© 2014. Published by The Company of Biologists Ltd | Disease Models & Mechanisms (2014) 7, 1275-1286 doi:10.1242/dmm.014720
RESEARCH ARTICLE
The Peutz-Jeghers kinase LKB1 suppresses polyp growth from intestinal cells of a proglucagon-expressing lineage in mice
ABSTRACT Liver kinase B1 (LKB1; also known as STK11) is a serine/threonine kinase and tumour suppressor that is mutated in Peutz-Jeghers syndrome (PJS), a premalignant syndrome associated with the development of gastrointestinal polyps. Proglucagon-expressing enteroendocrine cells are involved in the control of glucose homeostasis and the regulation of appetite through the secretion of gut hormones such as glucagon-like peptide-1 (GLP-1) and peptide tyrosine tyrosine (PYY). To determine the role of LKB1 in these cells, we bred mice bearing floxed alleles of Lkb1 against animals carrying Cre recombinase under proglucagon promoter control. These mice (GluLKB1KO) were viable and displayed near-normal growth rates and glucose homeostasis. However, they developed large polyps at the gastro-duodenal junction, and displayed premature mortality (death from 120 days of age). Histological analysis of the polyps demonstrated that they had a PJS-like appearance with an arborising smooth-muscle core. Circulating GLP-1 levels were normal in GluLKB1KO mice and the polyps expressed low levels of the peptide, similar to levels in the neighbouring duodenum. Lineage tracing using a Rosa26tdRFP transgene revealed, unexpectedly, that enterocytes within the polyps were derived from non-proglucagon-expressing precursors, whereas connective tissue was largely derived from proglucagon-expressing precursors. Developmental studies in wildtype mice suggested that a subpopulation of proglucagon-expressing cells undergo epithelial-mesenchymal transition (EMT) to become smooth-muscle-like cells. Thus, it is likely that polyps in the GluLKB1KO mice developed from a unique population of smoothmuscle-like cells derived from a proglucagon-expressing precursor. The loss of LKB1 within this subpopulation seems to be sufficient to drive tumorigenesis. KEY WORDS: Glucagon, LKB1, Peutz-Jeghers
INTRODUCTION
The tumour suppressor liver kinase B1 (LKB1), also known as serine/threonine kinase 11 (STK11), was first identified as a controller of zygote polarity in Caenorhabditis elegans (Kemphues 1 Department of Investigative Medicine, Imperial College London, London, W12 ONN, UK. 2Department of Surgery and Cancer, Imperial College London, London, W12 ONN, UK. 3Cambridge Institute for Medical Research, University of Cambridge, Hills Road, Cambridge, CB2 0XY, UK. 4Department of Cell Biology, Imperial College London, London, W12 ONN, UK. *Joint first authors; contributed equally to this manuscript ‡
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Received 1 November 2013; Accepted 3 September 2014
et al., 1988). LKB1 phosphorylates 13 members of the AMPactivated protein kinase (AMPK) family (Lizcano et al., 2004), including polarity-regulating kinase partitioning defective-1 (Par-1) and its mammalian homologue, microtubule-affinity regulating kinase-2 (MARK2) (Marx et al., 2010). The actions of LKB1 as a tumour suppressor thus seem to be due to its role in the control of cell polarity, as well as of cell growth, metabolism and survival. LKB1 is one of two key upstream regulators of classical AMPK complexes in mammalian cells. Activation of AMPK in response to metabolic stress restrains growth factor signalling by stimulating the tuberous sclerosis protein complex (TSC1-TSC2) (Inoki et al., 2003), leading to the inhibition of mammalian target of rapamycin (mTOR), and consequently to blockage of protein and lipid synthesis (Shackelford and Shaw, 2009). Consistent with these signalling roles, heterozygous mutation of the LKB1 gene in humans leads to the development of PeutzJeghers syndrome (PJS), a premalignant disorder characterised by the appearance of pigmentation around the lips, gastrointestinal polyps and an increased risk of all cancers (Boardman et al., 1998). Gastrointestinal polyps are the main clinical feature of PJS and these can grow to large sizes, leading to intestinal obstruction, intussusception, infarction and bleeding. A deeper understanding of how LKB1 restricts tumour formation, and the identification of the intestinal cell types most prone to transformation, are thus needed to allow the development of novel treatments for PJS, a disease for which there are presently no approved pharmaceutical strategies. Homozygous models of LKB1 deletion are