Polo-like Kinase Plk Research Articles

PLK-1 regulates MEX-1 polarization in the C. elegans zygote.

The one-cell C. elegans embryo undergoes an asymmetric cell division during which germline factors such as the RNA-binding proteins POS-1 and MEX-1 segregate to the posterior cytoplasm, leading to their asymmetric inheritance to the posterior germline daughter cell. Previous studies found that the RNA-binding protein MEX-5 recruits polo-like kinase PLK-1 to the anterior cytoplasm where PLK-1 inhibits the retention of its substrate POS-1, leading to POS-1 segregation to the posterior. In this study, we tested whether PLK-1 similarly regulates MEX-1 polarization. We find that both the retention of MEX-1 in the anterior and the segregation of MEX-1 to the posterior depend on PLK kinase activity and on the interaction between MEX-5 and PLK-1. Human PLK1 directly phosphorylates recombinant MEX-1 on 9 predicted PLK-1 sites in vitro, four of which were identified in previous phosphoproteomic analysis of C. elegans embryos. The introduction of alanine substitutions at these four PLK-1 phosphorylation sites (MEX-1(4A)) significantly weakened the inhibition of MEX-1 retention in the anterior, thereby weakening MEX-1 segregation to the posterior. In contrast, mutation of a predicted CDK1 phosphorylation site had no effect on MEX-1 retention or on MEX-1 segregation. MEX-1(4A) mutants are viable and fertile but display significant sterility and fecundity defects at elevated temperatures. Taken together with our previous findings, these findings suggest PLK-1 phosphorylation drives both MEX-1 and POS-1 polarization during the asymmetric division of the zygote.

Abstract 3266: Plk1 promotes Pdcd4 degradation to enhance enzalutamide resistance in prostate cancer

Abstract The androgen receptor (AR) signaling pathway plays critical role in the development of prostate cancer (PCa), including castration-resistant prostate cancer (CRPC). As a result, androgen signaling inhibitors (ASIs) like abiraterone and enzalutamide (ENZ) have emerged as the first-line treatment for CRPC. Nevertheless, the limited success of ASIs underscores the urgency of developing new approaches for managing CRPC patients who have become unresponsive to ASIs. Programmed cell death 4 (Pdcd4) is a tumor suppressor, which is frequently down-regulated during PCa progression. It has been shown that reduced Pdcd4 expression promotes both AR-dependent and AR-independent growth in PCa. In this study, we found that down-regulation of Pdcd4 led to acquisition of ENZ resistance in PCa. We also observed that down-regulation of Pdcd4 resulted in activation of mTORC2 activity. Additionally, knockdown of Rictor, a component of mTORC2 complex, enhanced sensitivity to ENZ treatment in PCa. These findings suggested that activation of mTORC2 due to Pdcd4 loss contributes to ASI resistance in PCa. To understand how Pdcd4 is down-regulated in the PCa, we identified that polo-like kinase (Plk1) phosphorylated Pdcd4 at Ser239. Phosphorylation of Pdcd4 by Plk1 led to ubiquitin-mediated proteasomal degradation of Pdcd4. Over-expression of the phosphomimic Pdcd4(S239D) in 22Rv1 cells promoted tumor cell growth in cultured cells and mice. Taken together, our data suggest that targeting Plk1/Pdcd4/mTORC2 signaling axis holds promise as a strategy to overcome ENZ resistance in PCa. Citation Format: Qing Wang, Qiongsi Zhang, Yanquan Zhang, Xiaoqi Liu, Hsin-Sheng Yang. Plk1 promotes Pdcd4 degradation to enhance enzalutamide resistance in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3266.

In silico study for discovering novel thiazole derivatives as anti-breast cancer agents (MCF-7)

The work validated the pharmacokinetic characteristics and docking approach, and discovered novel thiazole compounds with MCF-7 breast anti-cancer efficacy using quantitative structure-activity relationships (QSAR) models. Using an experimental dataset of 53 thiazole derivatives with IC50 values, QSAR models were developed using multivariate linear regression (QSARMLR) and artificial neural networks (QSARANN). Eight descriptions, with statistical values of R2train = 0.875 and Q2LOO = 0.834, comprised the successfully developed QSARMLR model. Based on the descriptors of the QSARMLR model, which has statistical values of R2 = 0.918, Q2test = 0.934, and Q2CV = 0.916, the QSARANN neural network model with the architectural network of I(8)-HL(9)-O(1) has also been constructed. Using the AD and outliers analysis, the models were utilized to forecast 120 new design derivatives based on the thiazole framework, and the IC50 values of 10 new thiazole derivatives were determined. Additionally, the derivatives were assessed for resistance to estrogen-positive breast cancer by docking them onto the Polo-like kinases (Plk1) receptor and screening them for pharmacokinetic features in accordance with Lipinski and Ghose guidelines. Consequently, it was discovered that the thiazole TAZ5 had promising derivative activity against the primary MCF-7 breast cancer cell line.

