Genetic control of apoptosis and tumourigenesis in murine models of intestinal neoplasia

Colorectal cancer is one of the most predominant cancers in the Western World. By the age of 70, 1 in 2 people will have a colon tumour. The genetic analysis of both spontaneous and hereditary forms of this disease have greatly added to our understanding of colorectal cancer, with mutations in a range of genes now strongly linked to neoplasia. In recent years, several different transgenic models of colorectal cancer have been generated. Key amongst these are strains mutant for the Ape (.Adenamatous Polyposis Coli) gene and strains mutant for different members of the mismatch repair (MMR) gene family, the majority of which show predisposition to intestinal neoplasia. In this thesis, these transgenic models are used in an attempt to systematically characterise the nature of the genetic control over a series of end points. These include the apoptotic response of enterocytes to cytotoxic agents; the effect of genotype upon clonogenic survival and mutation and ultimately the effect of genoytype upon the development of intestinal neoplasia.Previously analyses of enterocyte apoptosis had established roles for p53 and Msh2 following DNA damage of methylation type. This prompted an analysis in mice deficient for two other members of the MMR family, Mlhl and Pms2. Mlhl and Pms2 deficient mice were seen to have a significantly reduced apoptotic response to temozolomide, confirming again an association between the MMR family and apoptosis. However, both Mlhl'" and Pms2~/~ mice were found to possess a normal apoptotic response to high levels of the alkylating agent NMNU, even though they are deficient for functional MMR. This unexpected finding dissociates normal mismatch repair from MMR dependent apoptosis and raises fundamental questions about the nature of the death signal following damage of methylation type.Perturbations to the normal apoptotic response would be predicted to impact upon longer term survival as determined through the microcolony assay. Therefore clonogenic survival was examined using this approach in Msh2 and p53 null mice. Despite being necessary for apoptosis for all cytotoxic agents studied, loss ofp53 only led to an increased in survival following cisplatin treatment and not following NMNU or Nitrogen Mustard treatment.The above data was obtained from morphologically normal tissue. Therefore this analysis was extended to the apoptotic response within intestinal lesions. This displayed that there was both lesion type dependent differences and genotype dependent differences in the apoptotic response. As there were high basal levels of apoptosis in the smallest lesions whilst virtually no apoptosis in adenomas, this highlighted that loss of this apoptotic programme may be crucial to tumour progression.The battery of in vivo analyses used throughout this thesis were applied to a new candidate tumour suppressor, Mbd4. Mbd4 deficient mice have no overt phenotype, but fail to mediate normal apoptosis following a wide variety of DNA damage. Following cisplatin treatment, Mbd4 treatment confers increased clonogenic survival Surprisingly, Mbd4 mice are not characterised by an in increase in either spontaneous or induced mutation rate, but when crossed to ApcMm mice they accelerate tumour development. These studies demonstrate that Mbd4 is a central mediator of the response to DNA damage and that it functions as an intestinal tumour suppressor in the mouse.Finally the ability of aspirin to suppress intestinal neoplasia in murine models of colorectal cancer was examined. Numerous epidemological and animals studies have shown that Non-Steroidal Anti-Inflamatory Drugs (NSAIDS) are associated with 5 lower risks of colorectal cancer. However studies using aspirin in the ApcMm/+ mouse have yielded contrasting results. Here it is shown that aspirin does not reduce tumourigenesis when ApcMl + mice are put on diet containing aspirin post weaning. However when parents were put on aspirin, a significant suppression of tumourigenesis was observed in the min offspring. In fact there was incomplete penetrance of the ApcMm/+ phenotype (40%). To test whether in utero administration of aspirin could also suppress murine models of HNPCC, Msh2 deficient and (ApcMm/+, Msh2~/~) deficient mice were examined. In both cases a significant attenuation of tumourigenesis was observed. Taken together this raises the exciting prospect of prophylactic treatment of FAP and HNPCC patients and highlight the power of using transgenic models to investigate intestinal neoplasia.The interaction between Msh2 and p53 in tumourigenesis was also investigated. Both homozygosity and hemizygosity for p53 were found to dramatically accelerate tumourigenesis on a mismatch (Msh2) deficient background. Significantly, the levels of micro-satellite instability (MSI) were highest in tumours which were additionally heterozygous for p53. EMSA, Western and immunohistochemisty analysis of these tumours indicated retention of p53 function in at least a proportion of these tumours. Similar data were obtained from primary cultures, with again increased microsatellite instability and retained p53 functionality in cultures derived from p53 heterozygotes. Taken together, this data shows that hemizygosity for p53 increases microsatellite instability and that, at least in a percentage of tumours, complete loss of p53 is not a required event. These findings have particular relevance to our understanding of cross talk between p53 and MMR deficiency in human colorectal disease

