Expressional analysis of disease-relevant signalling-pathways in primary tumours and metastasis of head and neck cancers

Head and neck squamous cell carcinoma (HNSCC) often metastasize to lymph nodes resulting in poor prognosis for patients. Unfortunately, the underlying molecular mechanisms contributing to tumour aggressiveness, recurrences, and metastasis are still not fully understood. However, such knowledge is key to identify biomarkers and drug targets to improve prognosis and treatments. Consequently, we performed genome-wide expression profiling of 15 primary HNSSCs compared to corresponding lymph node metastases and non-malignant tissue of the same patient. Differentially expressed genes were bioinformatically exploited applying stringent filter criteria, allowing the discrimination between normal mucosa, primary tumours, and metastases. Signalling networks involved in invasion contain remodelling of the extracellular matrix, hypoxia-induced transcriptional modulation, and the recruitment of cancer associated fibroblasts, ultimately converging into a broad activation of PI3K/AKT-signalling pathway in lymph node metastasis. Notably, when we compared the diagnostic and prognostic value of sequencing data with our expression analysis significant differences were uncovered concerning the expression of the receptor tyrosine kinases EGFR and ERBB2, as well as other oncogenic regulators. Particularly, upregulated receptor tyrosine kinase combinations for individual patients varied, implying potential compensatory and resistance mechanisms against specific targeted therapies. Collectively, we here provide unique transcriptional profiles for disease predictions and comprehensively analyse involved signalling pathways in advanced HNSCC

