Chk1 and Wee1 control genotoxic-stress induced G2–M arrest in melanoma cells

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Chk1 and Wee1 control genotoxic-stress induced G2–M arrest in melanoma cells Julio Vera a,1, Yvonne Raatz b,1, Olaf Wolkenhauer c, Tina Kottek b, Animesh Bhattacharya b, Jan C. Simon b, Manfred Kunz b,⁎ a Laboratory of Systems Tumor Immunology, Department of Dermatology, University Hospital Erlangen and Friedrich-Alexander-University Erlangen-Nürnberg, Ulmenweg 18, 91054 Erlangen, Germany b Department of Dermatology, Venereology and Allergology, University of Leipzig, Philipp-Rosenthal-Str. 23, 04103 Leipzig, Germany c Department of Systems Biology & Bioinformatics, University of Rostock, Ulmenstrasse 69, 18057 Rostock, Germany

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Article history: Received 12 January 2015 Accepted 31 January 2015 Available online xxxx Keywords: Tumour biology Cell signalling Cell cycle Chk1 Wee1

a b s t r a c t In the present report, the role of ATR–Chk1–Wee1 and ATM–Chk2–p53–p21 pathways in stress-induced cell cycle control is analysed in melanoma cells. Treatment of p53 wild-type melanoma cells with the genotoxic agent doxorubicin induces G2–M arrest, inhibitory phosphorylation of cell cycle kinase Cdc2 (CDK1) and enhanced expression of p53/p21. Wee1 inhibition under doxorubicin pulse-treatment reduces G2– M arrest and induces apoptosis. Inhibition of upstream kinase Chk1 under doxorubicin treatment almost completely abolishes stress-induced G2–M arrest and induces enhanced apoptosis. Interestingly, Chk1 inhibition alone even further increases apoptosis. While Chk1 inhibition alone almost completely abolishes G0–G1 arrest, combined treatment with doxorubicin re-establishes G0–G1 arrest. Moreover, Chk1 inhibition alone induces only a slight p53/p21 induction, while a strong induction of both proteins is observed by the combination with doxorubicin. These findings are suggestive for a particular role of p53/p21 in G0–G1, and Chk1 in G0–G1 and G2–M arrest. In line with this, the p53-mutant SK-Mel-28 melanoma cells do not mount a significant G0–G1 arrest under combined doxorubicin and Chk1 inhibitor treatment but rather show extensive apoptosis. Moreover, knockdown of p21 dramatically reduces stress-induced G0–G1 arrest under doxorubicin and Chk1 inhibitor treatment accompanied by massive DNA damage and apoptosis induction. Treatment of melanoma cells with an inhibitor of Chk2 upstream kinase ATM and doxorubicin almost completely abolishes G0–G1 arrest. Taken together, both Chk1 and Wee1 are mediators of G2–M arrest, while p53, p21 and Chk1 are mediators of G0–G1 arrest in melanoma cells. Combined treatment with chemotherapeutic agents such as doxorubicin and Chk1 inhibitors may help to overcome apoptosis resistance of p53-proficient melanoma cells. But treatment with Chk1 inhibitor alone may even be more efficient. © 2015 Elsevier Inc. All rights reserved.

1. Introduction Malignant melanoma is a tumour of high metastatic potential and treatment resistance in the metastatic stage [1,2]. Overall response rates to classical chemotherapeutic agents had been disappointing in the past [3]. In more recent years, improved treatment response and overall survival of melanoma patients have been achieved in patients with the activating BRAF (V600E) mutation using specific BRAF inhibitors such as vemurafenib and dabrafenib [4]. However, activating mutations in the BRAF gene at the V600 position were found in only half of all cases. In more than 30% of all melanomas no as yet targetable genetic

⁎ Corresponding author. Tel.: +49 341 9718610; fax: +49 341 9718609. 1 Both authors contributed equally to this work.

alterations have been identified [5]. Even more important, the majority of patients with initial treatment success experience recurrences [4,6]. Thus, further molecular mechanisms involved in tumour growth and progression in melanoma besides activated oncogenes such as BRAF might serve as therapeutic targets. Well-controlled cell cycle checkpoints and DNA damage repair mechanisms are indispensible for growth and survival of normal as well as cancerous cells. The cell cycle is controlled by three major checkpoints, G1–S, intra-S and G2–M, which avoid pre-mature entry into cell cycle phases [7–9]. The central step of cell cycle progression from G2 into mitosis is mediated by activation of the mitosis-promoting cyclinB/Cdc2 complex. Cdc2 is negatively regulated by phosphorylation on the tyrosine 15 (Y15) residue by the upstream kinases Wee1 and Mik1, which cooperate in their inhibitory activity [10,11] or by checkpoint kinase 1 (Chk1)-mediated inhibition of Cdc25 phosphatases,