difficult to study because constitutive Lkb1−/− mice are not viable beyond embryonic day 10 (Jishage et al., 2002; Ylikorkala et al., 2001). In contrast, heterozygous deletion of a single Lkb1 allele leads to the appearance of PJS-like polyps after 5 months in mice (Bardeesy et al., 2002; Miyoshi et al., 2002). These polyps develop primarily at the gastroduodenal junction and have similar characteristics to polyps found in PJS in humans (Miyoshi et al., 2002). However, the cellular provenance of the intestinal polyps in this model has not been established definitively. Previous studies addressing this issue showed that mono- or biallelic deletion of Lkb1 from smooth muscle, using a conditional Lkb1 allele and recombination mediated by SM-CreERT2(ki), led to smaller polyps (~0.6 mm) than those seen in Lbk1+/− mice (1.5-10 mm), and not to large occluding polyps or to intestinal obstruction (Katajisto et al., 2008). By contrast, no polyps were observed after targeted gastrointestinal epithelial Lkb1 deletion using a Cyp1a1-specific inducible Cre recombinase (Shorning et al., 2009), suggesting that epithelial cells could play a minor role in polyp development in PJS. Enteroendocrine cells represent a small but significant population of cells within the gut. These cells make up less than 1% of all those within the intestine but are essential for gut physiology and, collectively, constitute the largest endocrine organ in the body. 1275
Disease Models & Mechanisms
Sagen Zac-Varghese1,*, Stefan Trapp2,*, Paul Richards3,*, Sophie Sayers4, Gao Sun4, Stephen R. Bloom1, Frank Reimann3, Fiona M. Gribble3 and Guy A. Rutter4,‡
TRANSLATIONAL IMPACT Clinical issue Peutz-Jeghers syndrome (PJS) is a pre-malignant syndrome that poses a considerable burden on health owing to the formation of gastrointestinal polyposis. This disease leads to premature mortality as a result of increased malignancy in all organs. Although mutations of the liver kinase B1 (LKB1) tumour suppressor gene have been characterised in PJS, the disease process towards malignancy is poorly understood. This makes early screening for cancer extremely difficult and currently there is no pharmaceutical treatment available for individuals with PJS. LKB1 influences several pathways that control cell growth, including AMP-activated protein kinase (AMPK) and mammalian target of rapamycin (mTOR) signalling. Understanding the role of LKB1 within different types of gastrointestinal cells is therefore of crucial importance for understanding the disease process and the pathways leading to malignancy. Previous research has studied the impact of LKB1 within the smooth muscle and the epithelium of the gastrointestinal tract. Enteroendocrine cells are specialised cells within the gut that allow the gut to communicate with the brain and the pancreas. Here, the authors studied the impact of deleting LKB1 within these cells to elucidate their role in PJS. Results In this study, the authors used mice in which LKB1 was knocked out by deleting the floxed Lkb1 alleles using Cre recombinase under the control of the proglucagon promoter. Deletion of LKB1 in proglucagonexpressing enteroendocrine cells led to the formation of large gastroduodenal polyps and premature mortality. These polyps had the appearance of PJS-like polyps, with an arborising smooth-muscle core. Proglucagon-expressing enteroendocrine cells were rare within the polyps. However, lineage tracing revealed that the connective tissue within the polyps was derived from proglucagon-expressing precursor cells, whereas villus-like cells were not. Lineage tracing in wild-type mice demonstrated that small numbers of proglucagon-expressing cells undergo epithelial-mesenchymal transition to become smooth-musclelike cells within the first 10 days of life. Implications and future directions These results suggest that LKB1 plays a role in the dysregulation of proglucagon-expressing enteroendocrine precursors towards tumorigenesis. Enteroendocrine cells are a minor cell population, making up less than 1% of the cellular content within the gut. However, deletion of LKB1 within these cells is sufficient to induce polyp formation, demonstrating their crucial importance in the development of PJS. LKB1 is an important determiner of gut cell fate, and targeting LKB1 or its downstream pathways could lead to the development of novel treatments for individuals with PJS. This work suggests that further studies are warranted in humans to assess the role of the enteroendocrine system in the pathogenesis of this disease.
