Dissertations / Theses on the topic 'Hepatocellular cancer, p21, p53'

Non melanoma skin cancer (NMSC) is a serious condition caused by chronic ultraviolet (UV) exposure that leads to DNA damage in skin. UV radiation has the potential to lead to DNA damage, which triggers biochemical pathways within a cell. The result is that the cell either undergoes cell cycle arrest, giving the cell time to repair DNA damage, or apoptosis. Sunscreen is the most commonly used treatment for preventing UV induced skin damage, but it involves a number of undesirable and toxic side effects including damaging the dermis, premature aging of skin and underweight child births. This has led to interest in finding safer alternatives to prevent UV damage without the negative side effects of sunscreen. In particular, bovine milk sphingomyelin (SM) is a compound that has the potential to protect against UV damage without any of the dangerous side effects of sunscreen. Here we present the use of SM for UV protection of human keratinocytes (KRTs) to prevent DNA mutations that result from UV exposure. In particular, analysis of the expression of DNA damage biomarkers p21 and p53 was done to determine the potential of SM to prevent DNA damage associated with UV exposure. Both non-SM treated KRTs and KRTs treated with 0.1% SM media 24 hours prior to UV radiation were fixed and IF-stained at 24 hours following 40 mJ/cm2 of UV exposure. Significant differences in both p21 and p53 were observed between the SM treated and non-SM treated cells at the UV dosage level (via t-test; p

Hepatocellular carcinoma (HCC) is an aggressive subtype of liver cancer with a poor prognosis. Currently, prognosis for HCC patients remains poor as few therapies are available. The clinical need for more effective HCC treatments remains unmet partially because HCC is genetically heterogeneous and HCC driver genes amenable to targeted therapy are largely unknown. Mutations in the TP53 gene are found in ~30% of HCC patients and confer poor prognosis to patients. Identifying genes whose depletion can inhibit HCC growth, and determining the mechanisms involved, will aid the development of targeted therapies for HCC patients. Therefore, the first half of this thesis focuses on identifying genes that are required for cell growth in HCC independent of p53 status. We performed a kinome-wide CRISPR screen to identify genes required for cell growth in three HCC cell lines: HepG2 (p53 wild-type), Huh7 (p53-mutant) and Hep3B (p53-null) cells. The kinome screen identified 31 genes that were required for cell growth in 3 HCC cell lines independent of TP53 status. Among the 31 genes, 8 genes were highly expressed in HCC compared to normal tissue and increased expression was associated with poor survival in HCC patients. We focused on TRRAP, a co-factor for histone acetyltransferases. TRRAP function has not been previously characterized in HCC. CRISPR/Cas9 mediated depletion of TRRAP reduced cell growth and colony formation in all three cell lines. Moreover, depletion of TRRAP reduced its histone acetyltransferase co-factors KAT2A and KAT5 at the protein level with no change at the mRNA level. I found that depletion of KAT5, but not KAT2A, reduced cell growth. Notably, inhibition of proteasome- and lysosome-mediated degradation failed to rescue protein levels of KAT2A and KAT5 in the absence of TRRAP. Moreover, tumor initiation in an HCC mouse model failed after CRISPR/Cas9 depletion of TRRAP due to clearance via macrophages and HCC cells depleted of TRRAP and KAT5 failed to grow as subcutaneous xenografts in vivo. RNA-seq and bioinformatic analysis of HCC patient samples revealed that TRRAP positively regulates expression of genes that are involved in mitotic progression. In HCC, this subset of genes is clinically relevant as they are overexpressed compared to normal tissue and high expression confers poor survival to patients. I identified TOP2A as one of the mitotic gene targets of the TRRAP/KAT5 complex whose inhibition greatly reduces proliferation of HCC cells. Given that this was the first time the TRRAP/KAT5 complex has been identified as a therapeutic target in HCC, the second half of this thesis focuses on identifying the mechanism via which depletion of this complex inhibits proliferation of HCC cells. I discovered that depletion of TRRAP, KAT5 and TOP2A reduced proliferation of HCC cells by inducing senescence. Typically, senescence is an irreversible state of cell cycle arrest at G1 that is due to activation of p53/p21 expression, phosphorylation of RB, and DNA damage. Surprisingly, induction of senescence after loss of TRRAP, KAT5 and TOP2A arrested cells during G2/M and senescence was independent of p53, p21, RB and DNA damage. In summary, this thesis identifies TRRAP as a potential oncogene in HCC. I identified a network of genes regulated by TRRAP and its-cofactor KAT5 that promote mitotic progression. Moreover, I demonstrated that disruption of TRRAP/KAT5 and its downstream target gene TOP2A result in senescence of HCC cells independent of p53 status. Taken together, this work suggests that targeting the TRRAP/KAT5 complex and its network of target genes is a potential therapeutic strategy for HCC patients.

This project was a step forward in discovering the potential role of intestinal cell kinase in prostate cancer development. Intestinal cell kinase was shown to be upregulated in prostate cancer cells and altered expression led to changes in key cell survival proteins. This study used in vitro experiments to monitor changes in cell growth, protein and RNA expression.

