The small-molecule inhibitor BI 2536 reveals novel insights into mitotic roles of polo-like kinase 1

BACKGROUND: The mitotic kinases, Cdk1, Aurora A/B, and Polo-like kinase 1 (Plk1) have been characterized extensively to further understanding of mitotic mechanisms and as potential targets for cancer therapy. Cdk1 and Aurora kinase studies have been facilitated by small-molecule inhibitors, but few if any potent Plk1 inhibitors have been identified. RESULTS: We describe the cellular effects of a novel compound, BI 2536, a potent and selective inhibitor of Plk1. The fact that BI 2536 blocks Plk1 activity fully and instantaneously enabled us to study controversial and unknown functions of Plk1. Cells treated with BI 2536 are delayed in prophase but eventually import Cdk1-cyclin B into the nucleus, enter prometaphase, and degrade cyclin A, although BI 2536 prevents degradation of the APC/C inhibitor Emi1. BI 2536-treated cells lack prophase microtubule asters and thus polymerize mitotic microtubules only after nuclear-envelope breakdown and form monopolar spindles that do not stably attach to kinetochores. Mad2 accumulates at kinetochores, and cells arrest with an activated spindle-assembly checkpoint. BI 2536 prevents Plk1's enrichment at kinetochores and centrosomes, and when added to metaphase cells, it induces detachment of microtubules from kinetochores and leads to spindle collapse. CONCLUSIONS: Our results suggest that Plk1's accumulation at centrosomes and kinetochores depends on its own activity and that this activity is required for maintaining centrosome and kinetochore function. Our data also show that Plk1 is not required for prophase entry, but delays transition to prometaphase, and that Emi1 destruction in prometaphase is not essential for APC/C-mediated cyclin A degradation.     

p53 is not directly relevant to the response of Polo-like kinase 1 inhibitors

Polo-like kinase 1 (Plk1) is elementary for cell proliferation and its deregulation is involved in tumorigenesis. Plk1 has been established as one of the most attractive targets for molecular cancer therapy. In fact, multiple small molecule inhibitors targeting either the kinase domain or the Polo-box binding domain (PBD) of Plk1 have been identified and intensively investigated. Intriguingly, Plk1 depletion affects more cancer cells than normal cells. It is also reported that the cytotoxicity induced by Plk1 inhibition is elevated in cancer cells with defective p53. The data lead to the hypothesis that p53 might be a predictive marker for the response of Plk1 inhibition. In this study, we demonstrate that there is no obvious different cytotoxic response between cancer cells with and without functional p53, including the isogenic colon cancer cell lines HCT116p53(+/+) and HCT116p53(-/-), breast cancer cell line MCF7, lung cancer cell line A549 and cervical carcinoma cell line HeLa, after treatment with either siRNA against Plk1, the kinase domain inhibitors BI 2536 and BI 6727 or the PBD inhibitor Poloxin. We suggest that the p53 status is not a predictor for the response of Plk1 inhibition, at least not directly. Yet, the long-term outcomes of losing p53, such as genome instability, could be associated with the cytotoxicity of Plk1 inhibition. Further studies are required to investigate whether other circumstances of cancer cells, such as DNA replication/damage stress, mitotic stress, and metabolic stress, which make possibly the survival of cancer cells more dependent on Plk1 function, are responsible for the sensitivity of Plk1 inhibition.     

BI 2536, a potent and selective inhibitor of polo-like kinase 1, inhibits tumor growth in vivo

Fine-mapping of the cell-division cycle, notably the identification of mitotic kinase signaling pathways, provides novel opportunities for cancer-drug discovery. As a key regulator of multiple steps during mitotic progression across eukaryotic species, the serine/threonine-specific Polo-like kinase 1 (Plk1) is highly expressed in malignant cells and serves as a negative prognostic marker in specific human cancer types . Here, we report the discovery of a potent small-molecule inhibitor of mammalian Plk1, BI 2536, which inhibits Plk1 enzyme activity at low nanomolar concentrations. The compound potently causes a mitotic arrest and induces apoptosis in human cancer cell lines of diverse tissue origin and oncogenome signature. BI 2536 inhibits growth of human tumor xenografts in nude mice and induces regression of large tumors with well-tolerated intravenous dose regimens. In treated tumors, cells arrest in prometaphase, accumulate phosphohistone H3, and contain aberrant mitotic spindles. This mitotic arrest is followed by a surge in apoptosis, detectable by immunohistochemistry and noninvasive optical and magnetic resonance imaging. For addressing the therapeutic potential of Plk1 inhibition, BI 2536 has progressed into clinical studies in patients with locally advanced or metastatic cancers.     

