NAP1L1: A Novel Human Colorectal Cancer Biomarker Derived From Animal Models of Apc Inactivation.

INTRODUCTION: Colorectal cancer (CRC) is the second leading cause of cancer death worldwide and most deaths result from metastases. We have analyzed animal models in which Apc, a gene that is frequently mutated during the early stages of colorectal carcinogenesis, was inactivated and human samples to try to identify novel potential biomarkers for CRC. MATERIALS AND METHODS: We initially compared the proteomic and transcriptomic profiles of the small intestinal epithelium of transgenic mice in which Apc and/or Myc had been inactivated. We then studied the mRNA and immunohistochemical expression of one protein that we identified to show altered expression following Apc inactivation, nucleosome assembly protein 1-like 1 (NAP1L1) in human CRC samples and performed a prognostic correlation between biomarker expression and survival in CRC patients. RESULTS: Nap1l1 mRNA expression was increased in mouse small intestine following Apc deletion in a Myc dependant manner and was also increased in human CRC samples. Immunohistochemical NAP1L1 expression was decreased in human CRC samples relative to matched adjacent normal colonic tissue. In a separate cohort of 75 CRC patients, we found a strong correlation between NAP1L1 nuclear expression and overall survival in those patients who had stage III and IV cancers. CONCLUSION: NAP1L1 expression is increased in the mouse small intestine following Apc inactivation and its expression is also altered in human CRC. Immunohistochemical NAP1L1 nuclear expression correlated with overall survival in a cohort of CRC patients. Further studies are now required to clarify the role of this protein in CRC.

3D imaging of colorectal cancer organoids identifies responses to Tankyrase inhibitors.

Aberrant activation of the Wnt signalling pathway is required for tumour initiation and survival in the majority of colorectal cancers. The development of inhibitors of Wnt signalling has been the focus of multiple drug discovery programs targeting colorectal cancer and other malignancies associated with aberrant pathway activation. However, progression of new clinical entities targeting the Wnt pathway has been slow. One challenge lies with the limited predictive power of 2D cancer cell lines because they fail to fully recapitulate intratumoural phenotypic heterogeneity. In particular, the relationship between 2D cancer cell biology and cancer stem cell function is poorly understood. By contrast, 3D tumour organoids provide a platform in which complex cell-cell interactions can be studied. However, complex 3D models provide a challenging platform for the quantitative analysis of drug responses of therapies that have differential effects on tumour cell subpopulations. Here, we generated tumour organoids from colorectal cancer patients and tested their responses to inhibitors of Tankyrase (TNKSi) which are known to modulate Wnt signalling. Using compounds with 3 orders of magnitude difference in cellular mechanistic potency together with image-based assays, we demonstrate that morphometric analyses can capture subtle alterations in organoid responses to Wnt inhibitors that are consistent with activity against a cancer stem cell subpopulation. Overall our study highlights the value of phenotypic readouts as a quantitative method to asses drug-induced effects in a relevant preclinical model.

APC2 is critical for ovarian WNT signalling control, fertility and tumour suppression.

BACKGROUND: Canonical WNT signalling plays a critical role in the regulation of ovarian development; mis-regulation of this key pathway in the adult ovary is associated with subfertility and tumourigenesis. The roles of Adenomatous polyposis coli 2 (APC2), a little-studied WNT signalling pathway regulator, in ovarian homeostasis, fertility and tumourigenesis have not previously been explored. Here, we demonstrate essential roles of APC2 in regulating ovarian WNT signalling and ovarian homeostasis. METHODS: A detailed analysis of ovarian histology, gene expression, ovulation and hormone levels was carried out in 10 week old and in aged constitutive APC2-knockout (Apc2-/-) mice (mixed background). Statistical significance for qRT-PCR data was determined from 95% confidence intervals. Significance testing was performed using 2-tailed Student's t-test, when 2 experimental cohorts were compared. When more were compared, ANOVA test was used, followed by a post-hoc test (LSD or Games-Howell). P-values of < 0.05 were considered statistically significant. RESULTS: APC2-deficiency resulted in activation of ovarian WNT signalling and sub-fertility driven by intra-ovarian defects. Follicular growth was perturbed, resulting in a reduced rate of ovulation and corpora lutea formation, which could not be rescued by administration of gonadotrophins. Defects in steroidogenesis and follicular vascularity contributed to the subfertility phenotype. Tumour incidence was assessed in aged APC2-deficient mice, which also carried a hypomorphic Apc allele. APC2-deficiency in these mice resulted in predisposition to granulosa cell tumour (GCT) formation, accompanied by acute tumour-associated WNT-signalling activation and a histologic pattern and molecular signature seen in human adult GCTs. CONCLUSIONS: Our work adds APC2 to the growing list of WNT-signalling members that regulate ovarian homeostasis, fertility and suppress GCT formation. Importantly, given that the APC2-deficient mouse develops tumours that recapitulate the molecular signature and histological features of human adult GCTs, this mouse has excellent potential as a pre-clinical model to study ovarian subfertility and transitioning to GCT, tumour biology and for therapeutic testing.

