Alteration of Epigenetic Regulation by Long Noncoding RNAs in Cancer

Long noncoding RNAs (lncRNAs) are important regulators of the epigenetic status of the human genome. Besides their participation to normal physiology, lncRNA expression and function have been already associated to many diseases, including cancer. By interacting with epigenetic regulators and by controlling chromatin topology, their misregulation may result in an aberrant regulation of gene expression that may contribute to tumorigenesis. Here, we review the functional role and mechanisms of action of lncRNAs implicated in the aberrant epigenetic regulation that has characterized cancer development and progression

Use of preclinical models for malignant pleural mesothelioma.

Malignant pleural mesothelioma (MPM) is an aggressive cancer most commonly caused by prior exposure to asbestos. Median survival is 12-18 months, since surgery is ineffective and chemotherapy offers minimal benefit. Preclinical models that faithfully recapitulate the genomic and histopathological features of cancer are critical for the development of new treatments. The most commonly used models of MPM are two-dimensional cell lines established from primary tumours or pleural fluid. While these have provided some important insights into MPM biology, these cell models have significant limitations. In order to address some of these limitations, spheroids and microfluidic chips have more recently been used to investigate the role of the three-dimensional environment in MPM. Efforts have also been made to develop animal models of MPM, including asbestos-induced murine tumour models, MPM-prone genetically modified mice and patient-derived xenografts. Here, we discuss the available in vitro and in vivo models of MPM and highlight their strengths and limitations. We discuss how newer technologies, such as the tumour-derived organoids, might allow us to address the limitations of existing models and aid in the identification of effective treatments for this challenging-to-treat disease.MS and JO are supported by BLF-Papworth Fellowships from the British Lung Foundation and the Victor Dahdaleh Foundation. MJG and HEF is supported by the British Lung Foundation and Wellcome Trust grant 206194. RCR is supported by the Cambridge Biomedical Research Centre, Cancer Research UK Cambridge Centre, British Lung Foundation and Royal Papworth Hospital. SJM is supported by the Medical Research Council, British Lung Foundation, Cambridge BRC, Royal Papworth Hospital, and the Alpha1-Foundatio