Combining p53 stabilizers with metformin induces synergistic apoptosis through regulation of energy metabolism in castration-resistant prostate cancer

Since altered energy metabolism is a hallmark of cancer, many drugs targeting metabolic pathways are in active clinical trials. The tumor suppressor p53 is often inactivated in cancer, either through downregulation of protein or loss-of-function mutations. As such, stabilization of p53 is considered as one promising approach to treat those cancers carrying wild type (WT) p53. Herein, SIRT1 inhibitor Tenovin-1 and polo-like kinase 1 (Plk1) inhibitor BI2536 were used to stabilize p53. We found that both Tennovin-1 and BI2536 increased the anti-neoplastic activity of metformin, an inhibitor of oxidative phosphorylation, in a p53 dependent manner. Since p53 has also been shown to regulate metabolic pathways, we further analyzed glycolysis and oxidative phosphorylation upon drug treatments. We showed that both Tennovin-1 and BI2536 rescued metformin-induced glycolysis and that both Tennovin-1 and BI2536 potentiated metformin-associated inhibition of oxidative phosphorylation. Of significance, castration-resistant prostate cancer (CRPC) C4-2 cells show a much more robust response to the combination treatment than the parental androgen-dependent prostate cancer LNCaP cells, indicating that targeting energy metabolism with metformin plus p53 stabilizers might be a valid approach to treat CRPC carrying WT p53.     

Polo-like kinase 1 inhibitors,mitotic stress and the tumor suppressor p53

Polo-like kinase 1 has been established as one of the most attractive targets for molecular cancer therapy. In fact, multiple small-molecule inhibitors targeting this kinase have been developed and intensively investigated. Recently, it has been reported that the cytotoxicity induced by Plk1 inhibition is elevated in cancer cells with inactive p53, leading to the hypothesis that inactive p53 is a predictive marker for the response of Plk1 inhibition. In our previous study based on different cancer cell lines, we showed that cancer cells with wild type p53 were more sensitive to Plk1 inhibition by inducing more apoptosis, compared with cancer cells depleted of p53. In the present work, we further demonstrate that in the presence of mitotic stress induced by different agents, Plk1 inhibitors strongly induced apoptosis in HCT116 p53^+/+ cells, whereas HCT116 p53^−/− cells arrested in mitosis with less apoptosis. Depletion of p53 in HCT116 p53^+/+ or U2OS cells reduced the induction of apoptosis. Moreover, the surviving HCT116 p53^−/− cells showed DNA damage and a strong capability of colony formation. Plk1 inhibition in combination with other anti-mitotic agents inhibited proliferation of tumor cells more strongly than Plk1 inhibition alone. Taken together, the data underscore that functional p53 strengthens the efficacy of Plk1 inhibition alone or in combination by strongly activating cell death signaling pathways. Further studies are required to investigate if the long-term outcomes of losing p53, such as low differential grade of tumor cells or defective DNA damage checkpoint, are responsible for the cytotoxicity of Plk1 inhibition.     

Inhibition of Polo-like Kinase 1 (Plk1) Enhances the Antineoplastic Activity of Metformin in Prostate Cancer

The widely used anti-diabetic drug metformin has been shown to exert strong antineoplastic actions in numerous tumor types, including prostate cancer (PCa). In this study, we show that BI2536, a specific Plk1 inhibitor, acted synergistically with metformin in inhibiting PCa cell proliferation. Furthermore, we also provide evidence that Plk1 inhibition makes PCa cells carrying WT p53 much more sensitive to low-dose metformin treatment. Mechanistically, we found that co-treatment with BI2536 and metformin induced p53-dependent apoptosis and further activated the p53/Redd-1 pathway. Moreover, we also show that BI2536 treatment inhibited metformin-induced glycolysis and glutamine anaplerosis, both of which are survival responses of cells against mitochondrial poisons. Finally, we confirmed the cell-based observations using both cultured cell-derived and patient-derived xenograft studies. Collectively, our findings support another promising therapeutic strategy by combining two well tolerated drugs against PCa proliferation and the progression of androgen-dependent PCa to the castration-resistant stage.     