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which remove the inhibitory phosphorylation on Cdc2. Both pathways Chk1–Cdc25 and Wee1–Cdc2 thus converge on Cdc2 [12]. At least in Xenopus laevis, Chk1 also directly phosphorylates and activates Wee1, but this had not yet been directly shown in mammalian cells [12]. Upstream activation of Chk1 is mediated by ataxia teleangiectasia mutated (ATM) and Rad3-related kinase (ATR) [13]. A substantial body of evidence has been provided that Wee1 plays an important role in the pathogenesis of different cancers and may be targeted using specific small molecule inhibitors [13]. In p53-deficient glioblastoma cells, Wee1 was one of the top over-expressed cell cycle molecules [14]. G2–M checkpoint control in these cells basically relied on Wee1 activity. Inhibition of Wee1 led to massive cell death under genotoxic stress. In another study, knockdown of Chk1 and Wee1, respectively, sensitised p53-deficient HeLa cells to stressinduced apoptosis mediated by the chemotherapeutic agent doxorubicin [15]. Moreover, 17-demethoxy-17-(2-propenylamino)geldanamycin (17-AAG), a chemical inhibitor of 90-kDa heat shock protein, decreased the half life of Wee1 and abrogated the G2–M checkpoint induced by treatment with SN-38, the active component of chemotherapeutic agent irinotecan, in p53-null HCT116 colon cancer cells [16]. The relevance of Wee1 for cancer cell survival under stress conditions was further emphasised in another study on ovarian cancers, which generally express only low levels of Kruppel-like factor 2 (KLF2), a negative regulator of Wee1. Re-introduction of KLF2 in different ovarian cancer cell lines repressed Wee1 expression and increased sensitivity to DNA damage-induced apoptosis [17]. Together, these findings support the notion that p53-deficient cells are largely dependent on Chk1- and Wee1-mediated G2–M checkpoint control. Consequently, this pathway was regarded as a therapeutic target for new treatment approaches in different cancers [13,18]. However, the situation for p53-proficient cancers is less clear. The impact of p53 on G2–M checkpoint control, Weel activity, Cdc2 Y15-phosphorylation and Cdc2 kinase activity was analysed in temperature-sensitive p53-mutant T-cell lymphoma cells [19]. In these experiments, temperature-dependent p53 activation resulted in the down-regulation of Weel expression, dephosphorylation of Cdc2 and enhanced apoptosis. Moreover, a parallel increase in Cdc2 kinase activity, which inactivates Wee1 via a negative feedback loop, was observed during p53-mediated apoptosis. Negative regulation of Weel expression and Cdc2 phosphorylation was also shown in cells from thymus tissues after whole body ionising radiation of p53+/+ mice, but not in cells from p53−/− mice. Based on these findings, it was concluded that p53-mediated G2–M checkpoint inactivation may contribute to the tumour suppressor activity and apoptosis via p53 pathways, although the precise mechanisms on how p53 downmodulates Wee1 remain to be defined. Interestingly, p53 has also been shown to downmodulate Chk1, which required p21 and retinoblastoma protein, giving p21 and Chk1 a complementary role in G2–M arrest [20]. Together, both p53 and Wee1 may be involved in G2–M checkpoint control. In line with this, evidence has been provided that G2–M checkpoint control in p53-proficient tumour cells was mediated by both p53 and Chk1 [21]. In the presence of wild-type p53, G2–M checkpoint control involved the Chk1 pathway, which may have therapeutic consequences regarding the use of Chk1 and Wee1 inhibitors in tumours. In the present report, the contribution of Chk1, Wee1, p53 and p21 to G2–M checkpoint and apoptosis control in melanoma cells under genotoxic stress was investigated. It is shown that melanoma cells activate the Chk1–Wee1–Cdc2 pathway to arrest cells in G2–M. Inhibition of Chk1 and Wee1, respectively, under genotoxic stress reduced G2–M arrest and induced apoptosis. Chk1 inhibition alone was even more effective in apoptosis induction. These findings are suggestive for Chk1 and Wee1 as central molecules in G2–M arrest and apoptosis protection in melanoma, which may open new therapeutic perspectives for this tumour.