Le suppresseur de tumeur p53 est un facteur de transcription impliqué dans la progression du cycle cellulaire et dans l'apoptose. Outre ses fonctions majeures, p53 régule également la migration et l'adhérence cellulaire qui sont deux évènements impliqués dans le processus métastatique. L'évolution maligne d'un carcinome peut aussi impliquer la répression transcriptionnelle de CDH1, qui code pour la E-cadhérine, protéine constitutive des jonctions adhérentes. Nous avons recherché si et comment p53 régule certains évènements moléculaires qui contrôle le processus métastatique. Nous montrons que la forme sauvage de p53 réprime directement la transcription de CDH1 dans des lignées cellulaires humaines de carcinome du colon (HCT116). Cette répression est associée à une expression de novo de la vimentine et à l'acquisition d'une morphologie plus fibroblastique. L'une des cibles transcriptionnelle majeure de p53, p21WAF1 court-circuite l'effet répresseur de p53 sur la transcription de CDH1. Trois mutants dominant-négatifs de p53 (R273H, R175H et V143A) répriment également la transcription de CDH1. De plus, l'expression stable du mutant V143A dans les cellules HCT116 p53-/- mime partiellement le phénotype observé suite l'accumulation aberrante de p53. De façon surprenante, ce phénotype mésenchymateux n'est pas associé à une augmentation des propriétés invasives. Ce travail implique p53 dans la régulation d'évènements moléculaires qui peuvent conduire à l'acquisition d'un phénotype mésenchymateux
The p53 tumour suppressor gene encodes a transcriptional regulator that monitor proliferation signals to prevent cells from uncontrolled growth. However, p53 has also alternative functions. Notably, loss of p53 favours cell migration and invasion, processes involved in tumour metastasis. Given that epithelial to mesenchymal transition (EMT) also increases cell migration by altering the cell phenotype and morphology, we hypothesized that p53 controls molecular alterations that mediate EMT during cancer progression. Analysis of E-cadherin promoter activity and chromatin immunoprecipitation identified p53 as a direct transcriptional repressor of E-cadherin in human colon carcinoma cells, HCT116. Aberrant levels of p53 disrupted E-Cadherin-based cell-cell contacts and induced a more mesenchymal phenotype with downregulation of E-Cadherin and induction of the mesenchymal gene, vimentin. In addition, p21Waf-1 impeded p53 transcriptional repression and restored in part cell to cell adhesion. Furthermore, HCT116p53-/- cells overexpressing dominant-negative form of p53 also displayed the EMT-like phenotype. Neither p53 nor mutant p53–mediated shift toward mesenchymal morphology led to an increase of cell invasiveness. This work and our previous finding of mutant p53-mediated cell invasion identify p53 as a novel regulator of EMT and offer new perspectives in the comprehension of metastasis

Cette thèse a eu pour objet d'évaluer différents marqueurs moléculaires susceptibles de prédire l'évolution d'une tumeur de vessie superficielle, afin de pouvoir proposer le traitement le plus conservateur possible. Nous avons démontré que l'antigène de prolifération Ki-67 est capable de prédire la récidive tumorale lorsque le diamètre de la tumeur de vessie initiale est supérieur à 3 cm, par contre la valeur pronostique des autres marqueurs moléculaires p53, MDM2, p21 n'était pas supérieure à celle des paramètres clinico-pathologiques actuellement validés. Les résultats discordants de la littérature sur l'expression en immuno-histochimie de p53 dans le cancer de vessie nous ont amenés à utiliser un test fonctionnel dans la levure susceptible de ne mettre en évidence que les mutations altérant la fonction de transcription de p53. Compte tenu de son caractère très spécifique, nous avons utilisé cette méthode pour apprécier le caractère prédictif de p53 dans la résistance à la BCG thérapie avec des résultats préliminaires encourageants. Enfin, l'étude du phénotype p21/MDM2/p53 de la tumeur a permis de mieux comprendre les interactions des protéines du cycle cellulaire impliquées dans les différentes étapes de la cancérogénèse vésicale. Ainsi, il semble bien que le phénotype p21-/MDM2-/p53+ soit associé aux lésions de haut grade cellulaire et donc aux tumeurs vésicales qui ont le plus mauvais pronostic.

Hepatocellular carcinoma (HCC) is a common malignancy of the liver that is one of the most frequent causes of cancer-related death in the world. Surgical resection and liver transplantation are the only curative options for HCC, and tumor invasion and metastasis render many patients ineligible for these treatments. Identification of the mechanisms that contribute to invasive and metastatic disease may enlighten therapeutic strategies for those not eligible for surgical treatments. In this dissertation, I describe two sets of experiments to elucidate mechanisms underlying HCC dissemination, involving the activities of Krüppel-like factor 6 and a particular p53 point mutation, R172H. Gene expression profiling of migratory HCC subpopulations demonstrated reduced expression of Krüppel-like factor 6 (KLF6) in invasive HCC cells. Knockdown of KLF6 in HCC cells increased cell transformation and migration. Single-copy deletion of Klf6 in a HCC mouse model results in increased tumor formation, increased metastasis to the lungs, and decreased survival, indicating that KLF6 suppresses both tumor formation and metastasis in HCC. To elucidate the mechanism of KLF6-mediated tumor and metastasis suppression, we performed gene expression profiling and ChIP-sequencing to identify direct transcriptional targets of KLF6 in HCC cells. This analysis revealed novel transcriptional targets of KLF6 in HCC including CDC42EP3 and VAV3, both of which are positive regulators of Rho family GTPases. Concordantly, KLF6 knockdown cells demonstrate increased activity of the Rho family GTPases RAC1 and CDC42, and RAC1 is required for migration induced following KLF6 knockdown. Moreover, VAV3 and CDC42EP3 are also required for enhanced cell migration in HCC cells with KLF6 knockdown. Together, this work describes a novel signaling axis through which KLF6-mediated repression of VAV3 and CDC42EP3 inhibits RAC1Gmediated HCC cell migration in culture, and potentially HCC metastasis in vivo. TP53 gene mutations are commonly found in HCC and are associated with poor prognosis. Prior studies have suggested that p53 mutants can display gain-of- function properties in other tumor types. Therefore, I sought to determine if a particular hotspot p53 mutation, p53R172H, provided enhanced, gain-of-function properties compared to p53 loss in HCC. In vitro, soft agar colony formation and cell migration is reduced upon knockdown of p53R172H, indicating that this mutation is required for transformation-associated phenotypes in these cells. However, p53R172H-expressing mice did not have enhanced tumor formation or metastasis compared to p53-null mice. These data suggest that p53R172H and p53 deletion are functionally equivalent in vivo, and that p53R172H is not a gain-of-function mutant in HCC. Inhibition of the related transcription factors p63 and p73 has been suggested as a potential mechanism by which mutant p53 exerts its gain-of-function effects. Analysis of p63 and p73 target genes demonstrated that they are similarly suppressed in p53-null and p53R172H-expressing HCC cell lines, suggesting a potential explanation for the phenotypes I observed in vivo and in vitro. Together, the studies described in this dissertation increase our understanding of the mechanisms underlying HCC progression and metastasis. Specifically, we find and characterize KLF6 as a novel suppressor of HCC metastasis, and determine the contribution of a common p53 point mutation in HCC. This work contributes to ongoing efforts to improve treatment options for HCC patients.

