Functional analysis for the inhibition of apoptosis in mitotic cells

Die Zelle repliziert ihre DNA während der S-Phase und segregiert sie dann später in der M Phase des Zellzykluses. Kommt es während dieser Prozesse zu DNA Schädigungen, arretiert die Zelle den Zellzyklus mit Hilfe spezifischer Kontrollmechanismen und versucht den Schaden zu beheben. Der DNA Kontrollpunkt wird bei DNA Schädigungen aktiviert, um mit Hilfe der DNA Reparatur den Schaden zu beheben und somit dafür zu sorgen, dass die DNA fehlerfrei repliziert werden kann. Der zweite Zellzyklus Kontrollpunkt, der Spindel Kontrollpunkt, stellt sicher, dass die Chromosomen während der M Phase unter gleicher Spannung innerhalb der Äquatorialplatte der Metaphase Spindel angeordnet sind. Kann in der Zeit, in der der Zellzyklus Kontrollpunkt aktiv ist, der Schaden nicht behoben werden, so wird Apoptose ausgelöst und die Zelle wird aus dem Zellverband entfernt. Krebszellen haben Strategien entwickelt, Zellzyklus Kontrollpunkte zu umgehen und darüber hinaus normalen Mechanismen der Apoptose zu entkommen. Die genauen molekularen Vorgänge der Deregulierung der Apoptose sind weitestgehend unaufgeklärt. Procaspase 8 ist ein wichtiges Schlüsselenzym des extrinsischen Apoptose Signalweges. Der extrinsische Signalweg wird extrazellulär über die Bindung von Liganden an ihre korrespondierenden Rezeptoren ausgelöst. In dieser Studie wird gezeigt, dass Procaspase 8 an Ser-387 in vitro als auch in vivo von Cdk1/Cyclin B1 phosphoryliert wird. Darüber hinaus zeigt diese Phosphorylierungsstelle die typische Struktur einer Bindungsstelle für Plk1, einer weiteren mitotischen Kinase. Die Interaktion von Procaspase 8 mit Cdk1/Cyclin B1 wird über die DE Domäne („death-effector-domain“ DED) von Procaspase 8 vermittelt. Wird Procaspase 8 an Ser 387 zu Alanin (S387A) mutiert, so wird die Phosphorylierung durch Cdk1/Cyclin B1 fast vollständig verhindert. Wird zudem diese Mutante (S387A) in humanen Krebszellen überexprimiert, so hemmt dies die Apoptose nach Stimulation des Fas Rezeptors. Wird umgekehrt Cyclin B1 mittels RNA Interferenz depletiert und dadurch Cdk1 nicht aktiviert, wird extrinsische Apoptose verstärkt. Diese Studie zeigt erstmals eine gezielte Inhibierung des extrinsischen Apoptose Signalweges durch mitotische Kinasen und schlägt ein Modell vor, in dem Serin/Threonin Kinasen extrinsische Apoptose inhibieren und somit der Tumorzelle ermöglichen, der Apoptose zu entkommen. Darüber hinaus wird ein neuartiger Mechanismus der Inhibition der autokatalytischen Spaltung von Procaspase 8 durch eine mitotische Kinase gezeigt.Caspase activation is a hallmark of apoptosis. However, the molecular mechanisms underlying the regulation of caspase-8 activation within the extrinsic death pathway are not well understood. In this study, we demonstrate that procaspase-8 is phosphorylated in mitotic cells by Cdk1/cyclin B1 on Ser-387, which is located at the N-terminus of the catalytic subunit p10. This phosphorylation of procaspase-8 on Ser-387 occurs in cancer cell lines as well as in primary breast tissues and lymphocytes. Furthermore, RNAi-mediated silencing of cyclin B1 or treatment with the Cdk1 inhibitor RO-3306 enhances the Fas-mediated activation and processing of procaspase-8 in mitotic cells. A non-phosphorylatable procaspase-8(S387A) facilitates Fas-induced apoptosis during mitosis. Our findings suggest that Cdk1/cyclin B1 activity shields human cells against extrinsic death stimuli and unravel the molecular details of the crosstalk between cell cycle and extrinsic apoptotic pathways. Finally, this new mechanism may also contribute to tumorigenesis