2. Materials and methods 2.1. Cell lines and drug treatment Three different human melanoma cell lines were used with a different p53 status (SK-Mel-28, p53-mutant; SK-Mel-147 and A375, p53 wild-type). The SK-Mel-147 cell line was kindly provided by M. Soengas, Department of Dermatology, University of Michigan, Ann Arbor, MI, U.S.A. [22]; SK-Mel-28 and A375 were kindly provided by J. Eberle, Department of Dermatology, Venereology and Allergology, University of Berlin, Charité, Campus Mitte, Berlin, Germany. Cell cultures were maintained in DMEM medium supplemented with 10% foetal calf serum and 100 μg penicillin/streptomycin ml−1. Treatment of cells was carried out 24 h after cell plating and at an approximately 70–80% cell confluency. Doxorubicin (Sigma-Aldrich, Taufkirchen, Germany) was dissolved in sterile water, while Wee1 inhibitor II (Chemicon, Millipore, Hofheim, Germany) and Chk1 inhibitor CHIR-124 (Axon Medchem, Groningen, The Netherlands) were dissolved in dimethyl sulfoxide (DMSO). Chk2 inhibitor II (cat. no. C3742, Sigma-Aldrich), ATM inhibitor 55933 (cat. no. S1092, Selleck Chemicals, Houston, TX, U.S.A.) and PLK inhibitor GW843682X (cat. no. G2171, Sigma-Aldrich) were also dissolved in DMSO. Cells were treated with 250 nM doxorubicin for 1 h to induce DNA damage. After drug removal, cells were rinsed and cultured until sampling. Treatment with Wee1 inhibitor II, CHIR-124, Chk1 and Chk2 inhibitor started 30 min prior induction of DNA damage, followed by continued incubation until sampling. Control cultures in experiments were treated with dimethyl sulfoxide only. Human dermal fibroblasts were isolated from forskin and maintained as decribed [23]. siRNAs directed against TP53 and CDKN1A (p21) as well as non-targeting siRNA were purchased from Ambion life technologies (Darmstadt, Germany), part number 4427038: siID TP53, s605; siID CDKN1A, s416). Cell transfection was performed 24 h before start of the experiments using Lipofectamine® 2000 (life technologies) as transfection reagent. 2.2. Quantitative immunoblotting Total cell extracts were prepared by incubating PBS-washed cell cultures for 20 min at 4 °C in lysis buffer containing 20 mM Tris HCl (pH 8), 137 mM NaCl, 10% glycerol, 1% NP-40, 2 mM EDTA, 1 mM sodium orthovanadate, 1 mM NaF, and protease inhibitor cocktail (Roche Diagnostics, Mannheim, Germany). Lysates were clarified by centrifugation, and the protein concentration of the supernatant was determined by BCA protein assay (Pierce; Rockford, Illinois, USA). 40 μg of lysate was denatured in 5× sample buffer containing 125 mM DTT. Proteins were separated on 9% or 12% SDS–polyacrylamide gels by electrophoresis and transferred to nitrocellulose membranes (Protran BA83, Whatman, Dassel, Germany) by electroblotting. Membranes were blocked for 1 h with Odyssey blocking buffer (LI-COR Biosciences; Bad Homburg, Germany) and incubated for 2 h with primary antibodies against p53 (554293, BD Biosciences, Heidelberg, Germany), p21 (BD556431, BD Biosciences), Wee1 (sc-5285, Santa Cruz Biotechnologies), phosphoTyr15 Cdc2 (sc-7989, Santa Cruz Biotechnology, Heidelberg, Germany), Cdc2 (Acris GmbH, Herford, Germany), cyclin B1 (sc-245, Santa Cruz Biotechnology), and β-actin (4967, Cell Signaling, New England Biolabs, Frankfurt a.M., Germany), phospho(γ)-H2AX (Ser139, sc-101696, Santa Cruz Biotechnology), and phospho-Rb (Ser780, sc-12901; Santa Cruz Biotechnology). Signal detection was performed by incubating membranes for 1 h with appropriate secondary IRDye 680 labelled goat anti-mouse or IRDye 800CW labelled goat anti-rabbit antibodies (LICOR Biosciences). Bands were imaged and quantified by Odyssey Infrared Imaging system (LI-COR Biosciences) with use of two-colour fluorescence detection at 700 and 800 nm. Densitometry values were normalised to β-actin values.