Hepatocellular carcinoma (HCC) is a common malignancy of the liver that is one of the most frequent causes of cancer-related death in the world. Surgical resection and liver transplantation are the only curative options for HCC, and tumor invasion and metastasis render many patients ineligible for these treatments. Identification of the mechanisms that contribute to invasive and metastatic disease may enlighten therapeutic strategies for those not eligible for surgical treatments. In this dissertation, I describe two sets of experiments to elucidate mechanisms underlying HCC dissemination, involving the activities of Krüppel-like factor 6 and a particular p53 point mutation, R172H. Gene expression profiling of migratory HCC subpopulations demonstrated reduced expression of Krüppel-like factor 6 (KLF6) in invasive HCC cells. Knockdown of KLF6 in HCC cells increased cell transformation and migration. Single-copy deletion of Klf6 in a HCC mouse model results in increased tumor formation, increased metastasis to the lungs, and decreased survival, indicating that KLF6 suppresses both tumor formation and metastasis in HCC. To elucidate the mechanism of KLF6-mediated tumor and metastasis suppression, we performed gene expression profiling and ChIP-sequencing to identify direct transcriptional targets of KLF6 in HCC cells. This analysis revealed novel transcriptional targets of KLF6 in HCC including CDC42EP3 and VAV3, both of which are positive regulators of Rho family GTPases. Concordantly, KLF6 knockdown cells demonstrate increased activity of the Rho family GTPases RAC1 and CDC42, and RAC1 is required for migration induced following KLF6 knockdown. Moreover, VAV3 and CDC42EP3 are also required for enhanced cell migration in HCC cells with KLF6 knockdown. Together, this work describes a novel signaling axis through which KLF6-mediated repression of VAV3 and CDC42EP3 inhibits RAC1Gmediated HCC cell migration in culture, and potentially HCC metastasis in vivo. TP53 gene mutations are commonly found in HCC and are associated with poor prognosis. Prior studies have suggested that p53 mutants can display gain-of- function properties in other tumor types. Therefore, I sought to determine if a particular hotspot p53 mutation, p53R172H, provided enhanced, gain-of-function properties compared to p53 loss in HCC. In vitro, soft agar colony formation and cell migration is reduced upon knockdown of p53R172H, indicating that this mutation is required for transformation-associated phenotypes in these cells. However, p53R172H-expressing mice did not have enhanced tumor formation or metastasis compared to p53-null mice. These data suggest that p53R172H and p53 deletion are functionally equivalent in vivo, and that p53R172H is not a gain-of-function mutant in HCC. Inhibition of the related transcription factors p63 and p73 has been suggested as a potential mechanism by which mutant p53 exerts its gain-of-function effects. Analysis of p63 and p73 target genes demonstrated that they are similarly suppressed in p53-null and p53R172H-expressing HCC cell lines, suggesting a potential explanation for the phenotypes I observed in vivo and in vitro. Together, the studies described in this dissertation increase our understanding of the mechanisms underlying HCC progression and metastasis. Specifically, we find and characterize KLF6 as a novel suppressor of HCC metastasis, and determine the contribution of a common p53 point mutation in HCC. This work contributes to ongoing efforts to improve treatment options for HCC patients.

Le suppresseur de tumeur p53 permet à la cellule de se défendre contre différentes formes de stress. Il joue un rôle de barrière s'opposant à la tumorigenèse: en effet la perte de p53 chez la souris prédispose grandement ces animaux à développer des tumeurs; de même le locus p53 est inactivé dans près de 50% des tumeurs humaines.

p53 constitue un facteur de transcription qui se lie à des séquences particulières de l'ADN et active l'expression des gènes adjacents. L'expression orchestrée de ces gènes conduit, directement ou indirectement et suivant le contexte cellulaire, soit à la mort de la cellule soit à l'inhibition de la division cellulaire.

Les mécanismes moléculaires médiant ces deux activités biologiques essentielles de p53, de même que les mécanismes influençant le choix de la réponse cellulaire, sont encore mal compris. L'importance de p53 dans ce choix reste également à démontrer.

Afin de contribuer à la compréhension de ces mécanismes, le modèle murin déficient pour Mdm4, un régulateur négatif de l'activité de p53, a été choisi. L'inactivation de Mdm4 chez la souris conduit en effet à l'activation ectopique de p53 in vivo et l'induction de deux types de réponse: apoptose dans le neuroépithélium et arrêt de la prolifération cellulaire dans les tissus non neuronaux. Le profil d'expression des gènes dans les tissus neuronaux et non neuronaux a donc été comparé entre embryons de souris sauvage et mdm4-/- par la technique d'hybridation de biopuces à ADN. Les résultats obtenus suggèrent que le type de réponse dépend du type cellulaire et non de p53 lui-même. En effet les profils d'expression des gènes dans les tissus neuronaux (conditions d'apoptose) et non neuronaux (conditions d'arrêt de la prolifération cellulaire) chez l'embryon de souris mdm4-/- sont comparables.

Nous nous sommes ensuite particulièrement intéressés à deux nouveaux gènes dont l'expression est augmentée dans les embryons mdm4-/-. Dans un premier temps, leur induction transcriptionnelle chez l'embryon de souris mdm4-/- a été confirmée par différentes techniques et il a été vérifié qu'ils constituaient tous deux des cibles directes de p53 induites suite à un stress génotoxique.

Le premier gène code Dapk1, une protéine suppresseur de tumeur pro-apoptotique présentant une activité de type sérine/thréonine kinase. Ce travail a permis d'établir que Dapk1 participait à une boucle de rétroaction du contrôle de l'activité de p53.