The Wae to repair: prostaglandin E2 (PGE2) triggers intestinal wound repair

Accurate wound repair is a crucial step to protect organisms from environmental damage, for example infection and toxin exposure. In this issue of The EMBO Journal, Miyoshi et al (2017) have elucidated a new mechanism underpinning this process within the intestine where mesenchymal prostaglandin E2 produced following damage drives intestinal regeneration

Loss of N-WASP drives early progression in an Apc model of intestinal tumourigenesis

N‐WASP (WASL) is a widely expressed cytoskeletal signalling and scaffold protein also implicated in regulation of Wnt signalling and homeostatic maintenance of skin epithelial architecture. N‐WASP mediates invasion of cancer cells in vitro and its depletion reduces invasion and metastatic dissemination of breast cancer. Given this role in cancer invasion and universal expression in the gastrointestinal tract, we explored a role for N‐WASP in the initiation and progression of colorectal cancer. While deletion of N‐wasp is not detectably harmful in the murine intestinal tract, numbers of Paneth cells increased, indicating potential changes in the stem cell niche and migration up the crypt‐villus axis was enhanced. Loss of N‐wasp promoted adenoma formation in an adenomatous polyposis coli (Apc) deletion model of intestinal tumourigenesis. Thus, we establish a tumour suppressive role of N‐WASP in early intestinal carcinogenesis despite its later pro‐invasive role in other cancers. Our study highlights that while the actin cytoskeletal machinery promotes invasion of cancer cells, it also maintains normal epithelial tissue function and thus may have tumour suppressive roles in pre‐neoplastic tissues

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

Wnt pathway deregulation is a common characteristic of many cancers. But only Colorectal Cancer predominantly harbours mutations in APC, whereas other cancer types (hepatocellular carcinoma, solid pseudopapillary tumours of 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 a 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 significantly higher expression of the β-catenin binding partner E-cadherin. This increased expression is associated with a higher number of E-cadherin:β-catenin complexes at the membrane. Reduction of E-cadherin synergised with an activating mutation of β-catenin so there was now a rapid CPC phenotype within the colon and SI. Thus there is a threshold of β-catenin that is required to drive transformation and E-cadherin can act as a buffer to prevent β-catenin accumulation

Histological phenotypic subtypes predict recurrence risk and response to adjuvant chemotherapy in patients with stage III colorectal cancer

Histological ‘phenotypic subtypes’ that classify patients into four groups (immune, canonical, latent and stromal) have previously been demonstrated to stratify survival in a stage I–III colorectal cancer (CRC) pilot cohort. However, clinical utility has not yet been validated. Therefore, this study assessed prognostic value of these subtypes in additional patient cohorts along with associations with risk of recurrence and response to chemotherapy. Two independent stage I–III CRC patient cohorts (internal and external cohort) were utilised to investigate phenotypic subtypes. The primary endpoint was disease‐free survival (DFS) and the secondary endpoint was recurrence risk (RR). Stage II–III patients, from the SCOT adjuvant chemotherapy trial, were utilised to further validate prognostic value and for exploratory analysis assessing associations with adjuvant chemotherapy. In an 893‐patient internal cohort, phenotypic subtype independently associated with DFS (p = 0.025) and this was attenuated in stage III patients (p = 0.020). Phenotypic subtype also independently associated with RR (p < 0.001) in these patients. In a 146‐patient external cohort, phenotypic subtype independently stratified patients by DFS (p = 0.028), validating their prognostic value. In 1343 SCOT trial patients, the effect of treatment type significantly depended on phenotypic subtype (pinteraction = 0.011). Phenotypic subtype independently associated with DFS in stage III patients receiving FOLFOX (p = 0.028). Furthermore, the immune subtype significantly associated with better response to FOLFOX compared to CAPOX adjuvant chemotherapy in stage III patients (p = 0.013). In conclusion, histological phenotypic subtypes are an effective prognostic classification in patients with stage III CRC that associates with risk of recurrence and response to FOLFOX adjuvant chemotherapy

Intravital FRAP imaging using an E-cadherin-GFP mouse reveals disease- and drug-dependent dynamic regulation of cell-cell junctions in live tissue