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2.3. Cell cycle analysis Cell cycle profiles were determined by propidium iodide staining of DNA content and flow cytometry. Cells were harvested by trypsinisation at indicated time points, washed with ice-cold PBS, fixed in ice-cold 70% ethanol for at least 30 min at −20 °C. To label DNA, cells were resuspended in PBS containing 50 μg/ml propidium jodide (PI) and 1 mg/ ml RNase, and incubated for 40 min at room temperature. Incorporation of PI into DNA was analysed by flow cytometry using a FACSCalibur® flow cytometer (BD Biosciences). Cell cycle distribution in G0–G1, S and G2–M phase was quantified and expressed as percentage of the total cell population. 2.4. Cell cycle synchronisation Synchronisation of cells at the G1–S boundary was achieved by a double thymidine block. Therefore, exponentially growing cells at 40– 50% confluence were treated with 2 mM thymidine (Sigma-Aldrich) for 18 h. Following three washes with PBS, the cells were cultured in normal growth medium for 8 h. Thereafter, the cells were cultured again in medium supplemented with 2 mM thymidine for 15 h, washed three times with PBS and released into normal medium. Cells were analysed at various time intervals and total cell extracts of protein were prepared for immunoblotting. 2.5. Statistics Differences in percentages of cells in different phases of cell cycle were analysed by unpaired t-test. P ≤ 0.05 was regarded as statistically significant. 3. Results 3.1. Genotoxic stress induces activation of Wee1–Cdc2 pathway in melanoma cells To analyse the pathways involved in G2–M arrest in melanoma cells, which might serve as future treatment targets, melanoma cell lines were treated with 250 nM of genotoxic-stress inducing agent doxorubicin for

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1 h followed by 23 h of culture under normal conditions without doxorubicin. In preliminary experiments, this pulse-treatment induced maximum G2–M arrest compared with different other concentrations (10 to 500 nM) and treatment durations (1 to 24 h). Doxorubicin treatment induced G2–M arrest in both melanoma cell lines SKMel-147 (p53 wild-type) and SK-Mel-28 (p53-mutant; carrying the functionally inactive TP53 p.R145L mutation) and control cells (benign fibroblasts). Although all cell types mounted a significant G2– M arrest, this arrest was most prominent in SK-Mel-28 cells, where it was accompanied by an almost complete loss of G0–G1 (Fig. 1A). Expression of cell cycle regulators p21, p53, Wee1, Cdc2, Cdc2-Y15p (pCdc2), and DNA damage marker γ-H2AX was analysed by immunoblotting. As shown in Fig. 1B, doxorubicin led to a reduction in Wee1 levels after 24 h, with maximum downregulation in SK-Mel-147 cells, which also showed significant downregulation of Cdc2 and Cdc2– Y15p (pCdc2). In contrast, Cdc2–Y15p was upregulated in SK-Mel-28 cells. p53 and p21 were both induced in SK-Mel-147 cells but, as expected, were either not induced in case of p53 in p53-mutant or not expressed in case of p21 in SK-Mel-28 melanoma cells. Although p53-proficient SK-Mel-147 cells showed similar reaction patterns compared with benign fibroblasts, almost complete downregulation of Wee1, Cdc2 and Cdc2–Y15p after 24 h was somewhat surprising (Fig. 1B). For a more detailed analysis of these findings, time course experiments were performed, and expression of p21, p53, Wee1, Cdc2Y15p and Cdc2 was analysed by immunoblotting (Fig. 2 and Supplemental Fig. 1). Doxorubicin treatment induced transient induction of Wee1 and phosphorylation Cdc2 at 5–7 h in SK-Mel-147. p53 expression was also induced with a maximum at 5–7 h and remained elevated until 24 h. In contrast, a constant increase in p21 was observed with maximum expression after 24 h. Similar results were obtained for A375, another p53-proficient melanoma cell line (Supplemental Fig. 1). In contrast, p53-mutant SK-Mel-28 cells showed constantly increasing Cdc2 phosphorylation after 5 h up to 24 h (Fig. 2B). Thus, Wee1–Cdc2 pathway is transiently activated in p53 wild-type melanoma cells, while p53–p21 shows prolonged induction. However, as shown in SK-Mel-28 cells, significant G2–M arrest was achieved also in the absence of functional p53–p21 pathway. Thus, p53 wild-type and p53-mutant cells seem to differ in their regulation of molecules involved in G2–M arrest.

Fig. 1. Activation of p53–p21 and Wee1–Cdc2 pathways in human fibroblasts, p53-mutated and p53 wild-type melanoma cell lines, respectively, under genotoxic stress. (A) Benign human fibroblasts, p53-mutant SK-Mel-28 and p53 wild-type SK-Mel-147 melanoma cell lines, respectively, were pulse-treated with 250 nM doxorubicin for 1 h. Twenty four hours later, cell cycle analysis was performed by flow cytometry after staining of melanoma cells with propidium iodide (PI). Diagrams show the percentage of cells in G0–G1 (2n) and G2–M (4n). Control, without treatment; Doxorubicin, doxorubicin treatment. (B) Parallel to cell cycle analysis as described in (A), whole cell extracts were subjected to quantitative immunoblotting for indicated proteins. Numbers above blots indicate fold changes of protein expression compared with controls.

