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Die Deregulation des Transkriptionsfaktors Myc ist ein zentraler Mechanismus in der kolorektalen Karzinogenese. Die Myc-Deletion in Tumormodellen hemmt das Wachstum von Kolonkarzinomen, somit stellt die Inaktivierung von Myc einen Ansatzpunkt in der Behandlung von kolorektalen Tumoren dar. Die direkte Inhibition von Myc ist schwierig, da Myc keine katalytische Aktivität besitzt und stattdessen für die Myc-Funktion nötige Protein-Protein- oder Protein-DNA-Interaktionen angegriffen werden müssen. Die E3-Ubiquitin-Ligase Huwe1 interagiert sowohl mit Myc als auch mit dem Myc-interagierenden Protein Miz1 und ist im Kolonkarzinom überexprimiert. Huwe1 ubiquitiniert Myc und induziert darüber dessen Transaktivierungsfunktion. Die Inaktivierung von Huwe1 ist somit eine vielversprechende Möglichkeit für die Inhibition der Myc-Funktion und die Therapie des Kolonkarzinoms.
In dieser Arbeit wird mittels shRNA-vermittelter Depletion von Huwe1 in Zellkulturexperimenten gezeigt, dass Huwe1 für die Proliferation von Kolonkarzinomzelllinien und für die Transaktivierung von Myc-Zielgenen benötigt wird. Mit zwei von Boehringer Ingelheim identifizierten niedermolekularen Huwe1-Inhibitoren (BI8622 und BI8626) ist es möglich, die Huwe1-Funktion spezifisch in Zellen zu blockieren. Die Huwe1-Inhibitoren induzieren einen Proliferationsarrest in kolorektalen Karzinomzelllinien, wohingegen die Substanzen auf embryonale Stammzellen keine Auswirkungen haben. Die Inaktivierung von Huwe1 führt zu einer Akkumulation von Miz1 an Promotoren Myc-aktivierter Zielgene und darüber zu einer vermehrten Bildung repressiver Myc/Miz1-Komplexe, was mit einer Deacetylierung von Histon H3 und einer transkriptionellen Repression Myc-gebundener Gene assoziiert ist. Miz1 akkumuliert nach Huwe1-Inhibition ebenso an direkten Miz1-Zielgenen, deren Expression bleibt aber unbeeinflusst. Diese Daten weisen darauf hin, dass eine kontinuierliche Degradierung von Miz1 durch Huwe1 zur Transaktivierung von Myc-Zielgenen in Kolonkarzinomzellen nötig ist. Damit wurde ein neuer Mechanismus identifiziert, über den Huwe1 die Myc-Transaktivierung reguliert und der eine tumorzellspezifische Repression der Myc-Funktion mit Hilfe von Huwe1-Inhibitoren ermöglicht.
The Notch signaling pathway is crucial for mammalian heart development. It controls cell-fate decisions, coordinates patterning processes and regulates proliferation and differentiation. Critical Notch effectors are Hey bHLH transcription factors (TF) that are expressed in atrial (Hey1) and ventricular (Hey2) cardiomyocytes (CM) and in the developing endocardium (Hey1/2/L). The importance of Hey proteins for cardiac development is demonstrated by knockout (KO) mice, which suffer from lethal cardiac defects, such as ventricular septum defects (VSD), valve defects and cardiomyopathy. Despite this clear functional relevance, little is known about Hey downstream targets in the heart and the molecular mechanism by which they are regulated.
Here, I use a cell culture system with inducible Hey1, Hey2 or HeyL expression to study Hey target gene regulation in HEK293 cells, in murine embryonic stem cells (ESC) and in ESC derived CM. In HEK293 cells, I could show that genome wide binding sites largely overlap between all three Hey proteins, but HeyL has many additional binding sites that are not bound by Hey1 or Hey2. Shared binding sites are located close to transcription start sites (TSS) where Hey proteins preferentially bind to canonical E boxes, although more loosely defined modes of binding exist. Additional sites only bound by HeyL are more scattered across the genome. The ability of HeyL to bind these sites depends on the C-terminal part of the protein. Although there are genes which are differently regulated by HeyL, it is unclear whether this regulation results from binding of additional sites by HeyL.