Inhibition of Plk1 represses androgen signaling pathway in castration-resistant prostate cancer

Prostate cancer (PCa) is the second leading cause of cancer-related death in males in the United States. Majority of prostate cancers are originally androgen-dependent and sensitive to androgen-deprivation therapy (ADT), however, most of them eventually relapse and progress into incurable castration-resistant prostate cancer (CRPC). Of note, the activity of androgen receptor (AR) is still required in CRPC stage. The mitotic kinase polo-like kinase 1 (Plk1) is significantly elevated in PCa and its expression correlates with tumor grade. In this study, we assess the effects of Plk1 on AR signaling in both androgen-dependent and androgen-independent PCa cells. We demonstrate that the expression level of Plk1 correlated with tumorigenicity and that inhibition of Plk1 caused reduction of AR expression and AR activity. Furthermore, Plk1 inhibitor BI2536 down-regulated SREBP-dependent expression of enzymes involved in androgen biosynthesis. Of interest, Plk1 level was also reduced when AR activity was inhibited by the antagonist MDV3100. Finally, we show that BI2536 treatment significantly inhibited tumor growth in LNCaP CRPC xenografts. Overall, our data support the concept that Plk1 inhibitor such as BI2536 prevents AR signaling pathway and might have therapeutic potential for CRPC patients.     

Unique subcellular distribution of phosphorylated Plk1 (Ser137 and Thr210) in mouse oocytes during meiotic division and pPlk1Ser137 involvement in spindle formation and REC8 cleavage

Polo-like kinase 1 (Plk1) is pivotal for proper mitotic progression, its targeting activity is regulated by precise subcellular positioning and phosphorylation. Here we assessed the protein expression, subcellular localization and possible functions of phosphorylated Plk1 (pPlk1^Ser137 and pPlk1^Thr210) in mouse oocytes during meiotic division. Western blot analysis revealed a peptide of pPlk1^Ser137 with high and stable expression from germinal vesicle (GV) until metaphase II (MII), while pPlk1^Thr210 was detected as one large single band at GV stage and 2 small bands after germinal vesicle breakdown (GVBD), which maintained stable up to MII. Immunofluorescence analysis showed pPlk1^Ser137 was colocalized with microtubule organizing center (MTOC) proteins, γ-tubulin and pericentrin, on spindle poles, concomitantly with persistent concentration at centromeres and dynamic aggregation between chromosome arms. Differently, pPlk1^Thr210 was persistently distributed across the whole body of chromosomes after meiotic resumption. The specific Plk1 inhibitor, BI2536, repressed pPlk1^Ser137 accumulation at MTOCs and between chromosome arms, consequently disturbed γ-tubulin and pericentrin recruiting to MTOCs, destroyed meiotic spindle formation, and delayed REC8 cleavage, therefore arresting oocytes at metaphase I (MI) with chromosome misalignment. BI2536 completely reversed the premature degradation of REC8 and precocious segregation of chromosomes induced with okadaic acid (OA), an inhibitor to protein phosphatase 2A. Additionally, the protein levels of pPlk1^Ser137 and pPlk1^Thr210, as well as the subcellular distribution of pPlk1^Thr210, were not affected by BI2536. Taken together, our results demonstrate that Plk1 activity is required for meiotic spindle assembly and REC8 cleavage, with pPlk1^Ser137 is the action executor, in mouse oocytes during meiotic division.     

Enabling and disabling polo-like kinase 1 inhibition through chemical genetics

Polo-like kinase 1 (Plk1) is a core regulator of cell division and an emerging target for cancer therapy. Pharmacologic inhibitors of Plk1 exist but affect other kinases, complicating their in vivo validation. To address this, we examined effects of two structurally unrelated Plk1 inhibitors (BI-2536 and TAL) against isogenic human cell lines that solely express wildtype (wt) or analogue-sensitive (as) Plk1 alleles. Unexpectedly, Plk1(as) cells displayed profound biochemical and functional resistance to both inhibitors. Cells that co-express Plk1(wt) and Plk1(as) exhibit loss-of-function phenotypes only when both kinase alleles are inhibited. Resistance to BI-2536 is linked to an intragenic suppressor mutation (C67V) that restores an otherwise invariant valine to the kinase active site. Structural modeling demonstrates that this mutation not only enables Plk1(as) to function in vivo but also occludes BI-2536 from the ATP-binding pocket. Our results reveal the molecular basis of Plk inhibitor selectivity and a potential mechanism for tumor cell resistance.     