Investigating the roles of RKIP and p53 in colorectal carcinoma

Raf Kinase Inhibitor Protein (RKIP) was originally described as an inhibitor of the Ras-Raf-MEK-ERK pathway, exerting its action by the physical inhibition of the interaction of Raf with MEK. It has subsequently been shown to play important roles in a number of other signalling pathways, including the NFκB pathway and in the stability of the mitotic spindle. Not surprisingly given that it impacts on many important signalling pathways RKIP levels have been shown to be important in the progression of a number of different cancers. RKIP expression is lost or decreased in a number of common human cancers and decreased still further in tumour metastases. One of the tumours in which RKIP is downregulated is colorectal cancer (CRC). Importantly it has been shown that not only is RKIP depleted in tumour tissue when compared with normal tissue but that the level of RKIP within a tumour is inversely correlated with the likelihood of metastatic relapse and with patient prognosis. Although we already have a number of very good prognostic indicators in CRC, one group of patients for whom new prognostic indicators would be useful are patients with Dukes B CRC. These are patients with locally advanced but non-metastatic disease and at present there is no firm consensus on their correct post-operative management. Therefore we set out to examine whether RKIP is a useful prognosticator in this particular group using a tissue microarray (TMA) with samples from over 200 patients with Dukes B CRC. The analysis revealed a strong inverse correlation between RKIP levels and disease specific survival. Moreover, in a multivariate analysis RKIP emerged as an independent prognostic indicator along with lympho-vascular invasion and peritoneal invasion, two well-known and powerful prognosticators. This allowed for the generation of a simple prognostic index, using information from the different independent indicators, allowing for improved patient risk stratification. This led us to examine whether RKIP could also function as a predictive marker in CRC. To do this we again used a TMA, this time consisting of a much larger cohort of patients across the whole range of tumour stages. The results confirmed the prognostic utility of RKIP and indicated that patients whose tumours have low levels of RKIP may derive a greater benefit from chemotherapy than those patients whose tumours have high levels, although this result did not reach statistical significance. In the second part of the thesis I have examined the effect of RKIP in previously characterised mouse models of CRC. To do this I have used a germline RKIP knockout mouse and in the first instance crossed it to the APC580S mouse. In this mouse APC is lost conditionally within the intestine and liver. RKIP knockout did not have any effect on the rate of tumourigenesis or on the invasiveness of tumours in this model. However, in the setting of acute homozygous deletion of APC, RKIP knockout resulted in a decrease in apoptoses in the small intestine and an increase in aberrant mitotic activity in the liver. To follow this up I have examined the effect of RKIP knockout in a mouse model of superficially invasive CRC, specifically to see if RKIP knockout can promote invasive and metastatic behaviour. In this model the APC580S mouse is crossed to mice which conditionally express oncogenic KRas. Although RKIP knockout did not result in an increase in invasive tumours in this model there was a shift in tumour location from the small intestine to the colon. This shift appeared to be due, at least in part to an increase in chromosomal instability in the tumours. The final aim of the thesis was to develop a mouse model of CRC which more closely recapitulates the late stages of the human disease, specifically invasion and metastasis. To do this we have crossed the APC580S mouse with either a conditional p53 knockout or with a mouse that conditionally expresses a point mutation of p53 (p53R172H). In human tumours the majority of abnormalities of p53 are point mutations that result in the production of mutant protein that accumulates in tumour cells. There is evidence that this mutant protein may have oncogenic properties beyond the simple loss of normal p53 protein function. Therefore we have also used this model to study the differing effects of p53 loss and point mutation in CRC. We found that mice homozygous for p53 deletion (p53fl/fl) and those expressing a single copy of the mutant allele with loss of the second copy (p53R172H/fl) developed invasive tumours with nearly 100% penetrance and indeed metastasis was observed. Remarkably, although mice that were heterozygous for p53 deletion (p53fl/+) only rarely developed invasive tumours almost 100% of mice expressing a single copy of the mutant allele (p53R172H/+) developed invasive tumours. We went on to show that the increase in invasion seen in this model is related to an increase in Wnt signalling, which is associated with increased expression of pro-invasive Wnt targets such as fascin. We also showed a novel pro-invasive role for ARF in this process. This is also an excellent model of Dukes B CRC and therefore the ideal model to test the effect of RKIP deletion on invasion and metastasis. These studies led us to examine the differences in effect between knockout and mutant p53 in another tumour model. In this we used a novel model of the aggressive tumour pleomorphic rhabdomyosarcoma to demonstrate that mutant p53 can both promote both tumourigenesis and metastasis more potently than p53 knockout. These studies have demonstrated the value of RKIP in the clinically important Dukes B CRC population and shown its possible utility as a predictive marker in this group. Although we have not seen an effect of RKIP knockout in traditional mouse models of CRC we have developed a novel model which closely recapitulates Dukes B CRC and may be useful in elucidating the effect of RKIP knockout. We have also used this model to gain novel insights into the invasive process, in particular into the role played by mutant p53

Investigating the functional significance of the upregulation of Cyclin D2 and p21 following Apc loss in vivo