Rapid activation of epithelial-mesenchymal transition drives PARP inhibitor resistance in Brca2-mutant mammary tumours.

Tumours defective in the DNA homologous recombination repair pathway can be effectively treated with poly (ADP-ribose) polymerase (PARP) inhibitors; these have proven effective in clinical trials in patients with BRCA gene function-defective cancers. However, resistance observed in both pre-clinical and clinical studies is likely to impact on this treatment strategy. Over-expression of phosphoglycoprotein (P-gp) has been previously suggested as a mechanism of resistance to the PARP inhibitor olaparib in mouse models of Brca1/2-mutant breast cancer. Here, we report that in a Brca2 model treated with olaparib, P-gp upregulation is observed but is not sufficient to confer resistance. Furthermore, resistant/relapsed tumours do not show substantial changes in PK/PD of olaparib, do not downregulate PARP1 or re-establish double stranded DNA break repair by homologous recombination, all previously suggested as mechanisms of resistance. However, resistance is strongly associated with epithelial-mesenchymal transition (EMT) and treatment-naïve tumours given a single dose of olaparib upregulate EMT markers within one hour. Therefore, in this model, olaparib resistance is likely a product of an as-yet unidentified mechanism associated with rapid transition to the mesenchymal phenotype.

TGFß inhibition restores a regenerative response in acute liver injury by suppressing paracrine senescence.

Liver injury results in rapid regeneration through hepatocyte proliferation and hypertrophy. However, after acute severe injury, such as acetaminophen poisoning, effective regeneration may fail. We investigated how senescence may underlie this regenerative failure. In human acute liver disease, and murine models, p21-dependent hepatocellular senescence was proportionate to disease severity and was associated with impaired regeneration. In an acetaminophen injury mouse model, a transcriptional signature associated with the induction of paracrine senescence was observed within 24 hours and was followed by one of impaired proliferation. In mouse genetic models of hepatocyte injury and senescence, we observed transmission of senescence to local uninjured hepatocytes. Spread of senescence depended on macrophage-derived transforming growth factor-ß1 (TGFß1) ligand. In acetaminophen poisoning, inhibition of TGFß receptor 1 (TGFßR1) improved mouse survival. TGFßR1 inhibition reduced senescence and enhanced liver regeneration even when delivered beyond the therapeutic window for treating acetaminophen poisoning. This mechanism, in which injury-induced senescence impairs liver regeneration, is an attractive therapeutic target for developing treatments for acute liver failure.

Oral Prion Neuroinvasion Occurs Independently of PrPC Expression in the Gut Epithelium.