Through the expression of proglucagon and prohormone convertase (PC)1/3, enteroendocrine L cells (and to a lesser extent K cells) synthesise and release hormones, including glucagon-like peptide-1 (GLP-1), GLP-2 and oxyntomodulin (OXM) (Habib et al., 2012). These hormones target receptors on pancreatic β-cells and in the central nervous system, thus regulating growth and metabolism (Diakogiannaki et al., 2012). Hormones released by enteroendocrine cells, including peptide tyrosine tyrosine (PYY), GLP-1, GLP-2 and OXM, are of considerable therapeutic interest due to their favourable influence on appetite, energy expenditure and glucose tolerance. GLP-1 is an incretin hormone, a hormone that stimulates the release of insulin from the pancreas. Thus, GLP-1 receptor agonists and inhibitors of GLP-1 hydrolysis are now front-line therapies for type 2 diabetes (T2D) (Campbell and Drucker, 2013; Nauck, 2011), and PYY and OXM analogues, which regulate appetite and control energy expenditure, are both currently being developed to treat obesity. Moreover, enhanced release of these endogenous hormones is strongly implicated in the beneficial effects of bariatric surgery on diabetes mellitus (Karra et al., 2010). Finally, 1276
Disease Models & Mechanisms (2014) doi:10.1242/dmm.014720
GLP-2 is a peptide hormone that is important in the turnover of epithelial cells (le Roux et al., 2010). Up to now, the role of LKB1 specifically in enteroendocrine cells has not been examined. We (Sun et al., 2010) and others (Fu et al., 2009; Granot et al., 2009) have previously shown that LKB1 is a powerful restrictor of pancreatic β-cell growth and development, and plays a role in the control of insulin secretion. We have also noted (Leclerc et al., 2011) that both LKB1 and AMPK are involved in the control of glucagon secretion. However, and in marked contrast to the effect of LKB1 deletion in β-cells, deletion in α-cells exerts little effect on α-cell proliferation or mass (G.S. and G.A.R., unpublished observation). To elucidate the role of LKB1 in proglucagon-expressing enteroendocrine cells, and the possible contribution of these cells or their progenitors in the formation of polyps in PJS, we have crossed mice bearing floxed Lkb1 alleles with mice bearing a proglucagonspecific Cre recombinase. This has allowed us to determine the consequences of the loss of Lkb1 gene function in cells expressing proglucagon, which include the enteroendocrine L cells (plus a subset of K cells), pancreatic α-cells, and GLP-1 neurons in the brainstem [notably the nucleus tractus solitarius (NTS)] and elsewhere in the central nervous system (Llewellyn-Smith et al., 2013). We show that Cre-recombinase-mediated deletion of Lkb1 from the enteroendocrine population causes the growth of large gastroduodenal polyps that eventually lead to death, most likely owing to intestinal obstruction. Lineage tracing suggests that transiently expressing proglucagon enteroendocrine precursor cells undergo epithelial-mesenchymal transition (EMT) to become smoothmuscle-like cells. Loss of LKB1 within these cells leads to dysregulated growth and tumorigenesis. The present study thus contributes towards increased understanding of the mechanism underlying the PJS phenotype. RESULTS Generation of GluLKB1KO mice