A rhodanine derivative as a potential antibacterial and anticancer agent: Crystal structure, spectral characterization, DFT calculations, Hirshfeld surface analysis, in silico molecular docking and ADMET studies

A novel rhodanine derivative, namely 5-(4-dimethylaminobenzylidene) rhodanine “C12H12N2OS2”, was successfully crystallized from condensation route and characterized by NMR, FT-IR, UV–Vis spectral methods as well as single-crystal X-ray diffraction. This rhodanine derivate crystallizes in the monoclinic P21/c space group, with the cell parameters: a = 3.93590(10), b = 11.2480(3), c = 26.6703(7) Å and β = 93.8530(10)°. The molecular structure displays intra- (C − S···H) and intermolecular (N − H···O) hydrogen-bonding interactions. Micro-spectroscopy performed on single-crystals of the studied compound revealed the first absorption transition at 2.25 eV, and a well-structured luminescence peaked at 2.01 eV (0.15 eV broad). Density functional theory (DFT) calculations allowed the structure optimization, the electronic properties, the IR-vibrational modes and frequencies as well as the 1H and 13C NMR chemical shifts’ calculation. Furthermore, time-dependent DFT (TD-DFT) calculations were performed for the vertical transition energies. Hirshfeld surface analysis (HSA) showed the presence of non-conventional C–H···H–C, C–H···π and π···lp interactions and π−π stacking. The anti-cancer and anti-bacterial activities of the studied compound towards the Polo-like kinase PLK1 and the Escherichia coli MurB enzymes were in silico evaluated by performing molecular docking simulations. The results suggest that the molecule can significantly inhibit the enzymes’ active sites. Additionally, the physicochemical and pharmacokinetic characteristics of the molecule were evaluated through absorption, distribution, metabolism, excretion and toxicity (ADMET) analysis, and the results ensure its good drug-likeness properties.

Polo-like kinase inhibitor BI2536 induces eryptosis.

BI2536 is potent inhibitor of polo-like kinases PLK1, 2, and 3. The inhibition of PLKs in nucleated cells induces apoptosis by perturbing the cell cycle with consequent engagement of mitotic catastrophe. BI2536 is being tested as chemotherapy in various phaseI/II/III clinical trials. Erythrocytes do not have anucleus; however, they may undergo programmed suicide with characteristic hallmarks including cell shrinkage and phosphatidylserine translocation to the cell surface. This particular death is baptized eryptosis. Our study explored whether BI2536 induces eryptosis. We used flow cytometry to access death in red blood cells. We analyzed the cellular volume, the intracellular calcium concentration, the cell surface phosphatidylserine exposure, and the ceramide abundance. In addition, we analyzed the effect of BI2536 on hemolysis. Our investigation showed that after 48 h of incubation with PLK inhibitor BI2536, erythrocytes lost volume and were positive for annexin‑V without any effect on hemolysis. Cells also showed an abundance of ceramide and an increase of intracellular calcium. All these finding suggest that BI2536 provokes eryptosis in red blood cells, ostensibly in part due to Ca2+ entry and ceramide accumulation.

Cortical microtubule pulling forces contribute to the union of the parental genomes in the Caenorhabditis elegans zygote.