The early replication of certain prion strains within Peyer's patches in the small intestine is essential for the efficient spread of disease to the brain after oral exposure. Our data show that orally acquired prions utilize specialized gut epithelial cells known as M cells to enter Peyer's patches. M cells express the cellular isoform of the prion protein, PrPC, and this may be exploited by some pathogens as an uptake receptor to enter Peyer's patches. This suggested that PrPC might also mediate the uptake and transfer of prions across the gut epithelium into Peyer's patches in order to establish infection. Furthermore, the expression level of PrPC in the gut epithelium could influence the uptake of prions from the lumen of the small intestine. To test this hypothesis, transgenic mice were created in which deficiency in PrPC was specifically restricted to epithelial cells throughout the lining of the small intestine. Our data clearly show that efficient prion neuroinvasion after oral exposure occurred independently of PrPC expression in small intestinal epithelial cells. The specific absence of PrPC in the gut epithelium did not influence the early replication of prions in Peyer's patches or disease susceptibility. Acute mucosal inflammation can enhance PrPC expression in the intestine, implying the potential to enhance oral prion disease pathogenesis and susceptibility. However, our data suggest that the magnitude of PrPC expression in the epithelium lining the small intestine is unlikely to be an important factor which influences the risk of oral prion disease susceptibility.IMPORTANCE The accumulation of orally acquired prions within Peyer's patches in the small intestine is essential for the efficient spread of disease to the brain. Little is known of how the prions initially establish infection within Peyer's patches. Some gastrointestinal pathogens utilize molecules, such as the cellular prion protein PrPC, expressed on gut epithelial cells to enter Peyer's patches. Acute mucosal inflammation can enhance PrPC expression in the intestine, implying the potential to enhance oral prion disease susceptibility. We used transgenic mice to determine whether the uptake of prions into Peyer's patches was dependent upon PrPC expression in the gut epithelium. We show that orally acquired prions can establish infection in Peyer's patches independently of PrPC expression in gut epithelial cells. Our data suggest that the magnitude of PrPC expression in the epithelium lining the small intestine is unlikely to be an important factor which influences oral prion disease susceptibility.

Mbd2 enables tumourigenesis within the intestine while preventing tumour-promoting inflammation.

Epigenetic regulation plays a key role in the link between inflammation and cancer. Here we examine Mbd2, which mediates epigenetic transcriptional silencing by binding to methylated DNA. In separate studies the Mbd2-/- mouse has been shown (1) to be resistant to intestinal tumourigenesis and (2) to have an enhanced inflammatory/immune response, observations that are inconsistent with the links between inflammation and cancer. To clarify its role in tumourigenesis and inflammation, we used constitutive and conditional models of Mbd2 deletion to explore its epithelial and non-epithelial roles in the intestine. Using a conditional model, we found that suppression of intestinal tumourigenesis is due primarily to the absence of Mbd2 within the epithelia. Next, we demonstrated, using the DSS colitis model, that non-epithelial roles of Mbd2 are key in preventing the transition from acute to tumour-promoting chronic inflammation. Combining models revealed that prior to inflammation the altered Mbd2-/- immune response plays a role in intestinal tumour suppression. However, following inflammation the intestine converts from tumour suppressive to tumour promoting. To summarise, in the intestine the normal function of Mbd2 is exploited by cancer cells to enable tumourigenesis, while in the immune system it plays a key role in preventing tumour-enabling inflammation. Which role is dominant depends on the inflammation status of the intestine. As environmental interactions within the intestine can alter DNA methylation patterns, we propose that Mbd2 plays a key role in determining whether these interactions are anti- or pro-tumourigenic and this makes it a useful new epigenetic model for inflammation-associated carcinogenesis. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Spatiotemporal regulation of liver development by the Wnt/ß-catenin pathway.

While the Wnt/ß-catenin pathway plays a critical role in the maintenance of the zonation of ammonia metabolizing enzymes in the adult liver, the mechanisms responsible for inducing zonation in the embryo are not well understood. Herein we address the spatiotemporal role of the Wnt/ß-catenin pathway in the development of zonation in embryonic mouse liver by conditional deletion of Apc and ß-catenin at different stages of mouse liver development. In normal development, the ammonia metabolising enzymes carbamoylphosphate synthetase I (CPSI) and Glutamine synthetase (GS) begin to be expressed in separate hepatoblasts from E13.5 and E15.5 respectively and gradually increase in number thereafter. Restriction of GS expression occurs at E18 and becomes increasingly limited to the terminal perivenous hepatocytes postnatally. Expression of nuclear ß-catenin coincides with the restriction of GS expression to the terminal perivenous hepatocytes. Conditional loss of Apc resulted in the expression of nuclear ß-catenin throughout the developing liver and increased number of cells expressing GS. Conversely, conditional loss of ß-catenin resulted in loss of GS expression. These data suggest that the Wnt pathway is critical to the development of zonation as well as maintaining the zonation in the adult liver.

