Molekularer Wirkmechanismus und Resistenzmechanismen der Mikrotubuli- stabilisierenden Substanz Sagopilone

Microtubule-stabilizing drugs, like taxanes are frequently used in the clinic for the treatment of lung, breast, ovary, head and neck carcinomas and melanomas. However, some tumors develop resistance which impairs the success of chemotherapy. Sagopilone, a novel microtubule-stabilizing drug from the class of epothilones, was selected to overcome the limitations associated with conventional tubulin-binding agents. The aim of this study was to investigate the mode of action of sagopilone and to elucidate molecular mechanisms of resistance. Sagopilone strongly inhibited cellular proliferation of various tumor cell lines in vitro. In contrast to paclitaxel, sagopilone inhibited the growth of multi drug transporter (MDR1) overexpressing cell lines, a common mechanism of resistance to paclitaxel. Depending on the drug concentration, Sagopilone mainly induced two different phenotypes. Low concentration treatment (2.5 nM) resulted in aberrant cell division, aneuploidy and cell cycle arrest in G1 whereas high concentration treatment (40 nM) led to mitotic arrest. These effects were similar to those observed with paclitaxel. However, unlike paclitaxel, sagopilone disturbed division of tumors cells already at the low concentration of 0.5 nM. The gene expression profiles of low and high concentration phenotypes were substantially different from each other. Low concentration sagopilone led to p53 (TP53) transactivation in A549 lung cancer cells leading to cell cycle arrest. Incubation with high concentration of sagopilone was associated with upregulation of several genes involved in spindle assembly checkpoint and mitosis, indicating mitotic arrest. For both phenotypes differences in the ability to undergo apoptosis were observed. Low concentration treated A549 cells showed markedly diminished apoptosis induction capability when compared to the high concentration sagopilone- treated cells. The knock-down of p21 (CDKN1A) and p53 in A549 cells led to abrogation of G1 arrest induced by low concentration sagopilone and to further progression of the cell cycle. This markedly increased the rate of apoptosis after low concentration sagopilone treatment. The relevance of the p53 transactivation was further demonstrated in in vivo A549 xenograft tumors which only moderately responded to sagopilone. These tumors showed the low concentration gene expression pattern similar to A549 cells in vitro as distinguished through upregulation of p53 target genes like p21. The gene expression of the cell cycle inhibitor p21 was induced more strongly in the four p53 wild type cell lines than the four p53 mutated cell lines. However, the p53 mutational status of the cell lines did not correlate with the sensitivity to sagopilone-induced apoptosis, indicating that additional mechanisms of resistance may exist. Herein it was demonstrated that paclitaxel and sagopilone have a similar mechanism of action, but the efficacy of sagopilone to inhibit tumor cell proliferation was higher. Sagopilone induced aberrant cell division and G1 arrest at low concentrations and mitotic arrest at high concentrations. The upregulation of p53 target genes and the induction of G1 arrest in response to low concentrations of sagopilone, as shown in A549 cells, may represent one cellular mechanism of resistance to the drug. The presented data could provide the basis for more extensive research as to whether the p53 mutational status can be used as a stratification marker for sagopilone response in the clinic and whether targeting p53 could increase the response to sagopilone.Mikrotubuli-stabilisierende Substanzen, wie beispielsweise Taxane, werden in der Klink häufig zur Behandlung von Lungen-, Brust-, Ovarial-, Kopf-Hals- Karzinomen oder Melanomen eingesetzt. Unter der Behandlung mit Taxanen entwickeln einige Tumore jedoch Resistenzen gegen diese Medikamente, was den Erfolg der Chemotherapie beeinträchtigt. Sagopilone, eine neue Mikrotubuli- stabilisierende Verbindung aus der Klasse der Epothilone, wurde entwickelt, um die Nachteile herkömmlicher Tubulin-bindender Substanzen zu überwinden. In dieser Doktorarbeit sollte die Wirkungsweise von Sagopilone auf Tumorzellen aufgeklärt werden und mögliche Resistenzmechanismen der Tumorzellen gegen Sagopilone untersucht werden. In vitro wird die Proliferation von verschiedenen Tumorzellen durch Sagopilone stark inhibiert. Im Gegensatz zu Paclitaxel hemmt Sagopilone auch die Proliferation von MDR1 (multi drug transporter) überexprimierenden Zellen, einem bekannten Resistenzmechanismus der Tumorzellen gegen Paclitaxel. Sagopilone induzierte zwei unterschiedliche Phänotypen. Geringe Konzentrationen lösten eine anomale Zellteilung aus, die zu Aneuploidie und Zellzyklusarrest in der G1 Phase führte. Hohe Konzentrationen führten zu einem Arrest des Zellzyklus in der Mitose. Diese Effekte sind denen Paclitaxels sehr ähnlich. Im Gegensatz zu Paclitaxel war bei Sagopilone jedoch schon bei sehr geringen Konzentrationen (0.5 nM) eine antiproliferative Wirkung festzustellen. Geringe und hohe Konzentrationen Sagopilone zeigten zudem ein unterschiedliches Genexpressionsprofil. Geringe Konzentrationen Sagopilone (2.5 nM) führten in den Lungenkarzinom Zellen A549 zu einer transkriptionellen Aktivierung von p53 (TP53) kontrollierten Genen. Hohe Konzentrationen Sagopilone (40 nM) führten zu einer Hochregulation von Genen, die im spindle assembly checkpoint und der Mitose eine Rolle spielen. Diese Phänotypen zeigten eine unterschiedlich starke Apoptose-Auslösung in A549 Zellen nach Sagopilone Behandlung. A549 Zellen, die mit einer geringen Konzentration Sagopilone behandelt wurde, wiesen signifikant geringere Apoptose-Raten auf, verglichen mit A549 Zellen, die mit hohen Konzentrationen Sagopilone behandelt wurden. Die Herunterregulation von p21 (CDKN1A) und p53 mittels shRNAs in A549 Zellen führte zur Aufhebung des G1 Zellzyklusarrests nach Behandlung mit geringen Konzentrationen Sagopilone. Dieser Effekt hatte signifikant höheren Apoptose-Raten von A549 Zellen nach der Behandlung mit geringen Konzentrationen Sagopilone zur Folge. Die Bedeutung der transkriptionellen p53 Aktivierung konnte zusätzlich an einem A549 Xenograft Mausmodell gezeigt werden, welches nur einen geringfügigen Rückgang im Tumorwachstum nach Sagopilone Gabe zeigte. Das Genexpressionsprofil dieser Tumore entsprach dem der A549 Zellen, die in vitro mit geringen Konzentrationen Sagopilone behandelt wurden und eine Apoptose-Resistenz aufwiesen. Eine unterschiedlich starke Induktion der Genexpression von p21 konnte in 4 verschieden Zelllinen mit normalem p53 im Gegensatz zu 4 Zelllinen mit mutiertem p53 festgestellt werden. Es konnte allerdings kein Zusammenhang zwischen der p21 Induktion und der Apoptose-Auslösung von p53 mutierten und normalen Zelllinen gefunden werden. In dieser Arbeit konnte gezeigt werden, dass Sagopilone und Paclitaxel einen ähnlichen Wirkmechanismus haben, die Effizienz von Sagopilone jedoch höher ist. Sagopilone induziert eine anomale Zellteilung und G1 Arrest bei geringen Konzentrationen und mitotischen Arrest bei hohen Konzentrationen. Die transkriptionelle Aktivierung von p53 und der G1 Arrest nach geringen Sagopilone Konzentrationen stellen einen möglichen Resistenzmechanismus dar. Die gezeigten Daten bilden eine Grundlage für weitergehende Forschungen, ob der p53 Status von Tumoren als Stratifikationsmarker für die Wirkung von Sagopilone benutzt werden kann