Additionally, Hey target gene regulation was studied in ESC and differentiated CM, which are more relevant for the observed cardiac phenotypes. ESC derived CM contract in culture and are positive for typical cardiac markers by qRT PCR and staining. According to these markers differentiation is unaffected by prolonged Hey1 or Hey2 overexpression. Regulated genes are largely redundant between Hey1 and Hey2. These are mainly other TF involved in e.g. developmental processes, apoptosis, cell migration and cell cycle. Many target genes are cell type specifically regulated causing a shift in Hey repression of genes involved in cell migration in ESC to repression of genes involved in cell cycle in CM.
The number of Hey binding sites is reduced in CM and HEK293 cells compared to ESC, most likely due to more regions of dense chromatin in differentiated cells. Binding sites are enriched at the proximal promoters of down-regulated genes, compared to up-or non-regulated genes. This indicates that up-regulation primarily results from indirect effects, while down-regulation is the direct results of Hey binding to target promoters. The extent of repression generally correlates with the amount of Hey binding and subsequent recruitment of histone deacetylases (Hdac) to target promoters resulting in histone H3 deacetylation.
However, in CM the repressive effect of Hey binding on a subset of genes can be annulled, likely due to binding of cardiac specific activators like Srf, Nkx2-5 and Gata4. These factors seem not to interfere with Hey binding in CM, but they recruit histone acetylases such as p300 that may counteract Hey mediated histone H3 deacetylation. Such a scenario explains differential regulation of Hey target genes between ESC and CM resulting in gene and cell-type specific regulation.
In contrast to c-Myc, a deregulated expression of the MYCN gene is restricted to human neuroendocrine tumours. In most cases, the excessive activity of N-Myc results from a MYCN amplification. In neuroblastoma, amplification of MYCN is a predictor of poor prognosis and resistance to therapy. The inability to target the N-Myc protein directly necessitates the search for alternative targets. This project aimed at identifying genes specifically required for growth and survival of cells that express high levels of N-Myc using high-throughput shRNA screening combined with next generation sequencing. The identification and analysis of these genes will shed light on functional interaction partners of N-Myc.
We screened a shRNA library containing 18,327 shRNAs and identified 148 shRNAs, which were selectively depleted in the presence of active N-Myc. In addition, shRNAs targeting genes that are involved in p53 and ARF turnover and apoptosis were depleted in the cell population during the screen. These processes are known to affect N-Myc-mediated apoptosis. Consequently, these results biologically validated the screen. The 148 shRNAs that showed a significant synthetic lethal interaction with high levels of N-Myc expression were further analysed using the bioinformatics program DAVID. We found an enrichment of shRNAs that target genes involved in specific biological processes. For example, we validated synthetic lethal interactions for genes such as, THOC1, NUP153 and LARP7, which play an important role in the process of RNA polymerase II-mediated transcription elongation. We also validated genes that are involved in the neddylation pathway.
In the screen we identified Cullin 3, which is a component of the BTB-CUL3-Rbx1 ubiquitin ligase that is involved in the turnover of Cyclin E. Depletion of cullin 3 and activation of N-Myc was found to synergistically increase Cyclin E expression to supraphysiological levels, inducing S-phase arrest and a strong DNA damage response.
Together with results from a proteomics analysis of N-Myc associated proteins, our results lead us to the following hypothesis: In a neuroblastoma cell, the high levels of N-Myc result in a conflict between RNA polymerase II and the replication machinery during S-phase. The newly identified interaction partners of N- Myc are required to solve this conflict. Consequently, loss of the interaction leads to a massive DNA damage and the induction of apoptosis. In addition, inhibition or depletion of the essential components of the neddylation pathway also results in an unresolvable problem during S-phase.