Regulation of inositol 1,4,5-trisphosphate receptor type 1 function during oocyte maturation by MPM-2 phosphorylation

Egg activation and further embryo development require a sperm-induced intracellular Ca²⁺ signal at the time of fertilization. Prior to fertilization, the egg's Ca²⁺ machinery is therefore optimized. To this end, during oocyte maturation, the sensitivity, i.e. the Ca²⁺ releasing ability, of the inositol 1,4,5-trisphosphate receptor type 1 (IP₃R1), which is responsible for most of this Ca²⁺ release, markedly increases. In this study, the recently discovered specific Polo-like kinase (Plk) inhibitor BI2536 was used to investigate the role of Plk1 in this process. BI2536 inactivates Plk1 in oocytes at the early stages of maturation and significantly decreases IP₃R1 phosphorylation at an MPM-2 epitope at this stage. Moreover, this decrease in Plk1-dependent MPM-2 phosphorylation significantly lowers IP₃R1 sensitivity. Finally, using in vitro phosphorylation techniques we identified T²⁶⁵⁶ as a major Plk1 site on IP₃R1. We therefore propose that the initial increase in IP₃R1 sensitivity during oocyte maturation is underpinned by IP₃R1 phosphorylation at an MPM-2 epitope(s).     

PLK1 inhibitors: setting the mitotic death trap

Polo-like kinase 1 (Plk1) regulates mitotic progression in all eukaryotes and has been implicated in the transformation of human cells. Analysis of the cytological and anti-tumor activities of BI 2536, a novel, selective pharmacological inhibitor of Plk1, has connected chemistry and biology to the bedside.     

Cotargeting Polo-Like Kinase 1 and the Wnt/β-Catenin Signaling Pathway in Castration-Resistant Prostate Cancer

The Wnt/β-catenin signaling pathway has been identified as one of the predominantly upregulated pathways in castration-resistant prostate cancer (CRPC). However, whether targeting the β-catenin pathway will prove effective as a CRPC treatment remains unknown. Polo-like kinase 1 (Plk1) is a critical regulator in many cell cycle events, and its level is significantly elevated upon castration of mice carrying xenograft prostate tumors. Indeed, inhibition of Plk1 has been shown to inhibit tumor growth in several in vivo studies. Here, we show that Plk1 is a negative regulator of Wnt/β-catenin signaling. Plk1 inhibition or depletion enhances the level of cytosolic and nuclear β-catenin in human prostate cancer cells. Furthermore, inhibition of Wnt/β-catenin signaling significantly potentiates the antineoplastic activity of the Plk1 inhibitor BI2536 in both cultured prostate cancer cells and CRPC xenograft tumors. Mechanistically, axin2, a negative regulator of the β-catenin pathway, serves as a substrate of Plk1, and Plk1 phosphorylation of axin2 facilitates the degradation of β-catenin by enhancing binding between glycogen synthase kinase 3β (GSK3β) and β-catenin. Plk1-phosphorylated axin2 also exhibits resistance to Cdc20-mediated degradation. Overall, this study identifies a novel Plk1-Wnt signaling axis in prostate cancer, offering a promising new therapeutic option to treat CRPC.     

The Plk1 inhibitor BI 2536 temporarily arrests primary cardiac fibroblasts in mitosis and generates aneuploidy in vitro

BI 2536 is a new anti-mitotic drug that targets polo-like kinase 1 (Plk1) and is currently under clinical development for cancer therapy. The effect of this drug on cancer cells has been extensively investigated, but information about the effects on primary dividing cells and differentiated non-dividing cells is scarce. We have investigated the effects of this drug on primary neonatal rat cardiac fibroblasts and on differentiated cardiomyocytes and explored the possibility to use this drug to enrich differentiated cell populations in vitro. BI 2536 had a profound effect on cardiac fibroblast proliferation in vitro and arrested these cells in mitosis with an IC50 of about 43 nM. Similar results were observed with primary human cells (HUVEC, IC50 = 30 nM), whereas the cancer cell line HeLa was more sensitive (IC50 of 9 nM). Further analysis revealed that prolonged mitotic arrest resulted in cell death for about 40% of cardiac fibroblasts. The remaining cells showed an interphase morphology with mostly multi- and micro-nucleated nuclei. This indicates that a significant number of primary fibroblasts are able to escape BI 2536 induced mitotic arrest and apparently become aneuploid. No effects were observed on cardiomyocytes and hypertrophic response (growth) upon endothelin-1 and phenylephrine stimulation was normal in the presence of BI 2536. This indicates that BI 2536 has no adverse effects on terminally differentiated cells and still allows proliferation independent growth induction in these cells. In conclusion, cardiomyocytes could be enriched using BI 2536, but the formation of aneuploidy in proliferating cells most likely limits this in vitro application and does not allow its use in putative cell based therapies.     