In order to generate GluLKB1KO mice deleted for both alleles of Lkb1 in proglucagon-expressing enteroendocrine cells, in pancreatic α-cells and in GLP-1+ neurons within the brainstem and elsewhere, we crossed Lkb1 floxed mice with animals carrying a Crerecombinase transgene under the control of the proglucagon promoter located within a bacterial artificial chromosome (BAC) (Parker et al., 2012). Deletion of LKB1 from proglucagon-expressing cells leads to premature mortality
GluLKB1KO mice developed normally and appeared similar to their heterozygous and wild-type littermates. Comparison of the growth curves of GluLKB1KO, heterozygous (GluLKB1fl/+) and wild-type mice revealed a trend towards lower body weights in the GluLKB1KO group, although this difference did not reach significance (as analysed by one-way ANOVA; Fig. 1A). Most of the conditional null animals appeared in good health and were well groomed. However, from around 120 days after birth, GluLKB1KO mice became bloated in appearance (Fig. 1E) and premature mortality was observed (Fig. 1B). Thus, 11/21 (52%) of the GluLKB1KO mice had died unexpectedly by day 164. The remainder (10/21) displayed symptoms of distress (bloating, weight loss or reduced activity) and were euthanized upon veterinary advice and subjected to post-mortem examination. By contrast, 1/16 (6.2%) wild-type mice died by 164 days of age. Heterozygous animals displayed an intermediate mortality, with 4/16 (25%) dying before day 200 (Fig. 1B).
Disease Models & Mechanisms
RESEARCH ARTICLE
RESEARCH ARTICLE
Disease Models & Mechanisms (2014) doi:10.1242/dmm.014720
GluLKB1KO mice develop large polyps at the gastro-duodenal junction
Pathological analysis of the gastrointestinal tract during post-mortem revealed that 8/10 homozygous GluLKB1KO mice examined developed gastro-duodenal polyps from around 120 days (107-164 days). These were not apparent earlier in life nor did they seem to cause any signs of obstruction or symptoms of distress until a few days prior to death, where weight loss was observed in some cases (6/13 animals; Fig. 1A). As a result, tumours were usually discovered on gross pathological analysis of the gastrointestinal tract during post-mortem (Fig. 1E,F), and were absent from wild-type
animals (Fig. 1C,D). In each case, the polyps were large [mean (±s.d.) length 15.8±7.6 mm; mean diameter 10.6±2.0 mm; n=5]. Polyps were either pedunculated (Fig. 1H) or sessile (Fig. 1G). The polyps were also hyperplastic and complex in structure (Fig. 1G,H,I). It is therefore likely that small-bowel obstruction caused by the polyps led to the weight loss and demise of GluLKB1KO animals. In contrast, we identified tumours in only one GluLKB1fl/+ mouse out of 16 examined. Detailed post-mortem analysis of elderly (471 day, n=3) GluLKB1fl/+ mice revealed that these mice had normal gastrointestinal tracts and no evident signs of polyp formation. In two of the GluLKB1KO mice examined, 1277
Disease Models & Mechanisms
Fig. 1. Decreased lifespan and development of gastro-duodenal polyps following Glu-Cre-dependent deletion of Lkb1. (A) Body weight changes in wild-type (n=10), GluLKB1fl/+ (n=11) and GluLKB1KO (n=13) mice. Analysis by one-way ANOVA did not demonstrate any significant difference between the three groups. (B) KaplanMeier survival curves for wild-type, GluLKB1fl/+ and GluLKB1KO mice. Vertical deflections on the graph represent censored data where the exact date of death was unknown owing to euthanasia of mice with ill health. GluLKB1KO mice (n=21; red), displayed significantly reduced lifespan and 100% of the cohort were deceased by day 164 (Log-rank Mantel-Cox test, P