Le deuxième gène identifié code la protéine Ptprv, un récepteur transmembranaire présentant une activité de type tyrosine phosphatase. En vue d'étudier la signification physiologique de l'induction transcriptionnelle de ptprv suite à l'activation de p53, des expériences effectuées à partir de matériel biologique issu de souris déficientes pour Ptprv ont été réalisées. Ces expériences confirment le rôle essentiel de Ptprv comme médiateur de l'arrêt du cycle cellulaire en phase G1 induit par p53 suite à un stress génotoxique, à la fois in vitro et in vivo. Par contre, Ptprv ne semble pas influencer l'apoptose induite suite à l'activation de p53. Ce travail a également permis d'établir le rôle essentiel de Ptprv dans la suppression de tumeurs induites chez la souris par activation constitutive de l'oncogène Ras.

Doctorat en sciences, Spécialisation biologie moléculaire
info:eu-repo/semantics/nonPublished

Hepatocellular cancer (HCC) is a highly prevalent neoplasm and common cause of death in patients with advanced liver disease and cirrhosis. While a growing number of molecular abnormalities have been identified, much is still unknown about its pathogenesis, particularly those early events that could lead to improved diagnosis and prognosis. The in vivo activity of KLF6, combined with its localization in humans on chromosomal locus 10P15, a commonly deleted region in many cancers, led to the evaluation of KLF6 as a bona fide tumor suppressor gene that might be lost and/or inactivated in human cancer. These efforts culminated in the discovery of KLF6 mutation and loss in a majority of human prostate cancers, a neoplasm evaluated initially because it reportedly harbored 10p deletions. Tumor-derived mutations of KLF6 were unable to upregulate p21, partially explaining the loss of growth suppression. Broader efforts at identifying both the prevalence of KLF6 inactivation in human cancer and the mechanisms of wild type KLF6 function have been remarkably fruitful, and validated by outside laboratories. KLF6 inactivation and/or loss have been identified in colorectal cancer, glioblastoma, ovarian cancer, and, most importantly, HCC. These exciting findings lead to key hypotheses that are tested in the experiments described in this study: KLF6 is a growth suppressor in normal hepatocytes, and its inactivation through loss, mutation or alternative splicing contributes to the development of HCC. To test these hypotheses 1. we correlated the relative expression of KLF6 by real-time PCR with histological progression from normal human liver to cirrhosis and HCC; 2. we replicated KLF6 dysregulation associated with human HCC in mouse models by determining the response of KLF6 +/- mice, which are phenotypically normal, to a chemical induced carcinogenic stress.

Hepatocellular carcinoma (HCC), or primary liver cancer, is the fifth most common cancer worldwide and the third most common cause of cancer mortality (El-Serag 2012). The development of HCC is thought to be a multi-staged process that involves several risk factors including the chronic hepatitis B and C infection, carcinogen exposure, metabolic disease, excessive alcohol consumption and male gender. Accumulation of genetic and epigenetic alterations with DNA-damaged hepatocytes can also contribute to the molecular pathogenesis of HCC. A better understanding of molecular mechanisms associated with HCC could ultimately improve our current strategies for screening and targeted therapy of this disease. Array comparative genomic hybridisation studies in murine diethylnitrosamine (DEN)-induced HCC have identified ccne1 (cyclin E) as a candidate gene associated in accelerated liver carcinogenesis (Teoh et al. 2008). In order to investigate the role of cyclin E and its impact on hepatocyte cell cycle regulation in early HCC development, we employed a well-known and highly reproducible rodent model of DEN-induced hepatocarcinogenesis. C57BL/6J male mice were injected with DEN (10 mg/kg i.p.), age day 12-15. In this model, male animals develop hepatocyte dysplasia at 6 mths and HCC in >90%. Transcript and protein expression of cyclin E was evident as early as 6 mths in dysplastic nodules (DNs), and significantly increased in HCCs. In contrast, there was little or undetectable cyclin E in normal liver and liver surrounding HCCs at all timepoints. Glutathione S transferase-pi form and cyclin E expression co-localised in DNs from liver in mice at 6 and 9 mths. Cyclin E/cdk2 kinase activity was also significantly upregulated in DNs, while increased proliferative activity by cyclin D1 and proliferating cell nuclear antigen (PCNA) in HCCs was observed at 9 mths, a timepoint where there was maximal p53 and p21 tumour suppressor expression. Aberrant cyclin E protein expression, including low molecular weight (LMW) isoforms were detected in HCCs and liver surrounding HCCs. Interestingly, sequencing analyses of p53 revealed a 1093-1361 nucleotide deletion in up to 90% of DNs, causing dysfunctional p53 nuclear localisation and export signalling. Because p53 directly signals to p21, co-immunoprecipitation studies were performed and revealed preferential binding of p21 to cyclin D1, rather than cyclin E, thereby allowing "escape" from the G1/S checkpoint. To directly test whether cyclin E regulates p53 expression in HCCs derived from DEN-treated male mice at 9 mths, we conducted cyclin E knockdown in primary HCC cells. This strategy resulted in increased p53 and p21 expression, as well as significant diminution of Bcl-xL, the p53-induced anti-apoptotic marker. Cell viability tetrazolium/formazan assay was significantly impaired in cyclin E RNAi targeted primary HCC cells. Conversely, chemical inhibition of p53 by pfithrin-α, augmented cyclin E, PCNA and Bcl-xL protein expression whilst cell viability was restored following co-treatment with MG-262, a 26S proteasome inhibitor. In contrast, overexpressing cyclin E in naïve primary hepatocytes enhanced PCNA expression, increased hepatocyte viability, downregulated p53 and its downstream signalling intermediate, p21. We next determined whether miRNA-34, a co-regulator of cyclin E and p53, was instrumental in the reciprocity between cyclin E and p53 as key "drivers"of hepatocarcinogenesis. Dysplastic liver and HCCs obtained from DEN-injected male mice were assayed for miR-34a,b,c. miR-34a and c were significantly upregulated in HCCs and dysplastic liver compared with normal liver. Similar trends were noted for miR-34a,b,c in human hepatitis C-related HCCs when compared with normal human liver. Importantly, this was associated with significantly enhanced cyclin E and p53 mRNA expression in human HCCs compared to normal and cirrhotic liver. In this murine model, there was disproportional and upregulation of functionally active cyclin E, miR-34a,c in DNs and early HCCs with congruent loss of p53 function associated with cell cycle checkpoint failure, diminished apoptosis and increased proliferative drive. In human HCV-related HCCs, miR-34a, p53 and cyclin E transcript levels were universally upregulated. When we performed in vitro experiments in murine primary hepatocytes and primary HCC cells, knocking down or overexpressing cyclin E did not affect miR-34 expression. However, stabilising p53 with MG-262 enhanced miR-34a,c expression (though not significant), whilst inhibiting p53 using pfithrin-α significantly reduced miR-34a,c. miR-34 may provide a plausible link to increased cyclin E expression, activity and increased proliferative drive in dysplastic and neoplastic liver in mice and humans. Gender disparity in human HCC is well described, with a strong male predominance. However, the role of sex hormones in hepatocarcinogenesis remains poorly defined. In order to determine if there are gender differences in the expression of cyclin E, effects on cell cycle regulators and tumour suppressors, dysplastic liver and HCCs were studied in DEN-treated C57BL/6J female mice. These mice displayed a significant reduction in dysplastic hepatocytes compared with intact DEN-treated males at 3, 6 mths, while HCC incidence, number and size of tumours were significantly diminished in females at up to 15 mths. In carcinogen-treated female mice, cyclin E (native and LMW isoforms) protein expression and kinase activity were reduced compared to males at 6-12 mths, with concomitant reduction in hepatocyte proliferation by PCNA and cyclin D1 expression. Unlike male mice, G1/S checkpoint is evident by robust p53-mediated apoptosis. To ascertain if these differences are attributable to the effects of oestradiol/progesterone (E/P) and/or testosterone, we conducted hormonal manipulation studies using the same carcinogen-model by performing ovariectomy in female animals and orchidectomy in male mice, in which some animals received E/P or testosterone supplementation. Castration of DEN-injected male mice resulted in a loss of cyclin E LMW isoforms compared to intact males, diminution of cyclin E kinase activity and phospho-retinoblastoma expression. There was also induction of p53-mediated apoptosis in dysplastic hepatocytes, leading to a reduction in number of DNs by 6 mths. These anti-proliferative and pro-apoptotic effects were magnified by E/P replacement in castrated DEN-treated males. In contrast, testosterone-replacement in ovariectomised-female mice exhibited accelerated hepatocarcinogenesis compared to intact female DEN-treated animals and displayed LMW cyclin E isoforms similar to those detected in DEN-injected intact males. In further analyses, there was increased oestrogen receptor-α (ERα) transcript and protein expression in HCCs derived from DEN-injected intact male mice, and in dysplastic liver from castrated male mice replaced with E/P. E/P replacement and testosterone withdrawal were associated with ERα expression, the loss of cyclin E LMW isoforms, intact cdk2 expression, functional G1/S checkpoint control and induction of p53-mediated apoptotic cell death in preneoplastic hepatocytes. Further, oestrogen (E2) stimulation had varying effects on cell cycle regulation and viability in primary hepatocytes and HCC cells. As there was little to no significant correlation between ERα and its downstream target, c-myc transcript levels, we propose that E2/ERα signalling may be operative via other pathways to subsequently activate p53. These findings open up tantalising avenues to further explore the inter-regulatory signalling pathways between E2/ERα, cell cycle regulators and the tumour suppressor, p53.