Funktionelle Analyse der onkologisch relevanten Protease Threonin-Aspartase 1

Krebs stellt eine der häufigsten Todesursachen in Europa dar. Grundlage für eine langfristige Verbesserung des Behandlungserfolgs ist ein molekulares Verständnis der Mechanismen, welche zur Krankheitsentstehung beitragen. In diesem Zusammenhang spielen Proteasen nicht nur eine wichtige Rolle, sondern stellen auch bei vielerlei Erkrankungen bereits anerkannte Zielstrukturen derzeitiger Behandlungsstrategien dar. Die Protease Threonin Aspartase 1 (Taspase1) spielt eine entscheidende Rolle bei der Aktivierung von Mixed Lineage Leukemia (MLL)-Fusionsproteinen und somit bei der Entstehung aggressiver Leukämien. Aktuelle Arbeiten unterstreichen zudem die onkologische Relevanz von Taspase1 auch für solide Tumore. Die Kenntnisse über die molekularen Mechanismen und Signalnetzwerke, welche für die (patho)biologischen Funktionen von Taspase1 verantwortlich sind, stellen sich allerdings noch immer als bruchstückhaft dar. Um diese bestehenden Wissenslücken zu schließen, sollten im Rahmen der Arbeit neue Strategien zur Inhibition von Taspase1 erarbeitet und bewertet werden. Zusätzlich sollten neue Einsichten in evolutionären Funktionsmechanismen sowie eine weitergehende Feinregulation von Taspase1 erlangt werden. Zum einen erlaubte die Etablierung und Anwendung eines zellbasierten Taspase1-Testsystem, chemische Verbindungen auf deren inhibitorische Aktivität zu testen. Überraschenderweise belegten solch zelluläre Analysen in Kombination mit in silico-Modellierungen eindeutig, dass ein in der Literatur postulierter Inhibitor in lebenden Tumorzellen keine spezifische Wirksamkeit gegenüber Taspase1 zeigte. Als mögliche Alternative wurden darüber hinaus Ansätze zur genetischen Inhibition evaluiert. Obwohl publizierte Studien Taspase1 als ααββ-Heterodimer beschreiben, konnte durch Überexpression katalytisch inaktiver Mutanten kein trans-dominant negativer Effekt und damit auch keine Inhibition des wildtypischen Enzyms beobachtet werden. Weiterführende zellbiologische und biochemische Analysen belegten erstmalig, dass Taspase1 in lebenden Zellen in der Tat hauptsächlich als Monomer und nicht als Dimer vorliegt. Die Identifizierung evolutionär konservierter bzw. divergenter Funktionsmechanismen lieferte bereits in der Vergangenheit wichtige Hinweise zur Inhibition verschiedenster krebsrelevanter Proteine. Da in Drosophila melanogaster die Existenz und funktionelle Konservierung eines Taspase1-Homologs postuliert wurde, wurde in einem weiteren Teil der vorliegenden Arbeit die evolutionäre Entwicklung der Drosophila Taspase1 (dTaspase1) untersucht. Obwohl Taspase1 als eine evolutionär stark konservierte Protease gilt, konnten wichtige Unterschiede zwischen beiden Orthologen festgestellt werden. Neben einem konservierten autokatalytischen Aktivierungsmechanismus besitzt dTaspase1 verglichen mit dem humanen Enzym eine flexiblere Substraterkennungs-sequenz, was zu einer Vergrößerung des Drosophila-spezifischen Degradoms führt. Diese Ergebnisse zeigen des Weiteren, dass zur Definition und Vorhersage des Degradoms nicht nur proteomische sondern auch zellbiologische und bioinformatische Untersuchungen geeignet und notwendig sind. Interessanterweise ist die differentielle Regulation der dTaspase1-Aktivität zudem auf eine veränderte intrazelluläre Lokalisation zurückzuführen. Das Fehlen von in Vertebraten hochkonservierten aktiven Kernimport- und nukleolären Lokalisationssignalen erklärt, weshalb dTaspase1 weniger effizient nukleäre Substrate prozessiert. Somit scheint die für die humane Taspase1 beschriebene Regulation von Lokalisation und Aktivität über eine Importin-α/NPM1-Achse erst im Laufe der Entwicklung der Vertebraten entstanden zu sein. Es konnte also ein bislang unbekanntes evolutionäres Prinzip identifiziert werden, über welches eine Protease einen Transport- bzw. Lokalisations-basierten Mechanismus zur Feinregulation ihrer Aktivität „von der Fliege zum Menschen“ nutzt. Eine weitere Möglichkeit zur dynamischen Funktionsmodulation bieten post-translationale Modifikationen (PTMs) der Proteinsequenz, zu welcher Phosphorylierung und Acetylierung zählen. Interessanterweise konnte für die humane Taspase1 über den Einsatz unabhängiger Methoden einschließlich massenspektrometrischer Analysen eine Acetylierung durch verschiedene Histon-Acetyltransferasen (HATs) nachgewiesen werden. Diese Modifikation erfolgt reversibel, wobei vor allem die Histon-Deacetylase HDAC1 durch Interaktion mit Taspase1 die Deacetylierung der Protease katalysiert. Während Taspase1 in ihrer aktiven Konformation acetyliert vorliegt, kommt es nach Deacetylierung zu einer Reduktion ihrer enzymatischen Aktivität. Somit scheint die Modulation der Taspase1-Aktivität nicht allein über intra-proteolytische Autoaktivierung, Transport- und Interaktionsmechanismen, sondern zudem durch post-translationale Modifikationen gesteuert zu werden. Zusammenfassend konnten im Rahmen dieser Arbeit entscheidende neue Einblicke in die (patho)biologische Funktion und Feinregulation der Taspase1 gewonnen werden. Diese Ergebnisse stellen nicht nur einen wichtigen Schritt in Richtung eines verbesserten Verständnis der „Taspase1-Biologie“, sondern auch zur erfolgreichen Inhibition und Bewertung der krebsrelevanten Funktion dieser Protease dar.Cancer represents the second leading cause of death in Europe. Key for the long-term improvement of clinical success is an understanding of the molecular mechanisms contributing to disease development and progression. In this context, proteases not only play an important role in (patho)biological processes, but are already clinically approved targets of current treatments. The protease threonine aspartase 1 (Taspase1) plays an essential role for the activation of Mixed Lineage Leukemia (MLL) fusionproteins and thus for the development of aggressive leukemias. Also, Taspase1-induced cleavage of the MLL protein plays an important physiological role in developmental and differentiation processes. In addition, recent insights strongly promote the oncological relevance of Taspase1 also for solid tumors. Besides MLL proteins a variety of Taspase1 bona fide target proteins have been identified and are predicted in different species. However, our knowledge about the molecular mechanisms being responsible for the (patho)biological functions of Taspase1, is still incomplete. Also, no effective Taspase1 inhibitor is available to date. In order to fill this existing knowledge gap, new Taspase1 interference strategies were developed and evaluated in the context of this thesis. Additionally, new insights into evolutionary and potential fine-regulation mechanisms of Taspase1 should be gained. The establishment and application of a cell-based Taspase1 assay enabled to test the inhibitory effect of chemical compounds on Taspase1´s activity. Surprisingly, cellular assay results as well as in silico modelling clearly demonstrated that previously postulated Taspase1 inhibitors were inactive in living tumor cells. As an alternative strategy, genetic inhibition of the protease was investigated. Although previous studies proposed that Taspase1 is active as an ααββ-heterodimer, overexpression of catalytic inactive mutants did not result in a trans dominant negative effect and thus, failed to inhibited the wildtype Taspase1 enzyme. Further biological and biochemical analyses proved for the first time that active Taspase1 exists predominantly as a monomer in living cells. Surprisingly, genetically enforced dimerization lead to inhibition of the protease suggesting to exploit the concept of chemical dimerizers as a potential inhibition strategy. In the past, the identification of evolutionary conserved or diverging functional mechanisms already provided important insights leading to the inhibition of different oncological relevant proteins. Since the existence and functional conservation of a Taspase1 homolog was postulated in Drosophila melanogaster, the function and evolutionary development of Drosophila Taspase1 (dTaspase1) was further examined. Although Taspase1 has been characterized as an evolutionary highly conserved protease, important differences could be demonstrated here for both orthologs. Besides a conserved autocatalytic activation mechanism depending on the essential nucleophile threonine195, dTaspase1 exhibits a more flexible substrate recognition sequence compared to human Taspase1 which leads to an enlarged degradome in Drosophila. Furthermore, the results demonstrated that for the definition and prediction of a degradome not only proteomic, but also biological as well as bioinformatic analyses are suitable and also necessary. Interestingly, the species-specific regulation of dTaspase1´s activity could be also attributed to differences in its intracellular localization. The lack of active nuclear and nucleolar targeting signals in dTaspase1, which are highly conserved in vertebrates, provides an explanation for the rather inefficient cleavage of nuclear substrates. Thus, the regulation of localization and activity via the importin-/NPM1-axis described for human Taspase1 has evolved during vertebrate development. Collectively, this thesis describes a hitherto unknown evolutionary principle how a protease exploits a transport-based mechanism to fine-tune its degradome and proteolytic activity “from fly to man”. Besides localization, post-translational modifications (PTMs) represent another mechanism allowing a dynamic regulation of protein functions. Important PTMs include phosphorylation and acetylation, which in contrast to kinases have not yet been identified for proteases. Interestingly, independent methods, including mass spectrometry analyses, demonstrated that Taspase1 is indeed acetylated by different histone acetyl transferases (HATs). This modification of lysine residues occurs reversible and deacetylation of Taspase1 is mediated by the binding of the histone deacetylase HDAC1. Functional analyses further indicate a post-translational fine-regulation of Taspase1’s proteolytic activity which has not been described for a protease so far. Whereas Taspase1 is acetylated in its active conformation, deacetylation results in decreased enzymatic activity. Thus, Taspase1´s activity could not only be regulated by intra-proteolytic self-activation, transport and interaction mechanisms, but also by post-translational modifications. These results enlarge our general understanding of Taspase1´s function, but additionally suggest to further investigate clinically approved HDAC inhibitors to rationally regulate Taspase1´s activity. In summary, essential new insights into the (patho)biological functions and regulation of Taspase1 could be gained during this thesis. These results do not only represent an important step towards an improved understanding of “Taspase1 biology”, but also for an effective inhibition and evaluation of its oncological relevance

