Molekularcytogenetische und molekulargenetische Untersuchung eines Patienten mit DiGeorge- verwandter Klinik und renalem Rhabdoidtumor

In dieser Diplomarbeit wird ein Patient mit mildem Phänotyp eines 22q11 Mikrodeletionssyndroms und einem malignen Rhabdoid Tumor in einer der beiden Nieren beschrieben. Der Patient zeigt ein angeborenes Rearrangement im Chromosomenband 22q11, welches in dieser Form noch nicht berichtet wurde. Es handelt sich um eine 2.8- Mb Mikroduplikation der bei VCFS/DGS typischerweise deletierten Region TDR. Dieser Duplikation ist eine unmittelbar telomerisch liegende Deletion von ebenfalls 2.8- Mb angeschlossen. Von der Deletion betroffen ist das Tumor Suppressor Gen SMARCB1, welches dadurch in den somatischen Zellen des Patieten in haploidem Zustand vorliegt. Die Eltern des Patienten zeigten einen normalen männlichen (46,XY) bzw. weiblichen (46,XX) Karyotyp. Mikrosatellitenanalysen bestätigten, dass sowohl die 2.8- Mb Duplikation, als auch die 2.8- Mb Deletion, de novo auf dem väterlich vererbten Chromosom entstanden sind. FISH und Array CGH Resultate zeigen übereinstimmend, dass die Bruchpunktregionen innerhalb der LCRs22 liegen, wodurch die Theorie unterstützt ist, dass nicht allelische homologe Rekombinationsereignisse (NAHR) während der väterlichen Meiose die Bildung dieses neuartigen Rearrangements mit hoher Wahrscheinlichkeit ausgelöst haben. Im malignen Rhabdoid Tumor des Patieten wurde im zweiten Allel des Tumor Suppressor Gens SMARCB1 eine 2- bp Deletion (c.664_665delCT oder c.666_667delCT) gefunden. Die dadurch entstandene Stop- Mutation führte zur homozygoten Inaktivierung von SMARCB1 und trägt mit großer Wahrscheinlichkeit zur Entstehung des malignen Rhabdoid- Tumors bei. (Biegel et al., 1990; Biegel, 2006)In dieser Diplomarbeit wird ein Patient mit mildem Phänotyp eines 22q11 Mikrodeletionssyndroms und einem malignen Rhabdoid Tumor in einer der beiden Nieren beschrieben. Der Patient zeigt ein angeborenes Rearrangement im Chromosomenband 22q11, welches in dieser Form noch nicht berichtet wurde. Es handelt sich um eine 2.8- Mb Mikroduplikation der bei VCFS/DGS typischerweise deletierten Region TDR. Dieser Duplikation ist eine unmittelbar telomerisch liegende Deletion von ebenfalls 2.8- Mb angeschlossen. Von der Deletion betroffen ist das Tumor Suppressor Gen SMARCB1, welches dadurch in den somatischen Zellen des Patieten in haploidem Zustand vorliegt. Die Eltern des Patienten zeigten einen normalen männlichen (46,XY) bzw. weiblichen (46,XX) Karyotyp. Mikrosatellitenanalysen bestätigten, dass sowohl die 2.8- Mb Duplikation, als auch die 2.8- Mb Deletion, de novo auf dem väterlich vererbten Chromosom entstanden sind. FISH und Array CGH Resultate zeigen übereinstimmend, dass die Bruchpunktregionen innerhalb der LCRs22 liegen, wodurch die Theorie unterstützt ist, dass nicht allelische homologe Rekombinationsereignisse (NAHR) während der väterlichen Meiose die Bildung dieses neuartigen Rearrangements mit hoher Wahrscheinlichkeit ausgelöst haben. Im malignen Rhabdoid Tumor des Patieten wurde im zweiten Allel des Tumor Suppressor Gens SMARCB1 eine 2- bp Deletion (c.664_665delCT oder c.666_667delCT) gefunden. Die dadurch entstandene Stop- Mutation führte zur homozygoten Inaktivierung von SMARCB1 und trägt mit großer Wahrscheinlichkeit zur Entstehung des malignen Rhabdoid- Tumors bei. (Biegel et al., 1990; Biegel, 2006