碩士
元培醫事科技大學
醫學檢驗生物技術系碩士班
105
In order to prevent cell transformation, when the tumor suppressor gene p53 mutates, it will cause cell proliferation and lead to tumor. P21 is a downstream regulator of p53, which is associated with colorectal cancer differentiation, depth of invasion, proliferation and metastasis. In recent years, various studies have pointed out that in the context of p53 mutation, p21 expression increased, but may cause tumor proliferation and metastasis. Litchi kernel extract is rich in polyphenols and is resistant to colon or rectal cancer. In this experiment, nude mice bearing the p53 mutant HT-29 cells transplanted with the litchi kernel extract showed a dose-dependent decrease in the expression of p53 and p21. However, in nude mice bearing tumors of SW480 cells, a dose-dependent decrease in p53 was seen. However, the performance of p21 was not significantly reduced compared to the results of HT-29. The results show that the litchi kernel extracts of HT-29 mutant p53 and p21 performance will have an inhibitory effect, and in cell experiments, the p53 mutant cell line HT-29 found p53 and p21 performance with the lychee kernel extract dose increases with a downward trend. On the p53-null mutant SW480, p53 was seen to decrease with increasing doses, but there was no significant reduction in the portion of p21. Based on all the above results of this experiment, we can confirm that the application of litchi nuclear extract in p53 mutant colorectal cancer has its influence, and can affect the expression of its downstream p21, we can expect by inhibiting the mutation in p53 and in Its absence of p21 environment, in order to achieve the effect of inhibiting colorectal cancer.

Zhang, Zhiyi.
Thesis (Ph.D.)--Chinese University of Hong Kong, 2011.
Includes bibliographical references (leaves ).
Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web.
Abstract also in Chinese.