Molecular mode of action and role of TP53 in the sensitivity to the Novel epothilone sagopilone (ZK-EPO) in A549 non-small cell lung cancer cells

Sagopilone, an optimized fully synthetic epothilone, is a microtubule-stabilizing compound that has shown high in vitro and in vivo activity against a broad range of human tumor models. We analyzed the differential mechanism of action of sagopilone in non-small cell lung cancer cell lines in vitro. Sagopilone inhibited proliferation of non-small cell lung cancer cell lines at lower nanomolar concentration. The treatment with sagopilone caused strong disturbances of cellular cytoskeletal organization. Two concentration-dependent phenotypes were observed. At 2.5 nM sagopilone or 4 nM paclitaxel an aneuploid phenotype occur whereas a mitotic arrest phenotype was induced by 40 nM sagopilone or paclitaxel. Interestingly, treatment with 2.5 nM of sagopilone effectively inhibited cell proliferation, but - compared to high concentrations (40 nM) - only marginally induced apoptosis. Treatment with a high versus a low concentration of sagopilone or paclitaxel regulates a non-overlapping set of genes, indicating that both phenotypes substantially differ from each other. Genes involved in G2/M phase transition and the spindle assembly checkpoint, like Cyclin B1 and BUBR1 were upregulated by treatment with 40 nM sagopilone. Unexpectedly, also genes involved in DNA damage response were upregulated under that treatment. In contrast, treatment of A549 cells with a low concentration of sagopilone revealed an upregulation of direct transcriptional target genes of TP53, like CDKN1A, MDM2, GADD45A, FAS. Knockdown of TP53, which inhibited the transcriptional induction of TP53 target genes, led to a significant increase in apoptosis induction in A549 cells when treated with a low concentration of sagopilone. The results indicate that activation of TP53 and its downstream effectors like CDKN1A by low concentrations of sagopilone is responsible for the relative apoptosis resistance of A549 cells and might represent a mechanism of resistance to sagopilone