PTEN loss and activation of K-RAS and ß-catenin cooperate to accelerate prostate tumourigenesis.

Aberrant phosphoinositide 3-kinase (PI3K), mitogen-activated protein kinase (MAPK) and WNT signalling are emerging as key events in the multistep nature of prostate tumourigenesis and progression. Here, we report a compound prostate cancer murine model in which these signalling pathways cooperate to produce a more aggressive prostate cancer phenotype. Using Cre-LoxP technology and the probasin promoter, we combined the loss of Pten (Ptenfl/fl ), to activate the PI3K signalling pathway, with either dominant stabilized ß-catenin [Catnb+/lox(ex3) ] or activated K-RAS (K-Ras+/V12 ) to aberrantly activate WNT and MAPK signalling, respectively. Synchronous activation of all three pathways (triple mutants) significantly reduced survival (median 96 days) as compared with double mutants [median: 140 days for Catnb+/lox(ex3) Ptenfl/fl ; 182 days for Catnb+/lox(ex3) K-Ras+/V12 ; 238 days for Ptenfl/fl K-Ras+/V12 ], and single mutants [median: 383 days for Catnb+/lox(ex3) ; 407 days for Ptenfl/fl ], reflecting the accelerated tumourigenesis. Tumours followed a stepwise progression from mouse prostate intraepithelial neoplasia to invasive adenocarcinoma, similar to that seen in human disease. There was significantly elevated cellular proliferation, tumour growth and percentage of invasive adenocarcinoma in triple mutants as compared with double mutants and single mutants. Triple mutants showed not only activated AKT, extracellular-signal regulated kinase 1/2, and nuclear ß-catenin, but also significantly elevated signalling through mechanistic target of rapamycin complex 1 (mTORC1). In summary, we show that combined deregulation of the PI3K, MAPK and WNT signalling pathways drives rapid progression of prostate tumourigenesis, and that deregulation of all three pathways results in tumours showing aberrant mTORC1 signalling. As mTORC1 signalling is emerging as a key driver of androgen deprivation therapy resistance, our findings are important for understanding the biology of therapy-resistant prostate cancer and identifying potential approaches to overcome this. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Oncogenic PIK3CA induces centrosome amplification and tolerance to genome doubling.

Mutations in PIK3CA are very frequent in cancer and lead to sustained PI3K pathway activation. The impact of acute expression of mutant PIK3CA during early stages of malignancy is unknown. Using a mouse model to activate the Pik3ca H1047R hotspot mutation in the heterozygous state from its endogenous locus, we here report that mutant Pik3ca induces centrosome amplification in cultured cells (through a pathway involving AKT, ROCK and CDK2/Cyclin E-nucleophosmin) and in mouse tissues, and increased in vitro cellular tolerance to spontaneous genome doubling. We also present evidence that the majority of PIK3CA H1047R mutations in the TCGA breast cancer cohort precede genome doubling. These previously unappreciated roles of PIK3CA mutation show that PI3K signalling can contribute to the generation of irreversible genomic changes in cancer. While this can limit the impact of PI3K-targeted therapies, these findings also open the opportunity for therapeutic approaches aimed at limiting tumour heterogeneity and evolution.

Analysis of Fascin-1 in Relation to Gleason Risk Classification and Nuclear ETS-Related Gene Status of Human Prostate Carcinomas: An Immunohistochemical Study of Clinically Annotated Tumours From the Wales Cancer Bank.

Although prostate-specific antigen (PSA) testing can identify early-stage prostate cancers, additional biomarkers are needed for risk stratification. In one study, high levels of the actin-bundling protein, fascin-1, were correlated with lethal-phase, hormone-refractory prostate cancer. Analyses of independent samples are needed to establish the value of fascin-1 as a possible biomarker. We examined fascin-1 by immunohistochemistry in tumour specimens from the Wales Cancer Bank in comparison with nuclear-located ETS-related gene (ERG), an emerging marker for aggressive prostate cancer. Fascin-1 was elevated in focal areas of a minority of tumours, yet fascin-1-positivity did not differentiate tumours of low-, intermediate-, or high-risk Gleason scores and did not correlate with PSA status or biochemical relapse after surgery. Stromal fascin-1 correlated with high Gleason score. Nuclear ERG was upregulated in tumours but not in stroma. The complexities of fascin-1 status indicate that fascin-1 is unlikely to provide a suitable biomarker for prediction of aggressive prostate cancers.