研究背景和目的
肝癌是非常高死亡率的恶性肿瘤之一。由于传统化疗方式的局限性，表观遗传治疗方法可能成为肝癌治疗的替代方法。研究报道ZBP-89诱导肝癌细胞Bak的表达，表观调控是否参与该诱导作用，目前仍然不清楚。
HDAC3被认为是化疗靶点和肝癌复发的肿瘤标记物。它常常在肝癌组织中高表达，对HDAC3的抑制作用可以增加肝癌的化疗效果。我们的研究表明ZBP-89可以降低肝癌细胞HDAC3的表达，但机制未明。蛋白的翻译后调控是细胞生化过程的重要调节因素。所以，研究调节HDAC3的降低途径对肝癌的发生和复发具有非常重要的研究意义。
本研究旨在研究ZBP-89调控Bak表达的表观遗传机制。同时，弄清楚DNA甲基化转移酶和组蛋白去乙酰化酶是否参与ZBP-89对Bak的调控作用，进一步阐明ZBP-89对HDAC3降低通路的机制。
方法和结果
肝癌病人组织蛋白分析表明，相对于癌旁组织，肝癌组织Bak和ZBP-89蛋白表达降低，而DNMT1和HDAC3表达升高。免疫共沉淀技术显示ZBP-89与HDAC3、 DNMT1结合，但不与HDAC4， DNMT3a和DNMT3b结合。相应地，HDAC3和 DNMT1免疫沉淀分析也显示三者形成免疫复合物。我们在肝癌细胞中过表达ZBP-89，验证它会不会影响HDACs和DNMTs的活性。实验结果表明过表达的ZBP-89抑制HDACs和DNMTs的活性。进一步发现ZBP-89调节的Bak表达可能是通过抑制HDACs活性和维持组蛋白H3和H4乙酰化水平实现的。另一方面，我们同样证明HDAC的抑制剂（HDACi）VPA和TSA可以诱导肝癌细胞Bak表达，此外，siRNA干扰HDAC3的表达同样可以诱导Bak表达。
对DNMT1表达的抑制和使用DNMT抑制剂（DNMTi）Zebularine也可以诱导Bak的表达。染色质免疫沉淀结果显示ZBP-89结合于Bak的启动子区域，从-3188bp到-3183bp，从-275到-49。 ZBP-89可以抑制DNMT的活性，那么ZBP-89是否会影响DNA中CpG岛甲基化状态和甲基化结合蛋白（MeCP2）的结合能力，这一点仍需要进一步证实。结果表明ZBP-89可以抑制MeCP2结合基因组DNA。为进一步揭示MeCP2是否由于启动子区域CpG岛去甲基化影响其结合能力，我们采用亚硫酸盐测序方法。测序结果显示ZBP-89过表达可以影响Bak启动子CpG岛的甲基化状态，并促进其去甲基化。
腺病毒介导的ZBP-89过表达降低HDAC3表达呈现剂量依赖性，然而HDAC3 的mRNA水平并没有受到ZBP-89的表达。免疫共沉淀方法和蛋白免疫印迹实验用于分析Pin1和HDAC3复合物，磷酸化IκB和HDAC3复合物的结合情况。结果表明Pin1结合HDAC3并促进HDAC3的减少。同时，HDAC3与磷酸的IκB结合并进入蛋白减少途径。
构建的mU6-siPin1表达质粒用于敲除肝癌细胞Pin1的表达，方法检测基因表达水平。Pin1的缺失表达阻碍ZBP-89介导的HDAC3降低。在Pin1 敲除细胞系 JB6 C141 Pin1⁻/⁻ 和Pin1过表达细胞系的研究，ZBP-89更加能促进Pin1⁺/⁺细胞中HDAC3减少，而对Pin1⁺/⁺的细胞则没那么明显。由此肯定了Pin1在ZBP-89介导的HDAC3降低中的重要作用。进一步研究发现， IκB激酶 (IKK)抑制剂，CAY10576，能抑制 ZBP-89介导的HDAC3的降低；而SN50, p65/p50人核抑制多肽，则不影响HDAC3的降低。研究结果证明HDAC3的降低依赖IκB通路，而不是NF- κB活性。
我们用人肝癌细胞的裸鼠移植瘤模型研究ZBP-89调控Bak表达的表观遗传机制，及其对肝癌的治疗效果。研究结果表明ZBP-89蛋白和组蛋白抑制剂VPA和DNA甲基化抑制zebularine都能抑制肿瘤的生长，并诱导肿瘤组织Bak表达及细胞凋亡。VPA和zebularine联合治疗的效果更好。研究也表明ZBP-89可以在体内降低HDAC3蛋白水平。
结论
本研究揭示了ZBP-89调节Bak蛋白表达和肝癌细胞凋亡的表观遗传机制。同时，进一步揭示ZBP-89联合Pin1经由IκB通路调节HDAC3降低的机制. 本研究为肝癌表观遗传学的治疗提供研究基础和科学依据。
Background
Hepatocellular carcinoma (HCC) is one of the most prevalent malignancies worldwide with a very high mortality. Because the success of the conventional therapies is limited, epigenetic therapy may represent an alternative for HCC management. ZBP-89 is known to induce Bak in HCC. However, it is unclear whether epigenetic mechanisms contribute to ZBP-89-mediated Bak.
Histone acetylase 3 (HDAC3) is realized as a chemotherapy target and a biomarker of recurrence in HCC. HDAC3 is frequently overexpressed in HCC and its inhibition enhances the efficacy of anti-HCC chemotherapy. The pilot data have indicated that ZBP-89 reduced HDAC3 in HCC but the mechanism responsible was unknown. The post-translational modification of proteins functions as a key regulatory factor in cellular physiological procedures, such as ubiquitinoylation degradation. As a biomarker of HCC development and recurrence, it is important to understand how ZBP-89 mediates the reduction of HDAC3.
This study focuses on if ZBP-89 regulates Bak expression through epigenetic mechanisms. It is designed to investigate whether DNA methyltransferases (DNMTs), histone acetylases (HDACs) are involved in regulation of ZBP-89-induced Bak expression. The study also elucidates the mechanism how ZBP-89 reduces the level of HDAC3 protein.
Methods and Results
The levels of Bak and ZBP-89 as shown on western blots were reduced but DNMT1 and HDAC3 were increased in HCC cancer tissues compared to the corresponding non-cancer tissues. Co-immunoprecipitation experiments showed that ZBP-89 bound to HDAC3 and DNMT1 but not other epigenetic enzymes, such as HDAC4, DNMT3a and DNMT3b. To clarify if ZBP-89 affects the activities of HDACs and DNMTs, ZBP-89 was overexpressed in HCC cells. Enzyme activities of HDACs and DNMTs were determined using relevant assay kits. Results showed that overexpressed ZBP-89 inhibited the activities of HDACs and DNMTs. Further experiments indicated that ZBP-89-mediated Bak up-regulation might contribute to maintenance of histone H3 and H4 acetylation through inhibition of HDACs activity. In another set of experiments, we also found an increased Bak expression in HCC cells when the cells were treated with HDAC inhibitors (HDACi) VPA and TSA. HDAC3 siRNA also increased Bak expression.
Both knockdown of DNMT1 expression and administration of DNMTs inhibitors (zebularine) induced Bak expression. Chromatin immunoprecipitation (ChIP) showed that ZBP-89 bound to Bak promoter at the region from -3188bp to -3183bp and from -275 to -49. As ZBP-89 inhibits DNMT activity, it is essential to know whether its inhibition affectes DNA CpG methylation status and methyl-CpG binding protein (MeCP) binding. The results showed that ZBP-89 overexpression inhibited MeCP2 binding to genomic DNA. The finding indicated that decreased MeCP2 binding to DNA might be due to decreased methyl-CpG number in Bak promoter, suggesting that ZBP-89 might affect CpG island methylation status. Therefore, the bisulfite modified DNA sequencing method was used to clarify if Bak promoter CpG island methylation status was altered after ZBP-89 overexpression. Results revealed that ZBP-89 overexpression could demethylate the CpG islands in Bak promoter.
ZBP-89 overexpression dose-dependently reduced the expression of HDAC3 at protein level but not at mRNA level. Co-immunoprecipitation and western blot methods were used to analyze Peptidyl-prolyl cis/trans isomerase 1 (Pin1) and HDAC3, phospho-I kappa B (pIκB), and the result revealed that HDAC3 could bound with either Pin1 or pIκB to promote the reduced expression of HDAC3.
Constructed mU6-siPin1 vector was used to knockdown Pin1 expression in HCC cells. We found that knockdown of Pin1 expression blocked ZBP-89-mediated HDAC3 reduction. Experiments performed in Pin1 allele-knockdown JB6 C141 Pin1⁻/⁻ and Pin1⁺/⁺ cells showed that the reduction of HDAC3 by ZBP-89 was greater in Pin1⁺/⁺ cells than in Pin1⁻/⁻ cells, confirming the role of Pin1 in ZBP-89-mediated HDAC3 reduction. Furthermore, the ZBP-89-mediated HDAC3 reduction was suppressed by CAY10576, an IκB kinase (IKK) activation inhibitor but not by SN50, a p65/p50 translocation inhibitor, suggesting that HDAC reduction may depend on IκB kinase rather than NF-κB activity.