Previously, we reported that the Polo-like kinase PLK-1 phosphorylates the single Caenorhabditis elegans lamin (LMN-1) to trigger lamina depolymerization during mitosis. We showed that this event is required to form a pronuclear envelope scission event that removes membranes on the juxtaposed oocyte and sperm pronuclear envelopes in the zygote, allowing the parental chromosomes to merge in a single nucleus after segregation (Velez-Aguilera et al., 2020). Here, we show that cortical microtubule pulling forces contribute to pronuclear envelopes scission by promoting mitotic spindle elongation, and conversely, nuclear envelopes remodeling facilitates spindle elongation. We also demonstrate that weakening the pronuclear envelopes via PLK-1-mediated lamina depolymerization, is a prerequisite for the astral microtubule pulling forces to trigger pronuclear membranes scission. Finally, we provide evidence that PLK-1 mainly acts via lamina depolymerization in this process. These observations thus indicate that temporal coordination between lamina depolymerization and mitotic spindle elongation facilitates pronuclear envelopes scission and parental genomes unification.

Role of Polo-like Kinases Plk1 and Plk4 in the Initiation of Centriole Duplication-Impact on Cancer.

Centrosomes nucleate and anchor microtubules and therefore play major roles in spindle formation and chromosome segregation during mitosis. Duplication of the centrosome occurs, similar to DNA, only once during the cell cycle. Aberration of the centrosome number is common in human tumors. At the core of centriole duplication is the conserved polo-like kinase 4, Plk4, and two structural proteins, STIL and Sas-6. In this review, I summarize and discuss developments in our understanding of the first steps of centriole duplication and their regulation.

Abstract 973: The selective polo-like kinase (Plk1) inhibitor onvansertib and the antiandrogen abiraterone synergistically kill cancer cells through disruption of mitosis independently of androgen receptor signaling

Abstract Abiraterone acetate, a widely-used standard of care for metastatic castrate-resistant prostate cancer (mCRPC), both abrogates androgen synthesis and inhibits the androgen receptor (AR). The AR is a central driver of prostate cancer, and abiraterone slows disease progression in the majority of men, however, extended treatment universally results in acquired resistance. We have observed that Plk1 inhibitors (Plk1i), including the clinically active, highly-selective and orally available Plk1i onvansertib, synergize with abiraterone in models of CRPC, providing a potential means to reverse or delay the onset of abiraterone resistance. Plk1 is a serine/threonine kinase that regulates entry into and progression through mitosis. In contrast to abiraterone, the AR-antagonist enzalutamide does not synergistically kill CRPC cells in combination with Plk1i. Moreover, we have found Plk1i-abiraterone synergy in AR-negative cancer cells demonstrating that this synergy is AR independent. A phase 2 clinical trial (NCT03414034) is currently testing combination of onvansertib and abiraterone in mCRPC patients with nascent abiraterone resistance. Preliminary efficacy, as measured by PSA decline or stabilization, was observed in a subset of patients, including those harboring the AR splice variant 7. To identify the mechanisms driving Plk1i-abiraterone synergy, prostate cancer cell lines showing, or not showing, synergy between these drugs (referred to as synergistic or non-synergistic cells, respectively), were treated with vehicle, abiraterone, enzalutamide, or onvansertib prior to RNA sequencing and Gene Set Variation Analysis (GSVA). In synergistic cells, a group of mitosis and mitotic spindle related gene sets were significantly upregulated by both abiraterone and onvansertib. These gene sets were not upregulated in non-synergistic cells, or by enzalutamide, indicating that abiraterone may target mitosis related genes or processes in an AR-independent manner. Live-cell microscopy showed that, while abiraterone does not cause gross mitotic arrest, it does impair mitotic spindle assembly. These mitotic gene sets were also relatively upregulated in untreated synergistic cells, suggesting that synergistic responses reflect baseline differences in molecular pathways. In summary, we have found that abiraterone impacts spindle assembly and sensitizes some cancer cells to Plk1i resulting in synergistic mitotic arrest and spindle assembly checkpoint dependent cell death. Baseline differences in these processes may predict synergy and be predicative of patient responses to the onvansertib + abiraterone combination. This hypothesis is being evaluated in patients currently enrolled in the clinical study of onvansertib + abiraterone. Citation Format: Jesse C. Patterson, Peter J. Croucher, Maya Ridinger, David J. Einstein, Andreas Varkaris, Steven P. Balk, Glenn J. Bubley, Mark G. Erlander, Michael B. Yaffe. The selective polo-like kinase (Plk1) inhibitor onvansertib and the antiandrogen abiraterone synergistically kill cancer cells through disruption of mitosis independently of androgen receptor signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 973.

