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

Efficient Wnt mediated intestinal hyperproliferation requires the cyclin D2-CDK4/6 complex

Inactivation of the gene encoding the adenomatous polyposis coli (APC) tumour suppressor protein is recognized as the key early event in the development of colorectal cancers (CRC). Apc loss leads to nuclear localization of beta-catenin and constitutive activity of the beta-catenin-Tcf4 transcription complex. This complex drives the expression of genes involved in cell cycle progression such as c-Myc and cyclin D2. Acute loss of Apc in the small intestine leads to hyperproliferation within the intestinal crypt, increased levels of apoptosis, and perturbed differentiation and migration. It has been demonstrated that c-Myc is a critical mediator of the phenotypic abnormalities that follow Apc loss in the intestine. As it may be difficult to pharmacologically inhibit transcription factors such as c-Myc, investigating more druggable targets of the Wnt-c-Myc pathway within the intestine may reveal potential therapeutic targets for CRC. Recent work in our laboratory has shown that the cyclin D2-cyclin-dependent kinase 4/6 (CDK4/6) complex promotes hyperproliferation in Apc deficient intestinal tissue and ApcMin/+ adenomas. We showed that the hyperproliferative phenotype associated with Apc loss in vivo was partially dependent on the expression of cyclin D2. Most importantly, tumour growth and development in ApcMin/+ mice was strongly perturbed in mice lacking cyclin D2. Furthermore, pharmacological inhibition of CDK4/6 suppressed the proliferation of adenomatous cells. This commentary discusses the significance of this work in providing evidence for the importance of the cyclin D2-CDK4/6 complex in colorectal adenoma formation. It also argues that inhibition of this complex may be an effective chemopreventative strategy in CRC

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

Constitutively active transforming growth factor β receptor 1 in the mouse ovary promotes tumorigenesis

Despite the well-established tumor suppressive role of TGFβ proteins, depletion of key TGFβ signaling components in the mouse ovary does not induce a growth advantage. To define the role of TGFβ signaling in ovarian tumorigenesis, we created a mouse model expressing a constitutively active TGFβ receptor 1 (TGFBR1) in ovarian somatic cells using conditional gain-of-function approach. Remarkably, these mice developed ovarian sex cord-stromal tumors with complete penetrance, leading to reproductive failure and mortality. The tumors expressed multiple granulosa cell markers and caused elevated serum inhibin and estradiol levels, reminiscent of granulosa cell tumors. Consistent with the tumorigenic effect, overactivation of TGFBR1 altered tumor microenvironment by promoting angiogenesis and enhanced ovarian cell proliferation, accompanied by impaired cell differentiation and dysregulated expression of critical genes in ovarian function. By further exploiting complementary genetic models, we substantiated our finding that constitutively active TGFBR1 is a potent oncogenic switch in mouse granulosa cells. In summary, overactivation of TGFBR1 drives gonadal tumor development. The TGFBR1 constitutively active mouse model phenocopies a number of morphological, hormonal, and molecular features of human granulosa cell tumors and are potentially valuable for preclinical testing of targeted therapies to treat granulosa cell tumors, a class of poorly defined ovarian malignancies

Opposing effects of TIGAR- and RAC1-derived ROS on Wnt-driven proliferation in the mouse intestine

Reactive oxygen species (ROS) participate in numerous cell responses, including proliferation, DNA damage, and cell death. Based on these disparate activities, both promotion and inhibition of ROS have been proposed for cancer therapy. However, how the ROS response is determined is not clear. We examined the activities of ROS in a model of Apc deletion, where loss of the Wnt target gene Myc both rescues APC loss and prevents ROS accumulation. Following APC loss, Myc has been shown to up-regulate RAC1 to promote proliferative ROS through NADPH oxidase (NOX). However, APC loss also increased the expression of TIGAR, which functions to limit ROS. To explore this paradox, we used three-dimensional (3D) cultures and in vivo models to show that deletion of TIGAR increased ROS damage and inhibited proliferation. These responses were suppressed by limiting damaging ROS but enhanced by lowering proproliferative NOX-derived ROS. Despite having opposing effects on ROS levels, loss of TIGAR and RAC1 cooperated to suppress intestinal proliferation following APC loss. Our results indicate that the pro- and anti-proliferative effects of ROS can be independently modulated in the same cell, with two key targets in the Wnt pathway functioning to integrate the different ROS signals for optimal cell proliferation