The Apc gene encodes the Adenomatous polyposis coli tumour suppressor protein, the germ line mutation of which characterizes Familial Adenomatous Polyposis (FAP), an autosomal syndrome characterized by multiple colorectal lesions. Inactivation of the Apc gene is recognized as a key early event in the development of colorectal cancers and leads to the deregulation of the Wnt pathway and the activation of TCF/LEF target genes. This project focuses on the proto-oncogene c-Myc as it is a key Wnt target gene which is activated following loss of Apc in vivo. This upregulation is noteworthy as c-Myc is implicated in stem cell survival, proliferation, apoptosis and tumourigenesis. Previous studies have shown c-Myc dependency for both apoptosis and proliferation following activation of the Wnt pathway, however little is known about the role c-Myc plays in inducing apoptosis following DNA damage in vivo. To study this I have conditionally deleted c-Myc from the intestinal epithelium and examined the response of intestinal enterocytes following DNA damage. Remarkably, following DNA damage, c-Myc deficient enterocytes were unable to upregulate p53 and induce apoptosis, which was mechanistically due to an upregulation of MDM2. Taken together, results from this study showed for the first time in vivo, a key role for c-Myc in inducing apoptosis following DNA damage through control of p53. Previous studies from this lab have shown that within the intestinal epithelium, c-Myc is absolutely required for the hyper-proliferative phenotype that is observed following loss of Apc. Therefore one of the key aims of this thesis is to look downstream of c-Myc in order to delineate how c-Myc induces and controls this proliferation. Given that one of the key postulated functions of c-Myc is the transcriptional repression of p21, this thesis examines this hypothesis by investigating the significance of the upregulation of p21 following c-Myc deletion in Apc deficient intestinal enterocytes. To do this, I have generated triple knockout (TKO) intestines by intercrossing p21 knockout mice to mice where we can conditionally delete both Apc and c-Myc within the murine intestinal epithelium. Surprisingly, the levels of proliferation were the same between double knockout Apc Myc and TKO intestines, which had markedly less proliferation than Apc deficient intestines. However, unlike double knockout enterocytes, TKO intestinal enterocytes no longer moved up the crypt-villus axis and failed to generate villus. To examine which of these phenomena were key to tumourigenesis (differentiation or proliferation), we investigated whether TKO intestines could form intestinal adenomas and found that even in the absence of p21, c-Myc deficient cells were unable to form tumours. Taken together we have identified a novel role for p21 in driving differentiation following Apc and Myc deletion. This is consistent with the expression of p21 in the normal crypt at the crypt villus junction. Remarkably this function of p21 is independent of its key role as a cell cycle inhibitor. Moreover, this study also examined the importance of the upregulation of the Cyclin D/CDK4 complexes following Apc loss and their role in c-Myc dependent proliferation. Results from these studies showed that Cyclin D2 is required for efficient proliferation immediately following loss of Apc as well as for tumourigeneis in the Apc Min/+ mouse. Taken together, results from these studies showed that the upregulation of Cyclin D2 and CDK4 are c-Myc dependent and that the upregulation of these complexes are key for Wnt driven proliferation and tumourigenesis. Lastly, in this study I have examined whether Apc loss within the intestinal epithelium, where it is a bona fide tumour suppressor gene, can provoke senescence, and compared this to the ability of Apc gene deletion to trigger senescence in the renal epithelium, where it is not mutated in human cancer. This study showed that deletion of Apc within the renal epithelium invoked a p21 dependent senescence response, and Apc deficient renal epithelial cells were cleared and very rarely initiated tumourigenesis. However, combined Apc and p21 gene deletion rapidly initiated tumourigenesis, with all mice developing renal carcinoma by 2 months of age. In contrast to Apc deficient intestinal epithelium, this process was unaffected by loss of c-Myc. However within the intestinal epithelium, deletion of Apc did not invoke senescence, but lead to a highly proliferative, p21 independent response. Combined Apc and p21 gene loss had no impact on either the short term phenotypes of Apc loss or upon tumourigenesis. Taken together these results show for the first time that Apc loss in vivo can invoke a senescence program but in a context dependent fashion. This implies escape from senescence is not a crucial pathway in colorectal cancers that are initiated by Apc loss, and goes to explain why renal carcinoma is not observed in FAP patients who are germline heterozygous for APC. Therefore the aims for this thesis are: • To investigate the role of c-Myc in inducing apoptosis within the intestinal crypt, and whether this is p21 dependent? • To investigate the role of p21 in causing senescence of Apc deficient cells, and whether this is c-Myc dependent? • To determine the functional importance of repression of p21 by c-Myc in Apc deficient cells. • To determine the significance of Cyclin D2 upregulation within Apc deficient cells

A genetic progression model of Braf(V600E)-induced intestinal tumorigenesis reveals targets for therapeutic intervention.

We show that BRAF(V600E) initiates an alternative pathway to colorectal cancer (CRC), which progresses through a hyperplasia/adenoma/carcinoma sequence. This pathway underlies significant subsets of CRCs with distinctive pathomorphologic/genetic/epidemiologic/clinical characteristics. Genetic and functional analyses in mice revealed a series of stage-specific molecular alterations driving different phases of tumor evolution and uncovered mechanisms underlying this stage specificity. We further demonstrate dose-dependent effects of oncogenic signaling, with physiologic Braf(V600E) expression being sufficient for hyperplasia induction, but later stage intensified Mapk-signaling driving both tumor progression and activation of intrinsic tumor suppression. Such phenomena explain, for example, the inability of p53 to restrain tumor initiation as well as its importance in invasiveness control, and the late stage specificity of its somatic mutation. Finally, systematic drug screening revealed sensitivity of this CRC subtype to targeted therapeutics, including Mek or combinatorial PI3K/Braf inhibition

Tumor Suppressor Genes

Functional evidence obtained from somatic cell fusion studies indicated that a group of genes from normal cells might replace or correct a defective function of cancer cells. Tumorigenesis that could be initiated by two mutations was established by the analysis of hereditary retinoblastoma, which led to the eventual cloning of RB1 gene. The two-hit hypothesis helped isolate many tumor suppressor genes (TSG) since then. More recently, the roles of haploinsufficiency, epigenetic control, and gene dosage effects in some TSGs, such as P53, P16 and PTEN, have been studied extensively. It is now widely recognized that deregulation of growth control is one of the major hallmarks of cancer biological capabilities, and TSGs play critical roles in many cellular activities through signaling transduction networks. This book is an excellent review of current understanding of TSGs, and indicates that the accumulated TSG knowledge has opened a new frontier for cancer therapies