Histone deacetylase inhibitors dysregulate DNA repair proteins and antagonize metastasis-associated processes

Purpose!#!We set out to determine whether clinically tested epigenetic drugs against class I histone deacetylases (HDACs) affect hallmarks of the metastatic process.!##!Methods!#!We treated permanent and primary renal, lung, and breast cancer cells with the class I histone deacetylase inhibitors (HDACi) entinostat (MS-275) and valproic acid (VPA), the replicative stress inducer hydroxyurea (HU), the DNA-damaging agent cis-platinum (L-OHP), and the cytokine transforming growth factor-β (TGFβ). We used proteomics, quantitative PCR, immunoblot, single cell DNA damage assays, and flow cytometry to analyze cell fate after drug exposure.!##!Results!#!We show that HDACi interfere with DNA repair protein expression and trigger DNA damage and apoptosis alone and in combination with established chemotherapeutics. Furthermore, HDACi disrupt the balance of cell adhesion protein expression and abrogate TGFβ-induced cellular plasticity of transformed cells.!##!Conclusion!#!HDACi suppress the epithelial-mesenchymal transition (EMT) and compromise the DNA integrity of cancer cells. These data encourage further testing of HDACi against tumor cells

Disease-relevant signalling-pathways in head and neck cancer : Taspase1’s proteolytic activity fine-tunes TFIIA function