Tumor inhibition by genomically integrated inducible RNAi-cassettes

RNA interference (RNAi) has emerged as a powerful tool to induce loss-of-function phenotypes by post-transcriptional silencing of gene expression. In this study we wondered whether inducible RNAi-cassettes integrated into cellular DNA possess the power to trigger neoplastic growth. For this purpose inducible RNAi vectors containing tetracycline (Tet)-responsive derivatives of the H1 promoter for the conditional expression of short hairpin RNA (shRNA) were used to target human polo-like kinase 1 (Plk1), which is overexpressed in a broad spectrum of human tumors. In the absence of doxycycline (Dox) HeLa clones expressing TetR, that carry the RNAi-cassette stably integrated, exhibited no significant alteration in Plk1 expression levels. In contrast, exposure to Dox led to marked downregulation of Plk1 mRNA to 3% and Plk1 protein to 14% in cell culture compared to mismatch shRNA/Plk1-expressing cells. As a result of Plk1 depletion cell proliferation decreased to 17%. Furthermore, for harnessing RNAi for silencing disease-related genes in vivo we transplanted inducible RNAi-HeLa cells onto nude mice. After administration of Dox knockdown of Plk1 expression was observed correlating to a significant inhibition of tumor growth. Taken together, our data revealed that genomically integrated RNAi-elements are suitable to hamper tumor growth by conditional expression of shRNA

Toxicity modelling of Plk1-targeted therapies in genetically engineered mice and cultured primary mammalian cells

High attrition rates of novel anti-cancer drugs highlight the need for improved models to predict toxicity. Although polo-like kinase 1 (Plk1) inhibitors are attractive candidates for drug development, the role of Plk1 in primary cells remains widely unexplored. Therefore, we evaluated the utility of an RNA interference-based model to assess responses to an inducible knockdown (iKD) of Plk1 in adult mice. Here we show that Plk1 silencing can be achieved in several organs, although adverse events are rare. We compared responses in Plk1-iKD mice with those in primary cells kept under controlled culture conditions. In contrast to the addiction of many cancer cell lines to the non-oncogene Plk1, the primary cells' proliferation, spindle assembly and apoptosis exhibit only a low dependency on Plk1. Responses to Plk1-depletion, both in cultured primary cells and in our iKD-mouse model, correspond well and thus provide the basis for using validated iKD mice in predicting responses to therapeutic interventions

Cdk1/Cyclin B1 Controls Fas-Mediated Apoptosis by Regulating Caspase-8 Activity▿

Caspase activation is a hallmark of apoptosis. However, the molecular mechanisms underlying the regulation of caspase-8 activation within the extrinsic death pathway are not well understood. In this study, we demonstrate that procaspase-8 is phosphorylated in mitotic cells by Cdk1/cyclin B1 on Ser-387, which is located at the N terminus of the catalytic subunit p10. This phosphorylation of procaspase-8 on Ser-387 occurs in cancer cell lines, as well as in primary breast tissues and lymphocytes. Furthermore, RNA interference-mediated silencing of cyclin B1 or treatment with the Cdk1 inhibitor RO-3306 enhances the Fas-mediated activation and processing of procaspase-8 in mitotic cells. A nonphosphorylatable procaspase-8 (S387A) facilitates Fas-induced apoptosis during mitosis. Our findings suggest that Cdk1/cyclin B1 activity shields human cells against extrinsic death stimuli and unravel the molecular details of the cross talk between cell cycle and extrinsic apoptotic pathways. Finally, this new mechanism may also contribute to tumorigenesis

A dimerization-dependent mechanism regulates enzymatic activation and nuclear entry of PLK1

