome is caused by a 40-kb upstream duplication that leads to increased and ectopic expression of the BMP antagonist GREM1 Nature Genetics, 44 (6), pp. 699-703. | Show Abstract | Read more

Hereditary mixed polyposis syndrome (HMPS) is characterized by apparent autosomal dominant inheritance of multiple types of colorectal polyp, with colorectal carcinoma occurring in a high proportion of affected individuals. Here, we use genetic mapping, copy-number analysis, exclusion of mutations by high-throughput sequencing, gene expression analysis and functional assays to show that HMPS is caused by a duplication spanning the 3′ end of the SCG5 gene and a region upstream of the GREM1 locus. This unusual mutation is associated with increased allele-specific GREM1 expression. Whereas GREM1 is expressed in intestinal subepithelial myofibroblasts in controls, GREM1 is predominantly expressed in the epithelium of the large bowel in individuals with HMPS. The HMPS duplication contains predicted enhancer elements; some of these interact with the GREM1 promoter and can drive gene expression in vitro. Increased GREM1 expression is predicted to cause reduced bone morphogenetic protein (BMP) pathway activity, a mechanism that also underlies tumorigenesis in juvenile polyposis of the large bowel. © 2012 Nature America, Inc. All rights reserved.

Galandiuk S, Rodriguez-Justo M, Jeffery R, Nicholson AM, Cheng Y, Oukrif D, Elia G, Leedham SJ, McDonald SA, Wright NA, Graham TA. 2012. Field cancerization in the intestinal epithelium of patients with Crohn's ileocolitis. Gastroenterology, 142 (4), pp. 855-864.e8. | Show Abstract | Read more

BACKGROUND & AIMS: Tumors that develop in patients with Crohn's disease tend be multifocal, so field cancerization (the replacement of normal cells with nondysplastic but tumorigenic clones) might contribute to intestinal carcinogenesis. We investigated patterns of tumor development from pretumor intestinal cell clones. METHODS: We performed genetic analyses of multiple areas of intestine from 10 patients with Crohn's disease and intestinal neoplasia. Two patients had multifocal neoplasia; longitudinal sections were collected from 3 patients. Individual crypts were microdissected and genotyped; clonal dependency analysis was used to determine the order and timing of mutations that led to tumor development. RESULTS: The same mutations in KRAS, CDKN2A(p16), and TP53 that were observed in neoplasias were also present in nontumor, nondysplastic, and dysplastic epithelium. In 2 patients, carcinogenic mutations were detected in nontumor epithelium 4 years before tumors developed. The same mutation (TP53 p.R248W) was detected at multiple sites along the entire length of the colon from 1 patient; it was the apparent founder mutation for synchronous tumors and multiple dysplastic areas. Disruption of TP53, CDKN2A, and KRAS were all seen as possible initial events in tumorigenesis; the sequence of mutations (the tumor development pathway) differed among lesions. CONCLUSIONS: Pretumor clones can grow extensively in the intestinal epithelium of patients with Crohn's disease. Segmental resections for neoplasia in patients with Crohn's disease might therefore leave residual pretumor disease, and dysplasia might be an unreliable biomarker for cancer risk. Characterization of the behavior of pretumor clones might be used to predict the development of intestinal neoplasia.

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Sakthianandeswaren A, Christie M, D'Andreti C, Tsui C, Jorissen RN, Li S, Fleming NI, Gibbs P, Lipton L, Malaterre J et al. 2011. PHLDA1 expression marks the putative epithelial stem cells and contributes to intestinal tumorigenesis Cancer Research, 71 (10), pp. 3709-3719. | Show Abstract | Read more

Studies employing mouse models have identified crypt base and position +4 cells as strong candidates for intestinal epithelial stem cells. Equivalent cell populations are thought to exist in the human intestine; however robust and specific protein markers are lacking. Here, we show that in the human small and large intestine, PHLDA1 is expressed in discrete crypt base and some position +4 cells. In small adenomas, PHLDA1 was expressed in a subset of undifferentiated and predominantly Ki-67-negative neoplastic cells, suggesting that a basic hierarchy of differentiation is retained in early tumorigenesis. In large adenomas, carcinomas, and metastases PHLDA1 expression became widespread, with increased expression and nuclear localization at invasive margins. siRNA-mediated suppression of PHLDA1 in colon cancer cells inhibited migration and anchorage-independent growth in vitro and tumor growth in vivo. The integrins ITGA2 and ITGA6 were downregulated in response to PHLDA1 suppression, and accordingly cell adhesion to laminin and collagen was significantly reduced. We conclude that PHLDA1 is a putative epithelial stem cell marker in the human small and large intestine and contributes to migration and proliferation in colon cancer cells. ©2011 AACR.

Gutierrez-Gonzalez L, Graham TA, Rodriguez-Justo M, Leedham SJ, Novelli MR, Gay LJ, Ventayol-Garcia T, Green A, Mitchell I, Stoker DL et al. 2011. The clonal origins of dysplasia from intestinal metaplasia in the human stomach. Gastroenterology, 140 (4), pp. 1251-1260.e1-6. | Show Abstract | Read more

BACKGROUND & AIMS: Studies of the clonal architecture of gastric glands with intestinal metaplasia are important in our understanding of the progression from metaplasia to dysplasia. It is not clear if dysplasias are derived from intestinal metaplasia or how dysplasias expand. We investigated whether cells within a metaplastic gland share a common origin, whether glands clonally expand by fission, and determine if such metaplastic glands are genetically related to the associated dysplasia. We also examined the clonal architecture of entire dysplastic lesions and the genetic changes associated with progression within dysplasia. METHODS: Cytochrome c oxidase-deficient (CCO⁻) metaplastic glands were identified using a dual enzyme histochemical assay. Clonality was assessed by laser capture of multiple cells throughout CCO⁻ glands and polymerase chain reaction sequencing of the entire mitochondrial DNA (mtDNA) genome. Nuclear DNA abnormalities in individual glands were identified by laser capture microdissection polymerase chain reaction sequencing for mutation hot spots and microsatellite loss of heterozygosity analysis. RESULTS: Metaplastic glands were derived from the same clone-all lineages shared a common mtDNA mutation. Mutated glands were found in patches that had developed through gland fission. Metaplastic and dysplastic glands can be genetically related, indicating the clonal origin of dysplasia from metaplasia. Entire dysplastic fields contained a founder mutation from which multiple, distinct subclones developed. CONCLUSIONS: There is evidence for a distinct clonal evolution from metaplasia to dysplasia in the human stomach. By field cancerization, a single clone can expand to form an entire dysplastic lesion. Over time, this field appears to become genetically diverse, indicating that gastric cancer can arise from a subclone of the founder mutation.

Epithelial cell dynamics and mutation in intestinal inflammation and colitis-associated cancer

Background. Inflammation and cancer are inextricably linked in numerous organs, and the intestinal epithelium is no exception. In health, intestinal epithelial cell-fate is strictly controlled by a balance of secreted signalling pathways as cells move along the intestinal crypt-villus axis. Differential expression of ligands, receptors and antagonists generates strictly regulated mucosal gradients, and the phenotypic response of a cell is determined by it’s position within these concentration ...

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