QSAR, molecular docking, design, and pharmacokinetic analysis of 2-(4-fluorophenyl) imidazol-5-ones as anti-breast cancer drug compounds against MCF-7 cell line

The anti-proliferative activities of Novel series of 2-(4-fluorophenyl) imidazol-5-ones against MCF-7 breast cancer cell line were explored via in-slico studies which includes Quantitative structure–activity relationship QSAR, molecular docking studies, designing new compounds, and analyzing the pharmacokinetics properties of the designed compounds. From the QSAR analysis, model number one emerged the best as seen from the arithmetic assessments of (R2) = 0.6981, (R2adj) = 0.6433, (Q2) = 0.5460 and (R2pred) of 0.5357. Model number one was used in designing new derivative compounds, with higher effectiveness against estrogen positive breast cancer (MCF-7 cell line). The Molecular docking studies between the derivatives and Polo-like kinases (Plk1) receptor proved that the derivatives of 2-(4-fluorophenyl) imidazol-5-ones bind tightly to the receptor, thou ligand 24 and 27 had the highest binding affinities of −8.8 and − 9.1 kcal/mol, which was found to be higher than Doxorubicin with a docking score of −8.0 kcal/mol. These new derivatives of 2-(4-fluorophenyl) imidazol-5-ones shall be excellent inhibitors against (plk1). The pharmacokinetics analysis performed on the new structures revealed that all the structures passed the test and also the Lipinski rule of five, and they could further proceed to pre-clinical tests. They both revealed a revolution in medicine for developing novel anti-breast cancer drugs against MCF-7 cell line.

PLK-1 promotes the merger of the parental genome into a single nucleus by triggering lamina disassembly.

Life of sexually reproducing organisms starts with the fusion of the haploid egg and sperm gametes to form the genome of a new diploid organism. Using the newly fertilized Caenorhabditis elegans zygote, we show that the mitotic Polo-like kinase PLK-1 phosphorylates the lamin LMN-1 to promote timely lamina disassembly and subsequent merging of the parental genomes into a single nucleus after mitosis. Expression of non-phosphorylatable versions of LMN-1, which affect lamina depolymerization during mitosis, is sufficient to prevent the mixing of the parental chromosomes into a single nucleus in daughter cells. Finally, we recapitulate lamina depolymerization by PLK-1 in vitro demonstrating that LMN-1 is a direct PLK-1 target. Our findings indicate that the timely removal of lamin is essential for the merging of parental chromosomes at the beginning of life in C. elegans and possibly also in humans, where a defect in this process might be fatal for embryo development.

A cryptic hydrophobic pocket in the polo-box domain of the polo-like kinase PLK1 regulates substrate recognition and mitotic chromosome segregation

The human polo-like kinase PLK1 coordinates mitotic chromosome segregation by phosphorylating multiple chromatin- and kinetochore-binding proteins. How PLK1 activity is directed to specific substrates via phosphopeptide recognition by its carboxyl-terminal polo-box domain (PBD) is poorly understood. Here, we combine molecular, structural and chemical biology to identify a determinant for PLK1 substrate recognition that is essential for proper chromosome segregation. We show that mutations ablating an evolutionarily conserved, Tyr-lined pocket in human PLK1 PBD trigger cellular anomalies in mitotic progression and timing. Tyr pocket mutations selectively impair PLK1 binding to the kinetochore phosphoprotein substrate PBIP1, but not to the centrosomal substrate NEDD1. Through a structure-guided approach, we develop a small-molecule inhibitor, Polotyrin, which occupies the Tyr pocket. Polotyrin recapitulates the mitotic defects caused by mutations in the Tyr pocket, further evidencing its essential function, and exemplifying a new approach for selective PLK1 inhibition. Thus, our findings support a model wherein substrate discrimination via the Tyr pocket in the human PLK1 PBD regulates mitotic chromosome segregation to preserve genome integrity.