Polo-like kinase 1 (PLK1) is a crucial regulator of cell cycle progression. It is established that the activation of PLK1 depends on the coordinated action of Aurora-A and Bora. Nevertheless, very little is known about the spatiotemporal regulation of PLK1 during G2, specifically, the mechanisms that keep cytoplasmic PLK1 inactive until shortly before mitosis onset. Here, we describe PLK1 dimerization as a new mechanism that controls PLK1 activation. During the early G2 phase, Bora supports transient PLK1 dimerization, thus fine-tuning the timely regulated activation of PLK1 and modulating its nuclear entry. At late G2, the phosphorylation of T210 by Aurora-A triggers dimer dissociation and generates active PLK1 monomers that support entry into mitosis. Interfering with this critical PLK1 dimer/monomer switch prevents the association of PLK1 with importins, limiting its nuclear shuttling, and causes nuclear PLK1 mislocalization during the G2-M transition. Our results suggest a novel conformational space for the design of a new generation of PLK1 inhibitors

Synthetic lethality in CCNE1-amplified high grade serous ovarian cancer through combined inhibition of Polo-like kinase 1 and microtubule dynamics

The taxanes are effective microtubule-stabilizing chemotherapy drugs that inhibit mitosis, induce apoptosis, and produce regression in a fraction of cancers that arise at many sites including the ovary. Novel therapeutic targets that augment taxane effects are needed to improve clinical chemotherapy response in CCNE1-amplified high grade serous ovarian cancer (HGSOC) cells. In this study, we conducted an siRNA-based kinome screen to identify modulators of mitotic progression in CCNE1-amplified HGSOC cells that may influence clinical paclitaxel response. PLK1 is overexpressed in many types of cancer, which correlates with poor prognosis. Here, we identified a novel synthetic lethal interaction of the clinical PLK1 inhibitor BI6727 and the microtubule-targeting drug paclitaxel in HGSOC cell lines with CCNE1-amplification and elucidated the underlying molecular mechanisms of this synergism. BI6727 synergistically induces apoptosis together with paclitaxel in different cell lines including a patient-derived primary ovarian cancer culture. Moreover, the inhibition of PLK1 reduced the paclitaxel-induced neurotoxicity in a neurite outgrowth assay. Mechanistically, the combinatorial treatment with BI6727/paclitaxel triggers mitotic arrest, which initiates mitochondrial apoptosis by inactivation of anti-apoptotic BCL-2 family proteins, followed by significant loss of the mitochondrial membrane potential and activation of caspase-dependent effector pathways. This conclusion is supported by data showing that BI6727/paclitaxel-co-treatment stabilizes FBW7, a component of SCF-type ubiquitin ligases that bind and regulate key modulators of cell division and growth including MCL-1 and Cyclin E. This identification of a novel synthetic lethality of PLK1 inhibitors and a microtubule-stabilizing drug has important implications for developing PLK1 inhibitor-based combination treatments in CCNE1-amplified HGSOC cells

Ligand stimulation of CD95 induces activation of Plk3 followed by phosphorylation of caspase-8

Upon interaction of the CD95 receptor with its ligand, sequential association of the adaptor molecule FADD (MORT1), pro-forms of caspases-8/10, and the caspase-8/10 regulator c-FLIP leads to the formation of a death-inducing signaling complex. Here, we identify polo-like kinase (Plk) 3 as a new interaction partner of the death receptor CD95. The enzymatic activity of Plk3 increases following interaction of the CD95 receptor with its ligand. Knockout (KO) or knockdown of caspase-8, CD95 or FADD prevents activation of Plk3 upon CD95 stimulation, suggesting a requirement of a functional DISC for Plk3 activation. Furthermore, we identify caspase-8 as a new substrate for Plk3. Phosphorylation occurs on T273 and results in stimulation of caspase-8 proapoptotic function. Stimulation of CD95 in cells expressing a non-phosphorylatable caspase-8-T273A mutant in a rescue experiment or in Plk3-KO cells generated by CRISPR/Cas9 reduces the processing of caspase-8 prominently. Low T273 phosphorylation correlates significantly with low Plk3 expression in a cohort of 95 anal tumor patients. Our data suggest a novel mechanism of kinase activation within the Plk family and propose a new model for the stimulation of the extrinsic death pathway in tumors with high Plk3 expression