HCC xenograft mouse model was used to support the involvement of epigenetic mechanism in ZBP-89-induced Bak expression and its therapeutic effects against HCC. Results showed that ZBP-89 as well as HDAC inhibitor valproic acid (VPA) or/and DNMT inhibitor zebularine stimulated Bak expression and induced apoptosis of tumor cells in an HCC xenograft mouse model, arresting tumor growth. In HCC xenografe model, treatment by injection of Ad-ZBP-89 viral expression vector mediated ZBP-89 expression decreased HDAC3 expression, but not HDAC4.
Conclusions
In conclusion, the study demonstrates a novel mechanism through which ZBP-89 mediates an epigenetic pathway to promote Bak expression, and induce apoptosis in HCC cells. It also reveals the mechanism of HDAC3 reduction by ZBP-89 is dependent on IκB, which requires the presence of Pin1. This pathway may help develop future epigenetic therapy against HCC.
Detailed summary in vernacular field only.
Detailed summary in vernacular field only.
Detailed summary in vernacular field only.
Detailed summary in vernacular field only.
Detailed summary in vernacular field only.
Detailed summary in vernacular field only.
Detailed summary in vernacular field only.
Detailed summary in vernacular field only.
Detailed summary in vernacular field only.
Detailed summary in vernacular field only.
Detailed summary in vernacular field only.
Detailed summary in vernacular field only.
Ye, Caiguo.
Thesis (Ph.D.)--Chinese University of Hong Kong, 2013.
Includes bibliographical references (leaves 123-140).
Abstracts also in Chinese.
Abstract --- p.i
摘要 --- p.v
Publications --- p.viii
Acknowledgements --- p.ix
Abbreviations --- p.xi
List of Tables --- p.xiii
List of figures --- p.xiv
Chapter Chapter One: --- General Introduction --- p.1
Chapter 1.1 --- Background --- p.2
Chapter 1.2 --- The complexity of HDAC family and functions --- p.3
Chapter 1.2.1 --- HDAC family --- p.4
Chapter 1.2.2 --- Multifunction of HDACs --- p.6
Chapter 1.3 --- HDACs and apoptosis --- p.6
Chapter 1.3.1 --- HDAC regulates apoptotic-related gene expression --- p.9
Chapter 1.3.2 --- HDACs regulate apoptosis through protein complexes --- p.18
Chapter 1.3.3 --- HDACs mediates non-histone deacetylation and apoptosis --- p.21
Chapter 1.3.4 --- HDACs degradation deficiency and apoptosis --- p.24
Chapter 1.4 --- DNMTs and epigenetic modification --- p.25
Chapter 1.4.1 --- DNMT family --- p.25
Chapter 1.4.2 --- CpG islands methylation and HCC --- p.26
Chapter 1.5 --- Perspectives --- p.28
Chapter Chapter Two: --- ZBP-89 up-regulates Bak expression through inhibition the activity of HDACs and DNMTs --- p.30
Chapter 2.1 --- Introduction --- p.31
Chapter 2.2 --- Materials and Methods --- p.33
Chapter 2.2.1 --- Hepatocellular carcinoma patient samples and cell lines --- p.33
Chapter 2.2.2 --- Chemicals and reagents --- p.34
Chapter 2.2.3 --- Cell proliferation --- p.34
Chapter 2.2.4 --- Adenovirus infection of cells --- p.35
Chapter 2.2.5 --- Apoptosis detection --- p.36
Chapter 2.2.6 --- Transfection of siRNA and plasmid --- p.36
Chapter 2.2.7 --- Co-immunoprecipitation (co-IP) --- p.37
Chapter 2.2.8 --- Western blotting --- p.37
Chapter 2.2.9 --- Immunohistochemistry and Immunofluorescence --- p.38
Chapter 2.2.10 --- Chromatin immunoprecipitation --- p.38
Chapter 2.2.11 --- Sodium bisulfite modified sequencing of Bak promoter --- p.40
Chapter 2.2.12 --- Histone deacetylase activity assay --- p.41
Chapter 2.2.13 --- DNA methyltransferases enzyme activity --- p.42
Chapter 2.2.14 --- Xenograft animal model --- p.43
Chapter 2.2.15 --- Statistical analysis --- p.43
Chapter 2.3 --- Results --- p.45
Chapter 2.3.1 --- ZBP-89 interacts with DNMT1 and HDAC3 --- p.45
Chapter 2.3.2 --- DNA methyltransferase-1 and histone deacetylase 3 are overexpressed in cancer tissues --- p.48
Chapter 2.3.3 --- Inhibition of HDACs and DNMTs induces Bak expression and apoptosis --- p.58
Chapter 2.3.4 --- Adenovirus mediated ZBP-89 expression inhibits HDACs activity --- p.65
Chapter 2.3.5 --- ZBP-89 suppresses DNMTs activity --- p.67
Chapter 2.3.6 --- Overexpressed ZBP-89 demethylates methyl-CpG islands --- p.69
Chapter 2.3.7 --- Downregulation of HDAC3 and DNMT1 enhances Bak expression --- p.74
Chapter 2.3.8 --- Xenograft nude mouse model reveals that Ad-ZBP-89 adenovirus diminishes tumor volume and induces Bak expression and apoptosis --- p.75
Chapter 2.4 --- Discussion --- p.81
Chapter Chapter Three: --- ZBP-89 targets IkappaB to reduce HDAC3 via a Pin1-dependent pathway --- p.86
Chapter 3.1 --- Introduction --- p.87
Chapter 3.2 --- Materials and Methods --- p.89
Chapter 3.2.1 --- Cell lines, chemicals and reagents --- p.89
Chapter 3.2.2 --- Transfection of siRNA plasmid --- p.89
Chapter 3.2.3 --- Plasmid extraction by mini-prep --- p.90
Chapter 3.2.4 --- Co-immunoprecipitation (co-IP) and Western blotting --- p.91
Chapter 3.2.5 --- Total RNA extraction --- p.92
Chapter 3.2.6 --- Reverse transcription and real-time PCR --- p.93
Chapter 3.2.7 --- Immunohistochemistry and Immunofluorescence --- p.94
Chapter 3.2.8 --- Xenograft animal model --- p.95
Chapter 3.2.9 --- Statistical analysis --- p.95
Chapter 3.3 --- Results --- p.97
Chapter 3.3.1 --- ZBP-89 overexpression diminishes HDAC3 expression but not HDAC4 --- p.97
Chapter 3.3.2 --- Knockdown of Pin1 blocks ZBP-89-mediated HDAC3 reduction --- p.99
Chapter 3.3.3 --- ZBP-89 reduces the level of IκB --- p.103
Chapter 3.3.4 --- IκB degradation inhibitors suppresses ZBP-89-meditaed HDAC3 reduction --- p.105
Chapter 3.3.5 --- ZBP-89 decreases HDAC3 but increases Bak in xenograft tumor tissues --- p.111
Chapter 3.4 --- Discussion --- p.115
Chapter Chapter Four: --- Conclusions and Future Perspectives --- p.119
Chapter 4.1 --- Summary of results --- p.120
Chapter 4.2 --- Conclusions --- p.121
Chapter 4.3 --- Future Perspectives --- p.121
References --- p.123