Switching of INCENP paralogs controls transitions in mitotic chromosomal passenger complex functions

ABSTRACTA single inner centromere protein (INCENP) found throughout eukaryotes modulates Aurora B kinase activity and chromosomal passenger complex (CPC) localization, which is essential for timely mitotic progression. It has been proposed that INCENP might act as a rheostat to regulate Aurora B activity through mitosis, with successively higher activity threshold levels for chromosome alignment, the spindle checkpoint, anaphase spindle transfer and finally spindle elongation and cytokinesis. It remains mechanistically unclear how this would be achieved. Here, we reveal that the urochordate, Oikopleura dioica, possesses two INCENP paralogs, which display distinct localizations and subfunctionalization in order to complete M-phase. INCENPa was localized on chromosome arms and centromeres by prometaphase, and modulated Aurora B activity to mediate H3S10/S28 phosphorylation, chromosome condensation, spindle assembly and transfer of the CPC to the central spindle. Polo-like kinase (Plk1) recruitment to CDK1 phosphorylated INCENPa was crucial for INCENPa-Aurora B enrichment on centromeres. The second paralog, INCENPb was enriched on centromeres from prometaphase, and relocated to the central spindle at anaphase onset. In the absence of INCENPa, meiotic spindles failed to form, and homologous chromosomes did not segregate. INCENPb was not required for early to mid M-phase events but became essential for the activity and localization of Aurora B on the central spindle and midbody during cytokinesis in order to allow abscission to occur. Together, our results demonstrate that INCENP paralog switching on centromeres modulates Aurora B kinase localization, thus chronologically regulating CPC functions during fast embryonic divisions in the urochordate O. dioica.Abbreviations: CCAN: constitutive centromere-associated network; CENPs: centromere proteins; cmRNA: capped messenger RNA; CPC: chromosomal passenger complex; INCENP: inner centromere protein; Plk1: polo-like kinase 1; PP1: protein phosphatase 1; PP2A: protein phosphatase 2A; SAC: spindle assembly checkpoint; SAH: single α-helix domain

Differential Requirements for Centrioles in Mitotic Centrosome Growth and Maintenance

Centrosomes, the predominant sites of microtubule nucleation and anchorage, coordinate spindle assembly and cell division in animal cells. At the onset of mitosis, centrioles accumulate microtubule-organizing pericentriolar material (PCM) in a process termed centrosome maturation. To what extent centrosome maturation depends on the continued activity of mitotic regulators or the presence of centrioles has hitherto been unclear. Using the C.elegans early embryo, we show that PCM expansion requires the Polo-like kinase PLK-1 and CEP192 (SPD-2 in C.elegans), but not its upstream regulator Aurora A (AIR-1), while maintenance of the PCM polymer depends exclusively on PLK-1. SPD-2 and PLK-1 are highly concentrated at centrioles. Unexpectedly, laser microsurgery reveals that while centrioles are required for PCM recruitment and centrosome structural integrity they are dispensable for PCM maintenance. We propose a model whereby centrioles promote centrosome maturation by recruiting PLK-1, but subsequent maintenance occurs via PLK-1 acting directly within the PCM.

Polo-like Kinase Couples Cytoplasmic Protein Gradients in the C. elegans Zygote

Intracellular protein gradients underlie essential cellular and developmental processes, but the mechanisms by which they are established are incompletely understood. During the asymmetric division of the C.elegans zygote, the RNA-binding protein MEX-5 forms an anterior-rich cytoplasmic gradient that causes the RNA-binding protein POS-1 to form an opposing, posterior-rich gradient. We demonstrate that the polo-like kinase PLK-1 mediates the repulsive coupling between MEX-5 and POS-1 by increasing the mobility of POS-1 in the anterior. PLK-1 is enriched in the anterior cytoplasm and phosphorylates POS-1, which is both necessary and sufficient to increase POS-1 mobility. Regulation of POS-1 mobility depends on both the interaction between PLK-1 and MEX-5 and between MEX-5 and RNA, suggesting that MEX-5 may recruit PLK-1 to RNA in the anterior. The low concentration of MEX-5/PLK-1 in the posterior cytoplasm provides a permissive environment for the retention of POS-1, which depends on POS-1 RNA binding. Our findings describe a novel reaction/diffusion mechanism in which the asymmetric distribution of cytoplasmic PLK-1 couples two RNA-binding protein gradients, thereby partitioning the cytoplasm.

Structural Basis of Wee Kinases Functionality and Inactivation by Diverse Small Molecule Inhibitors.