Suppression of Polo like kinase 1 (PLK1) by p21(Waf1) mediates the p53-dependent prevention of caspase-independent mitotic death

Polo-like kinase 1 (Plk1) plays key roles in many aspects of mitosis. We have previously shown that induction of p21^Waf1 by p53 is responsible for protection of cells against adriamycin-induced polyploidy formation and mitotic catastrophe. Here we show that adriamycin treatment suppressed Plk1 expression in a p53- and p21^Waf1-dependent manner. Ablation of p21^Waf1 inhibited the adriamycin-induced p53 activation, and this inhibition was alleviated by knockdown of Plk1, suggesting that p21^Waf1-dependent suppression of Plk1 expression is responsible for maintaining p53 activation during stress response. Plk1 associated with p53 and disrupted its interaction with target gene promoters in cells treated with adriamycin. Overexpression of Plk1 inhibited the p53-mediated prevention of caspase-independent mitotic death, but not polyploidy formation, in adriamycin-treated cells. Together our results indicate that suppression of Plk1 by p21^Waf1 is responsible for p53-dependent protection against adriamycin-induced caspase-independent mitotic death.     

Screening Compounds with a Novel High-Throughput ABCB1-Mediated Efflux Assay Identifies Drugs with Known Therapeutic Targets at Risk for Multidrug Resistance Interference

ABCB1, also known as P-glycoprotein (P-gp) or multidrug resistance protein 1 (MDR1), is a membrane-associated multidrug transporter of the ATP-binding cassette (ABC) transporter family. It is one of the most widely studied transporters that enable cancer cells to develop drug resistance. Reliable high-throughput assays that can identify compounds that interact with ABCB1 are crucial for developing new therapeutic drugs. A high-throughput assay for measuring ABCB1-mediated calcein AM efflux was developed using a fluorescent and phase-contrast live cell imaging system. This assay demonstrated the time- and dose-dependent accumulation of fluorescent calcein in ABCB1-overexpressing KB-V1 cells. Validation of the assay was performed with known ABCB1 inhibitors, XR9576, verapamil, and cyclosporin A, all of which displayed dose-dependent inhibition of ABCB1-mediated calcein AM efflux in this assay. Phase-contrast and fluorescent images taken by the imaging system provided additional opportunities for evaluating compounds that are cytotoxic or produce false positive signals. Compounds with known therapeutic targets and a kinase inhibitor library were screened. The assay identified multiple agents as inhibitors of ABCB1-mediated efflux and is highly reproducible. Among compounds identified as ABCB1 inhibitors, BEZ235, BI 2536, IKK 16, and ispinesib were further evaluated. The four compounds inhibited calcein AM efflux in a dose-dependent manner and were also active in the flow cytometry-based calcein AM efflux assay. BEZ235, BI 2536, and IKK 16 also successfully inhibited the labeling of ABCB1 with radiolabeled photoaffinity substrate [¹²⁵I]iodoarylazidoprazosin. Inhibition of ABCB1 with XR9576 and cyclosporin A enhanced the cytotoxicity of BI 2536 to ABCB1-overexpressing cancer cells, HCT-15-Pgp, and decreased the IC₅₀ value of BI 2536 by several orders of magnitude. This efficient, reliable, and simple high-throughput assay has identified ABCB1 substrates/inhibitors that may influence drug potency or drug-drug interactions and predict multidrug resistance in clinical treatment.     

Balancing the kinetochore ledger

Reduction of polo-like kinase-1 (Plk1) at kinetochores as cells progress from prometaphase to metaphase is surprising given that the kinase is thought to stabilize kinetochore-microtubule (kt-MT) attachments. In this issue, Liu et al. (2012. J. Cell Biol. doi:10.1083/jcb.201205090) demonstrate that kinetochore-associated Plk1 is a potent suppressor of microtubule plus-end dynamics. The authors propose that Plk1 activity facilitates the establishment of kt-MT attachments in prometaphase by stabilizing microtubules and that reduction of the kinase in metaphase promotes force generation by dynamic microtubules.     