Polo-like kinase 3 and phosphoT273 caspase-8 are associated with improved local tumor control and survival in patients with anal carcinoma treated with concomitant chemoradiotherapy

We have recently shown that caspase-8 is a new substrate of Polo-like kinase 3 (Plk3) that phosphorylates the protein on residue T273 thereby promoting its pro-apoptotic function. In the present study we aimed to investigate the clinical relevance of Plk3 expression and phosphorylation of caspase-8 at T273 in patients with anal squamous cell carcinoma (SSC) treated with 5-fluorouracil and mitomycin C-based chemoradiotherapy (CRT). Immunohistochemical detection of the markers was performed in pretreatment biopsy specimens of 95 patients and was correlated with clinical/histopathologic characteristics including HPV-16 virus load/p16INK4a expression and cumulative incidence of local and distant failure, cancer specific survival (CSS), and overall survival (OS). We observed significant positive correlations between Plk3 expression, pT273 caspase-8 signal, and levels of HPV-16 virus DNA load/p16INK4a detection. Patients with high scores of Plk3 and pT273 caspase-8 showed increased local control (p = 0.011; p = 0.001), increased CSS (p = 0.011; p = 0.013) and OS (p = 0.024; p = 0.001), while the levels of pT273 caspase-8 were significantly associated (p = 0.033) with distant metastases. In multivariate analyses Plk3 expression remained significant for local failure (p = 0.018), CSS (p = 0.016) and OS (p = 0.023). Moreover, a combined HPV16 DNA load and Plk3 or pT273 caspase-8 variable revealed a significant correlation to decreased local failure (p = 0.001; p = 0.009), increased CSS (p = 0.016; p = 0.023) and OS (p = 0.003; p = 0.003). In conclusion these data indicate that elevated levels of Plk3 and pT273 caspase-8 are correlated with favorable clinical outcome in patients with anal SCC treated with concomitant CRT

Polo-like kinase 3 and phosphoT273 caspase-8 are associated with improved local tumor control and survival in patients with anal carcinoma treated with concomitant chemoradiotherapy

We have recently shown that caspase-8 is a new substrate of Polo-like kinase 3 (Plk3) that phosphorylates the protein on residue T273 thereby promoting its pro-apoptotic function. In the present study we aimed to investigate the clinical relevance of Plk3 expression and phosphorylation of caspase-8 at T273 in patients with anal squamous cell carcinoma (SSC) treated with 5-fluorouracil and mitomycin C-based chemoradiotherapy (CRT). Immunohistochemical detection of the markers was performed in pretreatment biopsy specimens of 95 patients and was correlated with clinical/histopathologic characteristics including HPV-16 virus load/p16INK4a expression and cumulative incidence of local and distant failure, cancer specific survival (CSS), and overall survival (OS). We observed significant positive correlations between Plk3 expression, pT273 caspase-8 signal, and levels of HPV-16 virus DNA load/p16INK4a detection. Patients with high scores of Plk3 and pT273 caspase-8 showed increased local control (p = 0.011; p = 0.001), increased CSS (p = 0.011; p = 0.013) and OS (p = 0.024; p = 0.001), while the levels of pT273 caspase-8 were significantly associated (p = 0.033) with distant metastases. In multivariate analyses Plk3 expression remained significant for local failure (p = 0.018), CSS (p = 0.016) and OS (p = 0.023). Moreover, a combined HPV16 DNA load and Plk3 or pT273 caspase-8 variable revealed a significant correlation to decreased local failure (p = 0.001; p = 0.009), increased CSS (p = 0.016; p = 0.023) and OS (p = 0.003; p = 0.003). In conclusion these data indicate that elevated levels of Plk3 and pT273 caspase-8 are correlated with favorable clinical outcome in patients with anal SCC treated with concomitant CRT