E-cadherin-mediated cell-cell junctions play a prominent role in maintaining the epithelial architecture. The disruption or deregulation of these adhesions in cancer can lead to the collapse of tumor epithelia that precedes invasion and subsequent metastasis. Here we generated an E-cadherin-GFP mouse that enables intravital photobleaching and quantification of E-cadherin mobility in live tissue without affecting normal biology. We demonstrate the broad applications of this mouse by examining E-cadherin regulation in multiple tissues, including mammary, brain, liver, and kidney tissue, while specifically monitoring E-cadherin mobility during disease progression in the pancreas. We assess E-cadherin stability in native pancreatic tissue upon genetic manipulation involving Kras and p53 or in response to anti-invasive drug treatment and gain insights into the dynamic remodeling of E-cadherin during in situ cancer progression. FRAP in the E-cadherin-GFP mouse, therefore, promises to be a valuable tool to fundamentally expand our understanding of E-cadherin-mediated events in native microenvironments

Biological Misinterpretation of Transcriptional Signatures in Tumor Samples Can Unknowingly Undermine Mechanistic Understanding and Faithful Alignment with Preclinical Data

PURPOSE Precise mechanism-based gene expression signatures (GES) have been developed in appropriate in vitro and in vivo model systems, to identify important cancer-related signaling processes. However, some GESs originally developed to represent specific disease processes, primarily with an epithelial cell focus, are being applied to heterogeneous tumor samples where the expression of the genes in the signature may no longer be epithelial-specific. Therefore, unknowingly, even small changes in tumor stroma percentage can directly influence GESs, undermining the intended mechanistic signaling. EXPERIMENTAL DESIGN Using colorectal cancer as an exemplar, we deployed numerous orthogonal profiling methodologies, including laser capture microdissection, flow cytometry, bulk and multiregional biopsy clinical samples, single-cell RNA sequencing and finally spatial transcriptomics, to perform a comprehensive assessment of the potential for the most widely used GESs to be influenced, or confounded, by stromal content in tumor tissue. To complement this work, we generated a freely-available resource, ConfoundR; https://confoundr.qub.ac.uk/, that enables users to test the extent of stromal influence on an unlimited number of the genes/signatures simultaneously across colorectal, breast, pancreatic, ovarian and prostate cancer datasets. RESULTS Findings presented here demonstrate the clear potential for misinterpretation of the meaning of GESs, due to widespread stromal influences, which in-turn can undermine faithful alignment between clinical samples and preclinical data/models, particularly cell lines and organoids, or tumor models not fully recapitulating the stromal and immune microenvironment. CONCLUSIONS Efforts to faithfully align preclinical models of disease using phenotypically-designed GESs must ensure that the signatures themselves remain representative of the same biology when applied to clinical samples

The Glasgow Microenvironment Score associates with prognosis and adjuvant chemotherapy response in colorectal cancer

Background The Glasgow Microenvironment Score (GMS) combines peritumoural inflammation and tumour stroma percentage to assess interactions between tumour and microenvironment. This was previously demonstrated to associate with colorectal cancer (CRC) prognosis, and now requires validation and assessment of interactions with adjuvant therapy. Methods Two cohorts were utilised; 862 TNM I–III CRC validation cohort, and 2912 TNM II–III CRC adjuvant chemotherapy cohort (TransSCOT). Primary endpoints were disease-free survival (DFS) and relapse-free survival (RFS). Exploratory endpoint was adjuvant chemotherapy interaction. Results GMS independently associated with DFS (p = 0.001) and RFS (p < 0.001). GMS significantly stratified RFS for both low risk (GMS 0 v GMS 2: HR 3.24 95% CI 1.85–5.68, p < 0.001) and high-risk disease (GMS 0 v GMS 2: HR 2.18 95% CI 1.39–3.41, p = 0.001). In TransSCOT, chemotherapy type (pinteraction = 0.013), but not duration (p = 0.64) was dependent on GMS. Furthermore, GMS 0 significantly associated with improved DFS in patients receiving FOLFOX compared with CAPOX (HR 2.23 95% CI 1.19–4.16, p = 0.012). Conclusions This study validates the GMS as a prognostic tool for patients with stage I–III colorectal cancer, independent of TNM, with the ability to stratify both low- and high-risk disease. Furthermore, GMS 0 could be employed to identify a subset of patients that benefit from FOLFOX over CAPOX

Cost-Effectiveness of Pooled Nucleic Acid Amplification Testing for Acute HIV Infection after Third-Generation HIV Antibody Screening and Rapid Testing in the United States: A Comparison of Three Public Health Settings

Angela Hutchinson and colleagues conducted a cost-effectiveness analysis of pooled nucleic acid amplification testing following HIV testing and show that it is not cost-effective at recommended antibody testing intervals for high-risk persons except in very high-incidence settings

Aberrant epithelial GREM1 expression initiates colonic tumorigenesis from cells outside the stem cell niche

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