Mechanisms of starvation-induced p53 stabilization in hepatocytes

Der Transkriptionsfaktor p53 spielt ein zentrale Rolle in der Tumorunterdrückung. Aktiviert wird p53 von verschiedensten Stresssignalen, was zur Transkription von p53-Zielgenen führt. Trotz vieler Studien über p53, gibt es nur wenig Wissen über p53s physiologische Funktion. Wir konnten zeigen, dass obwohl die Anzahl der mRNA unverändert blieb, sich die Anzahl von p53 Protein in Lebern gefasteter Mäuse und in Hepatocyten massiv erhöhte. Das Ziel dieser Masterarbeit war deshalb die Aufklärung der p53 Stabilisierung, die durch Fasten ausgelöst wird. Wir vermuteten, dass das erhöhte Vorkommen von p53 durch AMPK verursacht wird. Der AMPK-Komplex beeinflusst die Bindung von p53 an seinen Regulator MDM2. Dadurch wird p53 aktiviert. Um diese Hypothese zu testen wurden Immunpräzipitationsexperiment (IP) durchgeführt. Dazu wurden zuerst p53 und MDM2 in Zelllinien überexprimiert. Nachdem stabile IP-Protokolle etabliert wurden, konnte die Bindung von ektopischen p53 an MDM2 gezeigt werden. Außerdem wurde die endogene Interaktion dieser Proteine in HepG2 Zellen untersucht. Die Isolierung von p53 oder MDM2 alleine wurde mit IP-Experimenten gezeigt, aber es konnte nur eine sehr schwache endogene Interaktion gezeigt werden. Weitere Versuche werden zeigen ob diese Beobachtungen durch technische Probleme verursacht wurden oder ob es tatsächlich eine biologische Relevanz gibt. Außerdem zeigten die letzten Experimente, dass die p53 Stabilisierung wahrscheinlich nicht nur durch AMPK Aktivierung, sondern auch durch eine Destabilisierung von MDM2 selbst verursacht werden.Des Weiteren wurde das CRISPR/Cas9 System verwendet um eine stabile p53 knock-out Zelllinie zu generieren. Diese Zellen dienen als Kontrolle bei Xenotransplantationsexperimenten in Mäusen um die Rolle von Fasten auf die Leberkrebsentstehung zu untersuchen.The transcription factor p53 plays a pivotal role in tumor suppression. p53 can be activated by a variety of stress signals inducing downstream transcriptional programs. Despite the excessive study of p53 there is relative few information about its role in non-cancerous cells. We could show that, while mRNA levels stayed unchanged, p53 protein levels increased under starvation in murine liver and in cultured hepatocytes.The aim of this master thesis was to elucidate the mechanisms behind this p53 protein stabilization under starvation. Based on preliminary experiments, we hypothesized that the increased p53 levels are mediated by AMPK signaling. The AMPK complex controls the binding of p53 to its negative regulator MDM2 and thereby activating p53s transactivation function. To test this hypothesis immunoprecipitation (IP) and co-IP experiments were established. First, p53 and MDM2 were overexpressed in cell lines followed by IP. After optimizing protocol parameters a robust co-pull-down of ectopic p53 and ectopic MDM2 could be shown. These experiments were repeated also in HepG2 cells to investigate the endogenous interaction of p53 and MDM2 under starvation. The pull-down of p53 or MDM2 alone was successful, but endogenous interaction could only be shown partially. It remains to be determined if these observations are caused by technical problems or if it has biological relevance. Furthermore, latest findings suggest that the p53 stabilization might not be only an effect of AMPK activation but also due to destabilization of MDM2 protein level under starvation. Additionally, the CRISPR/Cas9 system was used to derive stable p53 knock-out HepG2 clones, who will be used as isogenic controls in xenotransplantation experiments in mice to investigate the role of starvation and p53 in hepatocellular carcinoma development.presented by Christoph Nössing, BScKarl-Franzens-Universität Graz, Masterarbeit, 2017(VLID)342711

50 years on and still very much alive: ‘apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics’

No abstract available

ATG7 is a haploinsufficient repressor of tumor progression and promoter of metastasis

The role of autophagy in cancer is complex. Both tumor-promoting and tumor-suppressive effects are reported, with tumor type, stage and specific genetic lesions dictating the role. This calls for analysis in models that best recapitulate each tumor type, from initiation to metastatic disease, to specifically understand the contribution of autophagy in each context. Here, we report the effects of deleting the essential autophagy gene Atg7 in a model of pancreatic ductal adenocarcinoma (PDAC), in which mutant KrasG12D and mutant Trp53172H are induced in adult tissue leading to metastatic PDAC. This revealed that Atg7 loss in the presence of KrasG12D/+ and Trp53172H/+ was tumor promoting, similar to previous observations in tumors driven by embryonic KrasG12D/+ and deletion of Trp53. However, Atg7 hemizygosity also enhanced tumor initiation and progression, even though this did not ablate autophagy. Moreover, despite this enhanced progression, fewer Atg7 hemizygous mice had metastases compared with animals wild type for this allele, indicating that ATG7 is a promoter of metastasis. We show, in addition, that Atg7+/− tumors have comparatively lower levels of succinate, and that cells derived from Atg7+/− tumors are also less invasive than those from Atg7+/+ tumors. This effect on invasion can be rescued by ectopic expression of Atg7 in Atg7+/− cells, without affecting the autophagic capacity of the cells, or by treatment with a cell-permeable analog of succinate. These findings therefore show that ATG7 has roles in invasion and metastasis that are not related to the role of the protein in the regulation of autophagy

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

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