Hepatic p53 is regulated by transcription factor FOXO1 and acutely controls glycogen homeostasis

The tumor suppressor p53 is involved in the adaptation of hepatic metabolism to nutrient availability. Acute deletion of p53 in the mouse liver affects hepatic glucose and triglyceride metabolism. However, long-term adaptations upon the loss of hepatic p53 and its transcriptional regulators are unknown. Here we show that short-term, but not chronic, liver-specific deletion of p53 in mice reduces liver glycogen levels, and we implicate the transcription factor forkhead box O1 protein (FOXO1) in the regulation of p53 and its target genes. We demonstrate that acute p53 deletion prevents glycogen accumulation upon refeeding, whereas a chronic loss of p53 associates with a compensational activation of the glycogen synthesis pathway. Moreover, we identify fasting-activated FOXO1 as a repressor of p53 transcription in hepatocytes. We show that this repression is relieved by inactivation of FOXO1 by insulin, which likely mediates the upregulation of p53 expression upon refeeding. Strikingly, we find that high-fat diet-induced insulin resistance with persistent FOXO1 activation not only blunted the regulation of p53 but also the induction of p53 target genes like p21 during fasting, indicating overlapping effects of both FOXO1 and p53 on target gene expression in a context-dependent manner. Thus, we conclude that p53 acutely controls glycogen storage in the liver and is linked to insulin signaling via FOXO1, which has important implications for our understanding of the hepatic adaptation to nutrient availability

p53 as a Dichotomous Regulator of Liver Disease: The Dose Makes the Medicine

Lifestyle-related disorders, such as the metabolic syndrome, have become a primary risk factor for the development of liver pathologies that can progress from hepatic steatosis, hepatic insulin resistance, steatohepatitis, fibrosis and cirrhosis, to the most severe condition of hepatocellular carcinoma (HCC). While the prevalence of liver pathologies is steadily increasing in modern societies, there are currently no approved drugs other than chemotherapeutic intervention in late stage HCC. Hence, there is a pressing need to identify and investigate causative molecular pathways that can yield new therapeutic avenues. The transcription factor p53 is well established as a tumor suppressor and has recently been described as a central metabolic player both in physiological and pathological settings. Given that liver is a dynamic tissue with direct exposition to ingested nutrients, hepatic p53, by integrating cellular stress response, metabolism and cell cycle regulation, has emerged as an important regulator of liver homeostasis and dysfunction. The underlying evidence is reviewed herein, with a focus on clinical data and animal studies that highlight a direct influence of p53 activity on different stages of liver diseases. Based on current literature showing that activation of p53 signaling can either attenuate or fuel liver disease, we herein discuss the hypothesis that, while hyper-activation or loss of function can cause disease, moderate induction of hepatic p53 within physiological margins could be beneficial in the prevention and treatment of liver pathologies. Hence, stimuli that lead to a moderate and temporary p53 activation could present new therapeutic approaches through several entry points in the cascade from hepatic steatosis to HCC

Fasting improves therapeutic response in hepatocellular carcinoma through p53-dependent metabolic synergism

Cancer cells voraciously consume nutrients to support their growth, exposing metabolic vulnerabilities that can be therapeutically exploited. Here, we show in hepatocellular carcinoma (HCC) cells, xenografts, and patient-derived organoids that fasting improves sorafenib efficacy and acts synergistically to sensitize sorafenib-resistant HCC. Mechanistically, sorafenib acts noncanonically as an inhibitor of mitochondrial respiration, causing resistant cells to depend on glycolysis for survival. Fasting, through reduction in glucose and impeded AKT/mTOR signaling, prevents this Warburg shift. Regulating glucose transporter and proapoptotic protein expression, p53 is necessary and sufficient for the sorafenib-sensitizing effect of fasting. p53 is also crucial for fasting-mediated improvement of sorafenib efficacy in an orthotopic HCC mouse model. Together, our data suggest fasting and sorafenib as rational combination therapy for HCC with intact p53 signaling. As HCC therapy is currently severely limited by resistance, these results should instigate clinical studies aimed at improving therapy response in advanced-stage HCC