Matricellular protein SPARCL1 regulates tumor microenvironment-dependent endothelial cell heterogeneity in colorectal carcinoma.

Different tumor microenvironments (TMEs) induce stromal cell plasticity that affects tumorigenesis. The impact of TME-dependent heterogeneity of tumor endothelial cells (TECs) on tumorigenesis is unclear. Here, we isolated pure TECs from human colorectal carcinomas (CRCs) that exhibited TMEs with either improved (Th1-TME CRCs) or worse clinical prognosis (control-TME CRCs). Transcriptome analyses identified markedly different gene clusters that reflected the tumorigenic and angiogenic activities of the respective TMEs. The gene encoding the matricellular protein SPARCL1 was most strongly upregulated in Th1-TME TECs. It was also highly expressed in ECs in healthy colon tissues and Th1-TME CRCs but low in control-TME CRCs. In vitro, SPARCL1 expression was induced in confluent, quiescent ECs and functionally contributed to EC quiescence by inhibiting proliferation, migration, and sprouting, whereas siRNA-mediated knockdown increased sprouting. In human CRC tissues and mouse models, vessels with SPARCL1 expression were larger and more densely covered by mural cells. SPARCL1 secretion from quiescent ECs inhibited mural cell migration, which likely led to stabilized mural cell coverage of mature vessels. Together, these findings demonstrate TME-dependent intertumoral TEC heterogeneity in CRC. They further indicate that TEC heterogeneity is regulated by SPARCL1, which promotes the cell quiescence and vessel homeostasis contributing to the favorable prognoses associated with Th1-TME CRCs.

Epigenetic silencing of serine protease HTRA1 drives polyploidy.

BACKGROUND: Increased numbers and improperly positioned centrosomes, aneuploidy or polyploidy, and chromosomal instability are frequently observed characteristics of cancer cells. While some aspects of these events and the checkpoint mechanisms are well studied, not all players have yet been identified. As the role of proteases other than the proteasome in tumorigenesis is an insufficiently addressed question, we investigated the epigenetic control of the widely conserved protease HTRA1 and the phenotypes of deregulation. METHODS: Mouse embryonal fibroblasts and HCT116 and SW480 cells were used to study the mechanism of epigenetic silencing of HTRA1. In addition, using cell biological and genetic methods, the phenotypes of downregulation of HTRA1 expression were investigated. RESULTS: HTRA1 is epigenetically silenced in HCT116 colon carcinoma cells via the epigenetic adaptor protein MBD2. On the cellular level, HTRA1 depletion causes multiple phenotypes including acceleration of cell growth, centrosome amplification and polyploidy in SW480 colon adenocarcinoma cells as well as in primary mouse embryonic fibroblasts (MEFs). CONCLUSIONS: Downregulation of HTRA1 causes a number of phenotypes that are hallmarks of cancer cells suggesting that the methylation state of the HtrA1 promoter may be used as a biomarker for tumour cells or cells at risk of transformation.

Secreted HMGB1 from Wnt activated intestinal cells is required to maintain a crypt progenitor phenotype.