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Fig. 3. Influence of genotoxic stress and PLK1 inhibition on Wee1, Cdc2–Yp15 and Cdc2 expression in melanoma cells. (A) Melanoma cell line SK-Mel-147 was pulse-treated with 250 nM doxorubicin for 1 h and treated in parallel with PLK1 inhibitor GW843682X for 24 h. Whole cell extracts were prepared and subjected to quantitative immunoblotting for indicated proteins. Numbers above blots indicate fold changes of protein expression.

Fig. 2. Time course of p21, p53, Wee1, Cdc2–Yp15 and Cdc2 expression in p53 wild-type and p53-mutated melanoma cell lines under genotoxic stress. (A) p53 wild-type SKMel-147 and (B) p53-mutated SK-Mel-28 melanoma cell lines were pulse-treated with 250 nM doxorubicin for 1 h, and whole cell extracts were prepared at different time points and subjected to quantitative immunoblotting for indicated proteins. Numbers above blots indicate fold changes of protein expression.

To provide further evidence for the functional activity of p21, immunoblots were performed for phosphorylated retinoblastoma protein (pRB). p21 predominantly exerts its activity on cell cycle control via inhibition of phosphorylation of pRB. As shown in Supplemental Fig. 2, doxorubicin-induced p21 expression was paralleled by inhibition of pRB phosphorylation on Ser 780 in p53-proficient melanoma cell lines SK-Mel-147 and A375, and in human fibroblasts. Inhibition of pRB phosphorylation was not observed in SK-Mel-28 cells in the absence of p21. Thus, the data provided strong evidence that p21 is active under conditions of doxorubicin treatment of melanoma cells and exerts its function in p53-proficient melanoma cells at least in part via inhibition of pRB phosphorylation. To further substantiate the role of the Wee1–Cdc2 pathway in p53 wild-type melanoma cells, it was tested whether this pathway is also active during normal cell cycle progression. For this purpose, SK-Mel147 cells were first arrested in G1–S by double thymidine block. As shown in Supplemental Fig. 3, Y15p phosphorylation of Cdc2 increased up to 8 h after release of double thymidine block and decreased thereafter, parallel to the number of cells in G2–M. In contrast, expression of polo-like kinase 1 (PLK1), a major component of cell cycle re-entry after G2–M arrest, further increased after 8 h with highest expression when cells re-entered G0–G1. Similarly, cyclin B1, as a marker for mitotic cells, showed highest expression after 8 to 9 h. Overall, the expression patterns Cdc–Y15p and of Wee1 and correlated with the percentage of cells in G2–M and inversely correlated with the percentage of cells in G0–G1, suggestive for a regulatory activity of Wee1–Cdc2 in G2–M checkpoint control under normal conditions. Taken together, Wee1– Cdc2 pathway seems to exert cell cycle control of p53 wild-type melanoma cells under stress and normal conditions. 3.2. Polo-like kinase 1 (PLK1) is not responsible for Wee1 downregulation by genotoxic stress In a further series of experiments, the mechanisms underlying Wee1 downregulation and loss of Cdc2 phosphorylation under stress

conditions in p53 wild-type melanoma cells were analysed. Little is known about Wee1 regulation, but it is well-understood that PLK1 inactivates Wee1 by ubiquitination under normal conditions of G2–M transition. PLK1 may also inactivate Wee1 under genotoxic stress [24]. Melanoma cells were pulse-treated with doxorubicin and co-treated with PLK1 inhibitor GW843682X. As shown in Fig. 3, treatment with PLK1 inhibitor alone or in combination with doxorubicin reduced Wee1 expression and Cdc2 phosphorylation. Thus, PLK1 inhibitor could not counteract downmodulation of Wee1 and Cdc2–Y15p under genotoxic stress, as might have been expected. In contrast, combined treatment of doxorubicin and PLK1 inhibitor slightly increased Cdc2 expression compared with doxorubicin treatment alone. In line with this, doxorubicin treatment led to significant reduction in PLK1 expression in SK-Mel-147 melanoma cells (but not in SK-Mel-28 melanoma cells) after 24 h (Supplemental Fig. 2). This latter finding may be due to the fact that p53 is a known inhibitor of PLK1 transcription [25]. Taken together, these findings suggest that Wee1 and Cdc2–Yp15 downregulation in p53-proficient melanoma cells under genotoxic stress is independent of PLK1.