Evaluation of activity and combination strategies with the microtubule-targeting drug sagopilone in breast cancer cell lines

Sagopilone, a fully synthetic epothilone, is a microtubule-stabilizing agent optimized for high in vitro and in vivo activity against a broad range of tumor models, including those resistant to paclitaxel and other systemic treatments. Sagopilone development is accompanied by translational research studies to evaluate the molecular mode of action, to recognize mechanisms leading to resistance, to identify predictive response biomarkers, and to establish a rationale for combination with different therapies. Here, we profiled sagopilone activity in breast cancer cell lines. To analyze the mechanisms of mitotic arrest and apoptosis and to identify additional targets and biomarkers, an siRNA-based RNAi drug modifier screen interrogating 300 genes was performed in four cancer cell lines. Defects of the spindle assembly checkpoint (SAC) were identified to cause resistance against sagopilone-induced mitotic arrest and apoptosis. Potential biomarkers for resistance could therefore be functional defects like polymorphisms or mutations in the SAC, particularly in the central SAC kinase BUB1B. Moreover, chromosomal heterogeneity and polyploidy are also potential biomarkers of sagopilone resistance since they imply an increased tolerance for aberrant mitosis. RNAi screening further demonstrated that the sagopilone-induced mitotic arrest can be enhanced by concomitant inhibition of mitotic kinesins, thus suggesting a potential combination therapy of sagopilone with a KIF2C (MCAK) kinesin inhibitor. However, the combination of sagopilone and inhibition of the prophase kinesin KIF11 (EG5) is antagonistic, indicating that the kinesin inhibitor has to be highly specific to bring about the required therapeutic benefit

Molecular Mode of Action and Role of TP53 in the Sensitivity to the Novel Epothilone Sagopilone (ZK-EPO) in A549 Non-Small Cell Lung Cancer Cells

Sagopilone, an optimized fully synthetic epothilone, is a microtubule-stabilizing compound that has shown high in vitro and in vivo activity against a broad range of human tumor models. We analyzed the differential mechanism of action of sagopilone in non-small cell lung cancer cell lines in vitro. Sagopilone inhibited proliferation of non-small cell lung cancer cell lines at lower nanomolar concentration. The treatment with sagopilone caused strong disturbances of cellular cytoskeletal organization. Two concentration-dependent phenotypes were observed. At 2.5 nM sagopilone or 4 nM paclitaxel an aneuploid phenotype occur whereas a mitotic arrest phenotype was induced by 40 nM sagopilone or paclitaxel. Interestingly, treatment with 2.5 nM of sagopilone effectively inhibited cell proliferation, but - compared to high concentrations (40 nM) - only marginally induced apoptosis. Treatment with a high versus a low concentration of sagopilone or paclitaxel regulates a non-overlapping set of genes, indicating that both phenotypes substantially differ from each other. Genes involved in G2/M phase transition and the spindle assembly checkpoint, like Cyclin B1 and BUBR1 were upregulated by treatment with 40 nM sagopilone. Unexpectedly, also genes involved in DNA damage response were upregulated under that treatment. In contrast, treatment of A549 cells with a low concentration of sagopilone revealed an upregulation of direct transcriptional target genes of TP53, like CDKN1A, MDM2, GADD45A, FAS. Knockdown of TP53, which inhibited the transcriptional induction of TP53 target genes, led to a significant increase in apoptosis induction in A549 cells when treated with a low concentration of sagopilone. The results indicate that activation of TP53 and its downstream effectors like CDKN1A by low concentrations of sagopilone is responsible for the relative apoptosis resistance of A549 cells and might represent a mechanism of resistance to sagopilone