Inhibition of Plk1 induces mitotic infidelity and embryonic growth defects in developing zebrafish embryos

Polo-like kinase 1 (Plk1) is central to cell division. Here, we report that Plk1 is critical for mitosis in the embryonic development of zebrafish. Using a combination of several cell biology tools, including single-cell live imaging applied to whole embryos, we show that Plk1 is essential for progression into mitosis during embryonic development. Plk1 morphant cells displayed mitotic infidelity, such as abnormal centrosomes, irregular spindle assembly, hypercondensed chromosomes, and a failure of chromosome arm separation. Consequently, depletion of Plk1 resulted in mitotic arrest and finally death by 6 days post-fertilization. In comparison, Plk2 or Plk3 morphant embryos did not display any significant abnormalities. Treatment of embryos with the Plk1 inhibitor, BI 2536, caused a block in mitosis, which was more severe when used to treat plk1 morphants. Finally, using an assay to rescue the Plk1 morphant phenotype, we found that the kinase domain and PBD domains are both necessary for Plk1 function in zebrafish development. Our studies demonstrate that Plk1 is required for embryonic proliferation because its activity is crucial for mitotic integrity. Furthermore, our study suggests that zebrafish will be an efficient and economical in vivo system for the validation of anti-mitotic drugs.     

Centrosomal protein 55 (Cep55) stability is negatively regulated by p53 protein through Polo-like kinase 1 (Plk1)

Centrosomal protein 55 (Cep55), which is localized to the centrosome in interphase cells and recruited to the midbody during cytokinesis, is a regulator required for the completion of cell abscission. Up-regulation of Cep55 and inactivation of p53 occur in the majority of human cancers, raising the possibility of a link between these two genes. In this study we evaluated the role of p53 in Cep55 regulation. We demonstrated that Cep55 expression levels are well correlated with cancer cell growth rate and that p53 is able to negatively regulate Cep55 protein and promoter activity. Down-regulation of expression of Cep55 was accompanied by repression of polo-like kinase 1 (Plk1) levels due to p53 induction. Overexpression of Plk1 and knockdown of p53 expression both enhanced the post-translational protein stability of Cep55. BI 2356, a selective Plk1 inhibitor, however, prevented Cep55 accumulation in p53 knockdown cells while persistently keeping Plk1 levels elevated. Our results, therefore, indicate the existence of a p53-Plk1-Cep55 axis in which p53 negatively regulates expression of Cep55, through Plk1 which, in turn, is a positive regulator of Cep55 protein stability.     

Cell Surface Area and Membrane Folding in Glioblastoma Cell Lines Differing in PTEN and p53 Status

Glioblastoma multiforme (GBM) is characterized by rapid growth, invasion and resistance to chemo−/radiotherapy. The complex cell surface morphology with abundant membrane folds, microvilli, filopodia and other membrane extensions is believed to contribute to the highly invasive behavior and therapy resistance of GBM cells. The present study addresses the mechanisms leading to the excessive cell membrane area in five GBM lines differing in mutational status for PTEN and p53. In addition to scanning electron microscopy (SEM), the membrane area and folding were quantified by dielectric measurements of membrane capacitance using the single-cell electrorotation (ROT) technique. The osmotic stability and volume regulation of GBM cells were analyzed by video microscopy. The expression of PTEN, p53, mTOR and several other marker proteins involved in cell growth and membrane synthesis were examined by Western blotting. The combined SEM, ROT and osmotic data provided independent lines of evidence for a large variability in membrane area and folding among tested GBM lines. Thus, DK-MG cells (wild type p53 and wild type PTEN) exhibited the lowest degree of membrane folding, probed by the area-specific capacitance C _m = 1.9 µF/cm². In contrast, cell lines carrying mutations in both p53 and PTEN (U373-MG and SNB19) showed the highest C _m values of 3.7–4.0 µF/cm², which corroborate well with their heavily villated cell surface revealed by SEM. Since PTEN and p53 are well-known inhibitors of mTOR, the increased membrane area/folding in mutant GBM lines may be related to the enhanced protein and lipid synthesis due to a deregulation of the mTOR-dependent downstream signaling pathway. Given that membrane folds and extensions are implicated in tumor cell motility and metastasis, the dielectric approach presented here provides a rapid and simple tool for screening the biophysical cell properties in studies on targeting chemo- or radiotherapeutically the migration and invasion of GBM and other tumor types.