BACKGROUND AND AIMS: Colorectal cancer (CRC) arises via multiple genetic changes. Mutation of the tumour suppressor gene APC, a key regulator of Wnt signalling, is recognised as a frequent early driving mutation in CRC. We have previously shown that conditional loss of Apc within the murine small intestine (Apcfloxmice) results in acute Wnt signalling activation, altered crypt-villus architecture and many hallmarks of neoplasia. Our transctipomic profiling (Affymetrix Microarrays) and proteomic profiling (iTRAQ-QSTAR) of Apc-deficient intestine inferred the involvement of High Mobility Group Box 1 (Hmgb1) in CRC pathogenesis. Here we assess the contribution of HMGB1 to the crypt progenitor phenotype seen following Apc loss. RESULTS: Elevated HMGB1 was confirmed in intestinal epithelia and serum following conditional loss of Apc. Treatment of Apcflox mice with anti-HMGB1 neutralising antibody significantly reduced many of the crypt progenitor phenotypes associated with Apc loss; proliferation and apoptosis levels were reduced, cell differentiation was restored and the expansion of stem cell marker expression was eradicated. METHODS: Hmgb1 levels in intestinal epithelia and serum in Apcflox and ApcMin mice were assessed using qRT-PCR, Western blot and ELISA assays. The functional importance of elevated extracellular Hmgb1 was assessed using an anti-HMGB1 neutralising antibody in Apcflox mice. CONCLUSIONS: HMGB1 is expressed and secreted from intestinal epithelial cells in response to Wnt signalling activation. This secreted HMGB1 is required to maintain nearly all aspects of the crypt progenitor phenotype observed following Apc loss and add to the body of accumulating evidence indicating that targeting HMGB1 may be a viable novel therapeutic approach.

Vitamin D3 suppresses morphological evolution of the cribriform cancerous phenotype.

Development of cribriform morphology (CM) heralds malignant change in human colon but lack of mechanistic understanding hampers preventive therapy. This study investigated CM pathobiology in three-dimensional (3D) Caco-2 culture models of colorectal glandular architecture, assessed translational relevance and tested effects of 1,25(OH)2D3,theactive form of vitamin D. CM evolution was driven by oncogenic perturbation of the apical polarity (AP) complex comprising PTEN, CDC42 and PRKCZ (phosphatase and tensin homolog, cell division cycle 42 and protein kinase C zeta). Suppression of AP genes initiated a spatiotemporal cascade of mitotic spindle misorientation, apical membrane misalignment and aberrant epithelial configuration. Collectively, these events promoted "Swiss cheese-like" cribriform morphology (CM) comprising multiple abnormal "back to back" lumens surrounded by atypical stratified epithelium, in 3D colorectal gland models. Intestinal cancer driven purely by PTEN-deficiency in transgenic mice developed CM and in human CRC, CM associated with PTEN and PRKCZ readouts. Treatment of PTEN-deficient 3D cultures with 1,25(OH)2D3 upregulated PTEN, rapidly activated CDC42 and PRKCZ, corrected mitotic spindle alignment and suppressed CM development. Conversely, mutationally-activated KRAS blocked1,25(OH)2D3 rescue of glandular architecture. We conclude that 1,25(OH)2D3 upregulates AP signalling to reverse CM in a KRAS wild type (wt), clinically predictive CRC model system. Vitamin D could be developed as therapy to suppress inception or progression of a subset of colorectal tumors.

High Dose Atorvastatin Associated with Increased Risk of Significant Hepatotoxicity in Comparison to Simvastatin in UK GPRD Cohort.

BACKGROUND & AIMS: Occasional risk of serious liver dysfunction and autoimmune hepatitis during atorvastatin therapy has been reported. We compared the risk of hepatotoxicity in atorvastatin relative to simvastatin treatment. METHODS: The UK GPRD identified patients with a first prescription for simvastatin [164,407] or atorvastatin [76,411] between 1997 and 2006, but with no prior record of liver disease, alcohol-related diagnosis, or liver dysfunction. Incident liver dysfunction in the following six months was identified by biochemical value and compared between statin groups by Cox regression model adjusting for age, sex, year treatment started, dose, alcohol consumption, smoking, body mass index and comorbid conditions. RESULTS: Moderate to severe hepatotoxicity [bilirubin >60µmol/L, AST or ALT >200U/L or alkaline phosphatase >1200U/L] developed in 71 patients on atorvastatin versus 101 on simvastatin. Adjusted hazard ratio [AHR] for all atorvastatin relative to simvastatin was 1.9 [95% confidence interval 1.4-2.6]. High dose was classified as 40-80mg daily and low dose 10-20mg daily. Hepatotoxicity occurred in 0.44% of 4075 patients on high dose atorvastatin [HDA], 0.07% of 72,336 on low dose atorvastatin [LDA], 0.09% of 44,675 on high dose simvastatin [HDS] and 0.05% of 119,732 on low dose simvastatin [LDS]. AHRs compared to LDS were 7.3 [4.2-12.7] for HDA, 1.4 [0.9-2.0] for LDA and 1.5 [1.0-2.2] for HDS. CONCLUSIONS: The risk of hepatotoxicity was increased in the first six months of atorvastatin compared to simvastatin treatment, with the greatest difference between high dose atorvastatin and low dose simvastatin. The numbers of events in the analyses were small.