Head and neck cancer (HNC) is the seventh most common malignancy in the world and its prevailing form, the head and neck squamous cell carcinoma (HNSCC), is characterized as aggressive and invasive cancer type. The transcription factor II A (TFIIA), initially described as general regulator of RNA polymerase II-dependent transcription, is part of complex transcriptional networks also controlling mammalian head morphogenesis. Posttranslational cleavage of the TFIIA precursor by the oncologically relevant protease Taspase1 is crucial in this process. In contrast, the relevance of Taspase1-mediated TFIIA cleavage during oncogenesis of HNSCC is not characterized yet. Here, we performed genome-wide expression profiling of HNSCC which revealed significant downregulation of the TFIIA downstream target CDKN2A. To identify potential regulatory mechanisms of TFIIA on cellular level, we characterized nuclear-cytoplasmic transport and Taspase1-mediated cleavage of TFIIA variants. Unexpectedly, we identified an evolutionary conserved nuclear export signal (NES) counteracting nuclear localization and thus, transcriptional activity of TFIIA. Notably, proteolytic processing of TFIIA by Taspase1 was found to mask the NES, thereby promoting nuclear localization and transcriptional activation of TFIIA target genes, such as CDKN2A. Collectively, we here describe a hitherto unknown mechanism how cellular localization and Taspase1 cleavage fine-tunes transcriptional activity of TFIIA in HNSCC

Overexpression of the catalytically impaired Taspase1 T234V or Taspase1 D233A variants does not have a dominant negative effect in T(4;11) leukemia cells.

BACKGROUND: The chromosomal translocation t(4;11)(q21;q23) is associated with high-risk acute lymphoblastic leukemia of infants. The resulting AF4•MLL oncoprotein becomes activated by Taspase1 hydrolysis and is considered to promote oncogenic transcriptional activation. Hence, Taspase1's proteolytic activity is a critical step in AF4•MLL pathophysiology. The Taspase1 proenzyme is autoproteolytically processed in its subunits and is assumed to assemble into an αββα-heterodimer, the active protease. Therefore, we investigated here whether overexpression of catalytically inactive Taspase1 variants are able to interfere with the proteolytic activity of the wild type enzyme in AF4•MLL model systems. METHODOLOGY/FINDINGS: The consequences of overexpressing the catalytically dead Taspase1 mutant, Taspase1(T234V), or the highly attenuated variant, Taspase1(D233A), on Taspase1's processing of AF4•MLL and of other Taspase1 targets was analyzed in living cancer cells employing an optimized cell-based assay. Notably, even a nine-fold overexpression of the respective Taspase1 mutants neither inhibited Taspase1's cis- nor trans-cleavage activity in vivo. Likewise, enforced expression of the α- or β-subunits showed no trans-dominant effect against the ectopically or endogenously expressed enzyme. Notably, co-expression of the individual α- and β-subunits did not result in their assembly into an enzymatically active protease complex. Probing Taspase1 multimerization in living cells by a translocation-based protein interaction assay as well as by biochemical methods indicated that the inactive Taspase1 failed to assemble into stable heterocomplexes with the wild type enzyme. CONCLUSIONS: Collectively, our results demonstrate that inefficient heterodimerization appears to be the mechanism by which inactive Taspase1 variants fail to inhibit wild type Taspase1's activity in trans. Our work favours strategies targeting Taspase1's catalytic activity rather than attempts to block the formation of active Taspase1 dimers to interfere with the pathobiological function of AF4•MLL

Probing Taspase1 multimerization in living cells.

<p><b>A.</b> Heterocomplex formation of Taspase1 and Taspase1 variants shown by co-immunoprecipitation (IP). IPs of 293T cell extracts co-transfected with the indicated expression constructs were carried out using α-GFP Ab-coated magnetic beads and μ-MACS columns. Precipitated proteins were identified by immunoblot using the indicated antibodies. Input: Total amount of cell lysate. IP: immunoprecipitated proteins. *: GFP-degradation products <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034142#pone.0034142-Landgraf1" target="_blank">[33]</a>. <b>B.</b> Principle of the translocation based protein-protein interaction assay. The Tasp_Cyt fusion is composed of GFP, Taspase1 and a NES (?) and thus, continuously shuttling between the nucleus and the cytoplasm. The red-fluorescent Taspase1 variants (Tasp-mCherry prey) accumulate at the nucleus/nucleolus. Upon efficient protein-protein interaction, the GFP-tagged cytoplasmic Tasp_Cyt co-localizes with the Tasp-mCherry prey to the nucleus/nucleolus in living cells. <b>C.</b> Localization of indicated proteins in the absence of potential interaction partners. <b>D.</b> Neither co-expression of WT nor inactive Taspase1 variants resulted in strong nuclear/nucleolar translocation of Tasp_Cyt. Co-expression of NPM1-RFP, known to strongly interact with Taspase1, triggered nuclear/nucleolar translocation of Tasp_Cyt (positive control). In contrast, co-expression of the non-interacting nucleolar RevM10BL-RFP protein showed no effect (negative control) as visualized by fluorescence microscopy in living HeLa transfectants. Scale bars, 10 µm.</p