MiR-142-3p is downregulated in aggressive p53 mutant mouse models of pancreatic ductal adenocarcinoma by hypermethylation of its locus

Pancreatic ductal adenocarcinoma (PDAC) is an extremely aggressive disease with poor prognostic implications. This is partly due to a large proportion of PDACs carrying mutations in TP53, which impart gain-of-function characteristics that promote metastasis. There is evidence that microRNAs (miRNAs) may play a role in both gain-of-function TP53 mutations and metastasis, but this has not been fully explored in PDAC. Here we set out to identify miRNAs which are specifically dysregulated in metastatic PDAC. To achieve this, we utilised established mouse models of PDAC to profile miRNA expression in primary tumours expressing the metastasis-inducing mutant p53R172H and compared these to two control models carrying mutations, which promote tumour progression but do not induce metastasis. We show that a subset of miRNAs are dysregulated in mouse PDAC tumour tissues expressing mutant p53R172H, primary cell lines derived from mice with the same mutations and in TP53 null cells with ectopic expression of the orthologous human mutation, p53R175H. Specifically, miR-142-3p is downregulated in all of these experimental models. We found that DNA methyltransferase 1 (Dnmt1) is upregulated in tumour tissue and cell lines, which express p53R172H. Inhibition or depletion of Dnmt1 restores miR-142-3p expression. Overexpression of miR-142-3p attenuates the invasive capacity of p53R172H-expressing tumour cells. MiR-142-3p dysregulation is known to be associated with cancer progression, metastasis and the miRNA is downregulated in patients with PDAC. Here we link TP53 gain-of-function mutations to Dnmt1 expression and in turn miR-142-3p expression. Additionally, we show a correlation between expression of these genes and patient survival, suggesting that they may have potential to be therapeutic targets

Defining key concepts of intestinal and epithelial cancer biology through the use of mouse models

Over the past 20 years, huge advances have been made in modelling human diseases such as cancer using genetically modified mice. Accurate in vivo models are essential to examine the complex interaction between cancer cells, surrounding stromal cells, tumour-associated inflammatory cells, fibroblast and blood vessels, and to recapitulate all the steps involved in metastasis. Elucidating these interactions in vitro has inherent limitations, and thus animal models are a powerful tool to enable researchers to gain insight into the complex interactions between signalling pathways and different cells types. This review will focus on how advances in in vivo models have shed light on many aspects of cancer biology including the identification of oncogenes, tumour suppressors and stem cells, epigenetics, cell death and context dependent cell signalling

The role of mTOR-mediated signals during haemopoiesis and lineage commitment

The serine/threonine protein kinase mechanistic target of rapamycin (mTOR) has been implicated in the regulation of an array of cellular functions including protein and lipid synthesis, proliferation, cell size and survival. Here, we describe the role of mTOR during haemopoiesis within the context of mTORC1 and mTORC2, the distinct complexes in which it functions. The use of conditional transgenic mouse models specifically targeting individual mTOR signalling components, together with selective inhibitors, have generated a significant body of research emphasising the critical roles played by mTOR, and individual mTOR complexes, in haemopoietic lineage commitment and development. This review will describe the profound role of mTOR in embryogenesis and haemopoiesis, underscoring the importance of mTORC1 at the early stages of haemopoietic cell development, through modulation of stem cell potentiation and self-renewal, and erythroid and B cell lineage commitment. Furthermore, the relatively discrete role of mTORC2 in haemopoiesis will be explored during T cell development and B cell maturation. Collectively, this review aims to highlight the functional diversity of mTOR signalling and underline the importance of this pathway in haemopoiesis

Neutrophils: homing in on the myeloid mechanisms of metastasis

The metastasis cascade is complex and comprises several stages including local invasion into surrounding tissue, intravasation and survival of tumour cells in the circulation, and extravasation and colonisation of a distant site. It is increasingly clear that these processes are driven not only by signals within the tumour cells, but are also profoundly influenced by stromal cells and signals in the tumour microenvironment. Amongst the many cell types within the tumour microenvironment, immune cells such as lymphocytes, macrophages and neutrophils play a prominent role in tumour development and progression. Neutrophils, however, have only recently emerged as important players, particularly in metastasis. Here we review the current evidence suggesting a multi-faceted role for neutrophils in the metastatic cascade