Energy sensing and cancer: LKB1 function and lessons learnt from Peutz-Jeghers syndrome.

We describe in this review increasing evidence that loss of LKB1 kinase in Peutz-Jeghers syndrome (PJS) derails the existing natural balance between cell survival and tumour growth suppression. LKB1 deletion can plunge cells into an energy/oxidative stress-induced crisis which leads to the activation of alternative and often carcinogenic pathways to maintain cellular energy levels. It therefore appears that although LKB1 deficiency can suppress oncogenic transformation in the short term, it can ultimately lead to more progressed and malignant phenotypes by driving abnormal cell differentiation, genomic instability and increased tumour heterogeneity.

Protocols for Analyzing the Role of Paneth Cells in Regenerating the Murine Intestine using Conditional Cre-lox Mouse Models.

The epithelial surface of the mammalian intestine is a dynamic tissue that renews every 3 - 7 days. Understanding this renewal process identified a population of rapidly cycling intestinal stem cells (ISCs) characterized by their expression of the Lgr5 gene. These are supported by a quiescent stem cell population, marked by Bmi-1 expression, capable of replacing them in the event of injury. Investigating the interactions between these populations is crucial to understanding their roles in disease and cancer. The ISCs exist within crypts on the intestinal surface, these niches support the ISC in replenishing the epithelia. The interaction between active and quiescent ISCs likely involves other differentiated cells within the niche, as it has previously been demonstrated that the ''stemness'' of the Lgr5 ISC is closely tied to the presence of their neighboring Paneth cells. Using conditional cre-lox mouse models we tested the effect of deleting the majority of active ISCs in the presence or absence of the Paneth cells. Here we describe the techniques and analysis undertaken to characterize the intestine and demonstrate that the Paneth cells play a crucial role within the ISC niche in aiding recovery following substantial insult.

E-cadherin can limit the transforming properties of activating ß-catenin mutations.

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 ß-catenin (CTNNB1). We have compared the dynamics and the potency of ß-catenin mutations in vivo. Within the murine small intestine (SI), an activating mutation of ß-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 ß-catenin was unable to drive Wnt deregulation or induce the CPC phenotype. This ability of ß-catenin mutation to differentially transform the SI versus the colon correlated with higher expression of E-cadherin and a higher number of E-cadherin:ß-catenin complexes at the membrane. Reduction in E-cadherin synergised with an activating mutation of ß-catenin resulting in a rapid CPC phenotype within the SI and colon. Thus, there is a threshold of ß-catenin that is required to drive transformation, and E-cadherin can act as a buffer to sequester mutated ß-catenin.

Hepatic progenitor cells of biliary origin with liver repopulation capacity.

Hepatocytes and cholangiocytes self-renew following liver injury. Following severe injury hepatocytes are increasingly senescent, but whether hepatic progenitor cells (HPCs) then contribute to liver regeneration is unclear. Here, we describe a mouse model where the E3 ubiquitin ligase Mdm2 is inducibly deleted in more than 98% of hepatocytes, causing apoptosis, necrosis and senescence with nearly all hepatocytes expressing p21. This results in florid HPC activation, which is necessary for survival, followed by complete, functional liver reconstitution. HPCs isolated from genetically normal mice, using cell surface markers, were highly expandable and phenotypically stable in vitro. These HPCs were transplanted into adult mouse livers where hepatocyte Mdm2 was repeatedly deleted, creating a non-competitive repopulation assay. Transplanted HPCs contributed significantly to restoration of liver parenchyma, regenerating hepatocytes and biliary epithelia, highlighting their in vivo lineage potency. HPCs are therefore a potential future alternative to hepatocyte or liver transplantation for liver disease.