Senescence and immortality in hepatocellular carcinoma

Cataloged from PDF version of article.Cellular senescence is a process leading to terminal growth arrest with characteristic morphological features. This process is mediated by telomere-dependent, oncogene-induced and ROS-induced pathways, but persistent DNA damage is the most common cause. Senescence arrest is mediated by p16(INK4a)- and p21(Cip1)-dependent pathways both leading to retinoblastoma protein (pRb) activation. p53 plays a relay role between DNA damage sensing and p21(Cip1) activation. pRb arrests the cell cycle by recruiting proliferation genes to facultative heterochromatin for permanent silencing. Replicative senescence that occurs in hepatocytes in culture and in liver cirrhosis is associated with lack of telomerase activity and results in telomere shortening. Hepatocellular carcinoma (HCC) cells display inactivating mutations of p53 and epigenetic silencing of p16(INK4a). Moreover, they re-express telomerase reverse transcriptase required for telomere maintenance. Thus, senescence bypass and cellular immortality is likely to contribute significantly to HCC development. Oncogene-induced senescence in premalignant lesions and reversible immortality of cancer cells including HCC offer new potentials for tumor prevention and treatment. (C) 2008 Elsevier Ireland Ltd. All rights reserved

Genetic and epigenetic changes in primary metastatic and nonmetastatic colorectal cancer

Colorectal cancer (CRC) develops as multistep process, which involves genetic and epigenetic alterations. K-Ras, p53 and B-Raf mutations and RASSF1A, E-Cadherin and p16INK4A promoter methylation were investigated in 202 CRCs with and without lymph node and/or liver metastasis, to assess whether gene abnormalities are related to a metastogenic phenotype. K-Ras, B-Raf and p53 mutations were detected in 27, 3 and 32% of the cases, with K-Ras mutations significantly associated with metastatic tumour (P=0.019). RASSF1A, E-Cadherin and p16INK4A methylation was documented in 20, 44 and 33% of the cases with p16INK4A significantly associated with metastatic tumours (P=0.001). Overall, out of 202 tumours, 34 (17%) did not show any molecular change, 125 (62%) had one or two and 43 (21%) three or more. Primary but yet metastatic CRCs were prevalent in the latter group (P=0.023) where the most frequent combination was one genetic (K-Ras in particular) and two epigenetic alterations. In conclusion, this analysis provided to detect some molecular differences between primary metastatic and nonmetastatic CRCs, with K-Ras and p16INK4A statistically altered in metastatic tumours; particular gene combinations, such as coincidental K-Ras mutation with two methylated genes are associated to a metastogenic phenotype

MLL3 regulates the CDKN2A tumor suppressor locus in liver cancer

Mutations in genes encoding components of chromatin modifying and remodeling complexes are among the most frequently observed somatic events in human cancers. For example, missense and nonsense mutations targeting the mixed lineage leukemia family member 3 (MLL3, encoded by KMT2C) histone methyltransferase occur in a range of solid tumors, and heterozygous deletions encompassing KMT2C occur in a subset of aggressive leukemias. Although MLL3 loss can promote tumorigenesis in mice, the molecular targets and biological processes by which MLL3 suppresses tumorigenesis remain poorly characterized. Here, we combined genetic, epigenomic, and animal modeling approaches to demonstrate that one of the mechanisms by which MLL3 links chromatin remodeling to tumor suppression is by co-activating the Cdkn2a tumor suppressor locus. Disruption of Kmt2c cooperates with Myc overexpression in the development of murine hepatocellular carcinoma (HCC), in which MLL3 binding to the Cdkn2a locus is blunted, resulting in reduced H3K4 methylation and low expression levels of the locus-encoded tumor suppressors p16/Ink4a and p19/Arf. Conversely, elevated KMT2C expression increases its binding to the CDKN2A locus and co-activates gene transcription. Endogenous Kmt2c restoration reverses these chromatin and transcriptional effects and triggers Ink4a/Arf-dependent apoptosis. Underscoring the human relevance of this epistasis, we found that genomic alterations in KMT2C and CDKN2A were associated with similar transcriptional profiles in human HCC samples. These results collectively point to a new mechanism for disrupting CDKN2A activity during cancer development and, in doing so, link MLL3 to an established tumor suppressor network