3.3. Inhibition of Wee1 kinase in p53 wild-type melanoma cells under genotoxic stress leads to enhanced and prolonged p53 expression and impairs cell cycle arrest in G2–M To further analyse the functional contribution of Wee1–Cdc2 pathway to G2–M arrest in p53 wild-type melanoma cells under stress conditions, melanoma cell lines were pulse-treated with 250 nM doxorubicin for 1 h and Wee1 activity was inhibited by a chemical inhibitor (Wee1 inhibitor II). As shown in Fig. 4A, doxorubicin-induced Y15 phosphorylation of Cdc2 was inhibited by co-treatment with Wee1 inhibitor. Inhibition of Wee1 led to enhanced and prolonged p53 expression as compared with doxorubicin alone (Figs. 2 and 4). However, there was no enhanced p21 induction, as determined by quantitative immunoblotting. Treatment with Wee1 inhibitor alone slightly reduced Cdc2 baseline phosphorylation but had no effect on p53 or p21 expression (data not shown). Combined treatment of doxorubicin and Wee1 inhibitor enhanced DNA damage, as determined by γ-H2AX expression (Fig. 4B). Parallel performed cell cycle analysis showed that the percentage of cells arrested in G2–M after doxorubicin treatment was significantly reduced by Wee1 inhibition (Fig. 4C). Similar data were obtained for A375 cells (data not shown). At the same time, Wee1 inhibition increased the number of cells in G0–G1 under doxorubicin treatment. Thus, the Wee1–Cdc2 pathway plays a role in G2–M arrest in p53 wild-type melanoma cells under genotoxic stress, and Wee1 inhibition induces p53 expression/activation, inhibits G2–M arrest and enhances G0–G1 arrest in these cells. Combined treatment of doxorubicin and Wee1 inhibitor

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in p53-mutant SK-Mel-28 cells (for 48 h) almost completely abrogated G0–G1 (data not shown). As shown in Fig. 4C, combined treatment of doxorubicin and Wee1 inhibitor also induced enhanced apoptosis (as determined by the number of cells in sub-G1) compared with doxorubicin and Wee1 inhibitor alone (Fig. 4C). Thus, loss of G2–M arrest under genotoxic stress is accompanied by enhanced DNA damage induction and induction of apoptosis. Taken together, Wee1–Cdc2 pathway is required for G2–M arrest and apoptosis protection in p53-proficient melanoma cells. The induction of G0–G1 under doxorubicin/Wee1 inhibitor treatment may represent a fail-safe mechanism to protect melanoma cells from excessive apoptosis when the G2–M arrest is affected. 3.4. Inhibition of Chk1 impairs G2–M and G0–G1 arrest and induces apoptosis in melanoma cells To address the role of genotoxic stress pathways upstream of Wee1 for G2–M arrest, melanoma cells were treated with doxorubicin either alone or in combination with Chk1 inhibitor CHIR-124. As shown in Fig. 5A, treatment with CHIR-124 alone showed an only slight and delayed induction of p53 and p21 expression after 24 h. However, combined treatment with doxorubicin and CHIR-124, as well as doxorubicin treatment alone, induced rapid p53 and p21 induction with a very similar time course and similar levels of expression (Figs. 5B and 1). Chk1 inhibitor treatment alone resulted in dramatically enhanced DNA damage, compared with both other treatment conditions, as indicated by γ-H2AX expression (Fig. 5C). In subsequent experiments, it was tested how these treatments and expression patterns of cell cycle molecules translated into cell cycle regulation. As shown in Fig. 6A, doxorubicin-induced G2–M arrest was almost completely reversed by co-treatment with CHIR124, accompanied by enhanced apoptosis (cells in sub-G1). A significant G0–G1 arrest was still maintained under these conditions. In contrast, G0–G1 arrest was almost completely abolished by CHIR124 treatment alone, and the rate of apoptotic cells was even further enhanced. We reasoned that doxorubicin-induced p53/p21 expression might support G0–G1 arrest and confer relative apoptosis protection in melanoma cells under stress conditions induced by doxorubicin and CHIR-124. Thus, treatment of p53-proficient melanoma cells with Chk1 inhibitor alone might represent a more efficient treatment option than combined treatment with the genotoxic agent doxorubicin. Moreover, these findings were strongly suggestive for a particular role of p53/p21 in maintaining G0–G1 arrest in melanoma cells under genotoxic stress. To further substantiate these findings, cell cycle analyses were performed for p53-mutant SK-Mel-28 cells which lack p21 expression. As shown in Fig. 6B, combined treatment with doxorubicin and Chk1 inhibitor CHIR-124 induced a strong apoptotic response (more than 30% of cells). Treatment with CHIR-124 alone induced slightly lower numbers of apoptotic cells in SK-Mel-28 cells. Importantly, SK-Mel-28 cells were not able to establish a G0–G1 arrest by combined doxorubicin/ CHIR-124 treatment, but cells rather underwent extensive apoptosis. Taken together, these findings show that doxorubicin supports the establishment of G0–G1 arrest in p53-proficient melanoma cells when Chk1 activity is compromised, but not in p53-mutant melanoma cells. In a further series of experiments, p53 and p21 knockdown melanoma cells were used after transient transfection of melanoma cells with siRNAs directed against both proteins, and cells were treated with a combination of doxorubicin and CHIR-124. siRNA treatment significantly reduced protein expression of both proteins (Supplemental Fig. 4). As shown in Fig. 7A/B, after 48 h of treatment, G0–G1 arrest under doxorubicin and Chk1 inhibition was dramatically reduced by p53 and p21 knockdown. Thus, both p53 and p21 are involved in G0–G1 arrest under doxorubicin-induced stress in melanoma cells. Under the conditions of p21 knockdown, there was a dramatic induction of apoptosis (more than 60% of cells). The apoptosis rate was lower for p53