Members of the Wee family of kinases negatively regulate the cell cycle via phosphorylation of CDK1 and are considered potential drug targets. Herein, we investigated the structure-function relationship of human Wee1, Wee2, and Myt1 (PKMYT1). Purified recombinant full-length proteins and kinase domain constructs differed substantially in phosphorylation states and catalytic competency, suggesting complex mechanisms of activation. A series of crystal structures reveal unique features that distinguish Wee1 and Wee2 from Myt1 and establish the structural basis of differential inhibition by the widely used Wee1 inhibitor MK-1775. Kinome profiling and cellular studies demonstrate that, in addition to Wee1 and Wee2, MK-1775 is an equally potent inhibitor of the polo-like kinase PLK1. Several previously unrecognized inhibitors of Wee kinases were discovered and characterized. Combined, the data provide a comprehensive view on the catalytic and structural properties of Wee kinases and a framework for the rational design of novel inhibitors thereof.

Polo-like kinase-dependent phosphorylation of the synaptonemal complex protein SYP-4 regulates double-strand break formation through a negative feedback loop.

The synaptonemal complex (SC) is an ultrastructurally conserved proteinaceous structure that holds homologous chromosomes together and is required for the stabilization of pairing interactions and the completion of crossover (CO) formation between homologs during meiosis I. Here, we identify a novel role for a central region component of the SC, SYP-4, in negatively regulating formation of recombination-initiating double-strand breaks (DSBs) via a feedback loop triggered by crossover designation in C. elegans. We found that SYP-4 is phosphorylated dependent on Polo-like kinases PLK-1/2. SYP-4 phosphorylation depends on DSB formation and crossover designation, is required for stabilizing the SC in pachytene by switching the central region of the SC from a more dynamic to a less dynamic state, and negatively regulates DSB formation. We propose a model in which Polo-like kinases recognize crossover designation and phosphorylate SYP-4 thereby stabilizing the SC and making chromosomes less permissive for further DSB formation.

Cervical Cancer Growth Is Regulated by a c-ABL-PLK1 Signaling Axis.

The nonreceptor tyrosine kinase c-ABL controls cell growth but its contributions in solid tumors are not fully understood. Here we report that the Polo-like kinase PLK1, an essential mitotic kinase regulator, is an important downstream effector of c-ABL in regulating the growth of cervical cancer. c-ABL interacted with and phosphorylated PLK1. Phosphorylation of PLK1 by c-ABL inhibited PLK1 ubiquitination and degradation and enhanced its activity, leading to cell-cycle progression and tumor growth. Both c-ABL and PLK1 were overexpressed in cervical carcinoma. Notably, PLK1 tyrosine phosphorylation correlated with patient survival in cervical cancer. In a murine xenograft model of human cervical cancer, combination treatment with c-ABL and PLK1 inhibitors yielded additive effects on tumor growth inhibition. Our findings highlight the c-ABL-PLK1 axis as a novel prognostic marker and treatment target for human cervical cancers. Cancer Res; 77(5); 1142-54. ©2016 AACR.

Abstract 258: Is P53 the up-and-coming predictive biomarker for volasertib treatment in NSCLC

Abstract Introduction: Polo-like kinase (Plk1), a key regulator of cell division, is considered as an attractive target for mitotic intervention. The observed Plk1 overexpression in several tumor types, including non-small cell lung cancer (NSCLC), has led to the development of small molecule inhibitors. Moreover, previous studies suggested an interplay between Plk1 and the tumor suppressor P53. This led to the hypothesis that the P53 status might be predictive for the response to Plk1 inhibition. Therefore, we investigated the cytotoxic effect of volasertib in NSCLC cell lines with a different P53 background, under normoxia and hypoxia. Material and methods: A549 (TP53 wild type) was transduced with a P53 shRNA lentiviral vector to obtain the P53 deficient sub-cell line A549-920 or with an empty vector as control (A549-NTC). In addition, NCI-H1975 cells were used as a TP53 mutant (R273H) cell line. Cells were treated with 0-85nM volasertib (24-72h). Cell survival was assessed using the sulphorhodamine B assay and IC50-values were calculated using WinNonlin software. The effect of Plk1 inhibition (0-20nM) on cell cycle distribution (24h) and apoptosis induction (48h) was determined flow cytometrically. In addition, we investigated the potential of volasertib to inhibit cell migration using a Transwell system. All experiments were performed under both normoxia and hypoxia (<0.1% O2). Results: Plk1 inhibition established a dose-dependent growth inhibition under both normoxia and hypoxia. Interestingly, a reduced sensitivity to volasertib treatment (24h) was observed in P53 deficient cells (A549-920, IC50: 27.59±5.77 nM) compared to P53 wild type cells (A549-NTC, IC50: 17.87±0.40 nM) (p<0.001). Except for the NCI-H1975 cells, a decreased effect of volasertib was observed under hypoxia (p<0.001). After treatment, a G2/M phase block was induced in all cell lines (p<0.004), albeit more pronounced in A549-920 cells compared to A549-NTC cells (p = 0.06). Under hypoxia, this mitotic arrest was only detected using high volasertib concentrations (12.5–20nM) (p<0.04). Furthermore, a sub-G1 peak could be observed after treatment with high volasertib concentrations, suggesting apoptosis induction. Indeed, an increase in PI-stained cells was detected 48h after Plk1 inhibition in all cell lines (p<0.042). Remarkably, induction of cell death was higher in P53 wild type cells compared to P53 deficient cells (p<0.02). Transwell experiments, investigating the potential of volasertib to inhibit cell migration, are currently being analyzed. Conclusion: Our results show that there is a difference in response to Plk1 inhibition in cancer cells with and without functional P53. While P53 deficient/mutant cells were mostly arrested in the G2/M phase, cell death was induced in P53 wild type cells. These data lead to the hypothesis that P53 might be a predictive marker for response to Plk1 inhibition. Citation Format: Jolien Van den Bossche, Filip Lardon, Christophe Deben, Ines De Pauw, Vanessa Deschoolmeester, Evelien Smits, Pol Specenier, Patrick Pauwels, Marc Peeters, An Wouters. Is P53 the up-and-coming predictive biomarker for volasertib treatment in NSCLC. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 258.