Complementary omics strategies to dissect p53 signaling networks under nutrient stress

Signaling trough p53is a major cellular stress response mechanism and increases upon nutrient stresses such as starvation. Here, we show in a human hepatoma cell line that starvation leads to robust nuclear p53 stabilization. Using BioID, we determine the cytoplasmic p53 interaction network within the immediate-early starvation response and show that p53 is dissociated from several metabolic enzymes and the kinase PAK2 for which direct binding with the p53 DNA-binding domain was confirmed with NMR studies. Furthermore, proteomics after p53 immunoprecipitation (RIME) uncovered the nuclear interactome under prolonged starvation, where we confirmed the novel p53 interactors SORBS1 (insulin receptor signaling) and UGP2 (glycogen synthesis). Finally, transcriptomics after p53 re-expression revealed a distinct starvation-specific transcriptome response and suggested previously unknown nutrient-dependent p53 target genes. Together, our complementary approaches delineate several nodes of the p53 signaling cascade upon starvation, shedding new light on the mechanisms of p53 as nutrient stress sensor. Given the central role of p53 in cancer biology and the beneficial effects of fasting in cancer treatment, the identified interaction partners and networks could pinpoint novel pharmacologic targets to fine-tune p53 activity

Complementary omics strategies to dissect p53 signaling networks under nutrient stress

Signaling trough p53is a major cellular stress response mechanism and increases upon nutrient stresses such as starvation. Here, we show in a human hepatoma cell line that starvation leads to robust nuclear p53 stabilization. Using BioID, we determine the cytoplasmic p53 interaction network within the immediate-early starvation response and show that p53 is dissociated from several metabolic enzymes and the kinase PAK2 for which direct binding with the p53 DNA-binding domain was confirmed with NMR studies. Furthermore, proteomics after p53 immunoprecipitation (RIME) uncovered the nuclear interactome under prolonged starvation, where we confirmed the novel p53 interactors SORBS1 (insulin receptor signaling) and UGP2 (glycogen synthesis). Finally, transcriptomics after p53 re-expression revealed a distinct starvation-specific transcriptome response and suggested previously unknown nutrient-dependent p53 target genes. Together, our complementary approaches delineate several nodes of the p53 signaling cascade upon starvation, shedding new light on the mechanisms of p53 as nutrient stress sensor. Given the central role of p53 in cancer biology and the beneficial effects of fasting in cancer treatment, the identified interaction partners and networks could pinpoint novel pharmacologic targets to fine-tune p53 activity

Fasting improves therapeutic response in hepatocellular carcinoma through p53-dependent metabolic synergism

Cancer cells voraciously consume nutrients to support their growth, exposing metabolic vulnerabilities that can be therapeutically exploited. Here, we show in hepatocellular carcinoma (HCC) cells, xenografts, and patient-derived organoids that fasting improves sorafenib efficacy and acts synergistically to sensitize sorafenib-resistant HCC. Mechanistically, sorafenib acts noncanonically as an inhibitor of mitochondrial respiration, causing resistant cells to depend on glycolysis for survival. Fasting, through reduction in glucose and impeded AKT/mTOR signaling, prevents this Warburg shift. Regulating glucose transporter and proapoptotic protein expression, p53 is necessary and sufficient for the sorafenib-sensitizing effect of fasting. p53 is also crucial for fasting-mediated improvement of sorafenib efficacy in an orthotopic HCC mouse model. Together, our data suggest fasting and sorafenib as rational combination therapy for HCC with intact p53 signaling. As HCC therapy is currently severely limited by resistance, these results should instigate clinical studies aimed at improving therapy response in advanced-stage HCC