博士
國立臺灣師範大學
生命科學系
104
Lung cancer is the leading cause of cancer mortality worldwide. NSCLC accounts for about 75% to 80% of lung cancer cases. Chemotherapy adverse side effects and resistance to current anticancer agents have been the pressing problems in the success of lung cancer therapy. Anticancer drugs are in urgent need especially for lung cancer. A program to develop a new anti-lung cancer agent by screening novel synthetic compounds was initiated. EMMQ was selected by MTT cell viability assay. The cell growth rate of A549 and H460 NSCLC cells was reduced by a low concentration of EMMQ treatment. Our study revealed that EMMQ induced apoptosis in NSCLC cells with wild-type p53, while the drug is less potent to against p53-null cells. The study elucidated that EMMQ-induced apoptosis is was mediated through the intrinsic pathway involving DNA damage, activation of p53, interference of mitochondrial ΔΨm that led to release of cytochrome c and down regulation Bcl-2, activation of caspase family proteins, and finally cleavage of PARP polymerase cleavage. In vivo study showed that EMMQ reduced tumorigenesis and significantly suppressed growth rate of xenograft tumors in nude mice. In addition, metastasis studies demonstrated that EMMQ may inhibit wild-type p53 cells migration at low concentration. In conclusion, EMMQ was demonstrated as an effective p53 regulator in NSCLC cells. Our findings indicate that EMMQ may serve as a promising new and potential therapeutic agent for human lung cancer. Human liver cancer is the fifth most frequently diagnosed cancer worldwide. The important limitation in the clinical battle against this tumor is its marked intrinsic and acquired refractivity to the available chemotherapies. Anticancer agents effective against chemo-resistant cells are greatly needed for liver cancer treatment. Previously, our study have identified EMMQ as an effective drug in the treatment of lung cancer cells in vitro and in animal models. In this work, results show EMMQ treatment may inhibit cell growth and induce apoptosis in HCC. EMMQ induced apoptosis in HCC cells with wild-typed p53, and is less potent in cells with mutant p53 and in p53-null. The study also demonstrated that EMMQ induces apoptosis through two major pathways. First, the compound induced cell death through the intrinsic pathway by first damaging DNA increasing expression of p53 and γ-H2AX and decreasing cyclin D1 and CDK 2, finally leading to G1 arrest in HepG2 cells as studied cell cycle. Second, the tumor suppressor gene p53 was activated following a reduction of ΔΨm, ROS generation and down-regulating Akt, Bcl-2, Bax, cytochrome c, caspase-3 and cleavage of PARP, the critical events leading to cell death in HepG2 cells treated with EMMQ. The in vitro findings indicate that EMMQ is a promising candidate for the treatment of liver cancer.