RB/PLK1-dependent induced pathway by SLAMF3 expression inhibits mitosis and control hepatocarcinoma cell proliferation.

Polo-like kinase PLK1 is a cell cycle protein that plays multiple roles in promoting cell cycle progression. Among the many roles, the most prominent role of PLK1 is to regulate the mitotic spindle formation checkpoint at the M-phase. Recently we reported the expression of SLAMF3 in Hepatocytes and show that it is down regulated in tumor cells of hepatocellular carcinoma (HCC). We also show that the forced high expression level of SLAMF3 in HCC cells controls proliferation by inhibiting the MAPK ERK/JNK and the mTOR pathways. In the present study, we provide evidence that the inhibitory effect of SLAMF3 on HCC proliferation occurs through Retinoblastoma (RB) factor and PLK1-dependent pathway. In addition to the inhibition of MAPK ERK/JNK and the mTOR pathways, expression of SLAMF3 in HCC retains RB factor in its hypophosphorylated active form, which in turn inactivates E2F transcription factor, thereby repressing the expression and activation of PLK1. A clear inverse correlation was also observed between SLAMF3 and PLK expression in patients with HCC. In conclusion, the results presented here suggest that the tumor suppressor potential of SLAMF3 occurs through activation of RB that represses PLK1. We propose that the induction of a high expression level of SLAMF3 in cancerous cells could control cellular mitosis and block tumor progression.

Parkin New Cargos: a New ROS Independent Role for Parkin in Regulating Cell Division.

Cell cycle progression requires the destruction of key cell cycle regulators by the multi-subunit E3 ligase called the anaphase promoting complex (APC/C). As the cell progresses through the cell cycle, the APC/C is sequentially activated by two highly conserved co-activators called Cdc20 and Cdh1. Importantly, APC/CCdc20 is required to degrade substrates in G2/M whereas APCCdh1 drives the cells into G1. Recently, Parkin, a monomeric E3 ligase that is required for ubiquitin-mediated mitophagy following mitochondrial stress, was shown to both bind and be activated by Cdc20 or Cdh1 during the cell cycle. This mitotic role for Parkin does not require an activating phosphorylation by its usual kinase partner PINK. Rather, mitotic Parkin activity requires phosphorylation on a different serine by the polo-like kinase Plk1. Interestingly, although ParkinCdc20 and ParkinCdh1 activity is independent of the APC/C, it mediates degradation of an overlapping subset of substrates. However, unlike the APC/C, Parkin is not necessary for cell cycle progression. Despite this, loss of Parkin activity accelerates genome instability and tumor growth in xenograft models. These findings provide a mechanism behind the previously described, but poorly understood, tumor suppressor role for Parkin. Taken together, studies suggest that the APC/C and Parkin have similar and unique roles to play in cell division, possibly being dependent upon the different subcellular address of these two ligases.