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knockdown cells (45%). Overall, baseline and induced apoptosis rates in transfected cells were higher than in non-transfected cells, likely due to transfection conditions (Figs. 6, 7). p53 knockdown under the conditions of combined treatment induced massive DNA damage (Supplemental Fig. 4). Taken together, inhibition of Chk1 under genotoxic stress abolished G2–M arrest and induced apoptosis in melanoma cells, while Chk1 inhibitor alone was even more efficient regarding apoptosis induction in p53-deficient melanoma cells, apparently due to a failure to mount a significant G0–G1 arrest under these conditions. G0–G1 arrest under these conditions is largely dependent on p53/p21. 3.5. ATM–Chk2–p53–p21 pathway is not required for G2–M arrest in melanoma cells To address the role of ATM–Chk2, two well-known upstream activators of p53–p21 stress pathway, in DNA damage response in p53 wildtype melanoma cells, both proteins were inhibited by chemical inhibitors. As shown in Fig. 8, inhibition of Chk2 by Chk2 Inh II and ATM by KU55933 under doxorubicin-induced stress had no significant effects on G2–M arrest. In contrast, ATM inhibition by KU55933 even further enhanced the number of cells in G2–M. Chk2 inhibition under doxorubicin-induced stress significantly reduced the number of cells in G0–G1 (Fig. 8A), and G0–G1 arrest was almost completely abrogated by ATM inhibitor KU55933 (Fig. 8B). Inhibition of ATM by KU55933 induced high levels of apoptosis parallel to the abrogation of the G0–G1 arrest. Taken together, in p53 wild-type melanoma cells, G2–M arrest remained undisturbed by inhibition of the ATM–Chk2 pathway in case of functional ATR–Chk1 and Wee1–Cdc2 pathways. However, a significant number of cells underwent apoptosis after ATM inhibition, parallel to a dramatic reduction of cells in G0–G1, arguing for an exit of cells from cell cycle during G1–S towards apoptosis. Thus, both ATM and Chk2 seem to be dispensable for G2–M arrest in p53 wild-type melanoma cells under stress conditions used in our experiments. Supplemental Fig. 5 provides a schematic representation of the pathway regulation of ATR/ATM stress pathways under doxorubicin in melanoma. 3.6. Inhibition of ATR–Chk1 and ATM–Chk2 pathways under stress conditions has little effects on fibroblast apoptosis In a series of control experiments using benign fibroblasts, the majority of experimental conditions were recapitulated for these cells in order to evaluate the potential side-effects of the treatment conditions in humans. Benign fibroblasts showed a reduction in G0–G1 phase after 24 h by CHIR-124 treatment alone and a slight reduction in G2–M by the combined treatment of doxorubicin and CHIR-124 regarding G2–M arrest (Supplemental Fig. 6). However, the effects were much less impressive as compared with melanoma cells. Similarly, the reduction in G0–G1 arrest under combined treatment with doxorubicin and ATM inhibitor KU55933 was less dramatic. The most important finding was that fibroblasts only slightly increased the apoptosis rate (cells in sub-G1) under single and combined treatment. Cells in subG1 under different conditions were always below 10% (Supplemental Fig. 6). Thus fibroblasts appear to mount a more robust cell cycle arrest at G–G1 and G2–M and are less sensitive for apoptosis induction under the mentioned treatment conditions. 4. Discussion In the present study, G2–M checkpoint control was analysed in melanoma cells under genotoxic stress conditions. It is demonstrated that members of the ATR–Chk1–Wee1–Cdc2 pathway are involved in stress-induced G2–M arrest and apoptosis protection of melanoma cells. In contrast, the ATM–Chk2–p53–p21 pathway seems to play no role in G2–M arrest induced by doxorubicin pulse-treatment but protects melanoma cells from apoptosis by induction of G0–G1 arrest.

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Fig. 4. Time course of p53 and p21 expression and cell cycle distribution of melanoma cells after exposure to genotoxic stress and Wee1 inhibition. (A/B) SK-Mel-147 melanoma cell line was pulse-treated with 250 nM doxorubicin for 1 h and with Wee1 Inh II for 24 h. Whole cell extracts were prepared at different time points and subjected to quantitative immunoblotting for indicated proteins. (C) Cell cycle analysis from cells in (A/B) was performed after 48 h by flow cytometry after staining of melanoma cells with propidium iodide (PI). Diagrams show the percentage of cells in G2–M (4n). Wee1 Inh II, Wee1 inhibitor II. Numbers above blots indicate fold changes of protein expression. Data are given as mean +/− SD of three independent experiments; unpaired t-test was used to analyse statistical significance. *, P value ≤ 0.05; **, P value ≤0.01; ***, P value ≤0.001; n.s., not significant. Doxo, doxorubicin.

Fig. 5. Time course of p53 and p21 expression in melanoma cells after exposure to genotoxic stress and Chk1 inhibition. (A-C) Melanoma cell line SK-Mel-147 was pulsetreated with 250 nM doxorubicin for 1 h and with Chk1 inhibitor CHIR-124 for 24 h. Whole cell extracts were prepared at different time points and subjected to quantitative immunoblotting for p53, p21 and γ-H2AX.

Interestingly, enhanced induction of p53 was observed when Wee1 kinase was inhibited under doxorubicin-induced genotoxic stress. However, induction of p53 under these conditions did not enhance G2–M arrest, but rather enhanced G0–G1 arrest. Inhibition of Chk1 under genotoxic stress dramatically reduced G2–M arrest and induced apoptosis. Interestingly, Chk1 inhibition alone almost completely inhibited both G2–M and G0–G1 arrest, leading to further enhanced apoptosis. p53/p21 knockdown or absence of p21 in p53-mutant melanoma cells in these experiments reduced G0–G1 arrest, further supporting the role of p53/p21 in G0–G1 arrest in melanoma cells under genotoxic stress. Finally, inhibition of the ATM–Chk2 pathway under stress conditions dramatically reduced G0–G1 arrest and induced enhanced apoptosis, but had no influence on G2–M arrest. Together, the presented findings emphasise the role of Wee1 and Chk1 in G2–M checkpoint control in melanoma, which might be of future therapeutic relevance as inhibitors of this pathway have already been tested in clinical trials of different other cancers [13,18,26]. A majority of cancers have a defective p53 tumour suppressor pathway, and it is widely accepted that p53-defective tumour cells rely on the ATR–Chk1–Cdc25/Wee1–Cdc2 pathway for establishment of a G2– M arrest as a central mechanism for cell cycle control and DNA repair under genotoxic stress [9,25]. Inhibition of ATR pathway in these cells sensitises malignant tumours to chemotherapeutic agents [13,18,19, 26,27]. In a recent study on p53-deficient glioblastoma cells, Wee1 topped the ranking of overexpressed kinases [14]. Inhibition of Wee1 by siRNA or a small molecule inhibitor in the glioblastoma cell line U251MG exposed to DNA damaging agents resulted in abrogation of the G2–M arrest and cell death. Although not directly shown for eukaryotic cells, a plethora of experimental evidence suggests that ATR–Chk1 pathway may exert its effects via phosphorylation of both Cdc25 and Wee1 [12]. In the present report, Wee1 significantly contributed to G2–M arrest in p53 wild-type (SK-Mel-147, A375) melanoma cells. Wee1 inhibition

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Fig. 6. Cell cycle analysis of melanoma cells under genotoxic stress and Chk1 inhibition. (A/B) Melanoma cell lines SK-Mel-147 and SK-Mel-28 were pulse-treated with 250 nM doxorubicin for 1 h and with Chk1 inhibitor CHIR-124 for 48 h. Cell cycle analysis was performed by flow cytometry after staining of melanoma cells with propidium iodide (PI). Data are given as mean +/− SD of three independent experiments; unpaired t-test was used to analyse statistical significance. *, P value ≤0.05; **, P value ≤0.01; ***, P value ≤0.001; n.s., not significant. Doxo, doxorubicin.

reduced G2–M arrest in these cells and led to enhanced apoptosis and p53 induction. Thus, Wee1 is involved in G2–M arrest and apoptosis protection in p53-proficient melanoma cells. The decision between cell cycle arrest and DNA repair or apoptosis seems to depend on the level and duration of p53 expression as described in a series of earlier

reports [28–31]. In the present analysis, p53 appears to support apoptosis and at the same time G0–G1 arrest. Downregulation of Wee1 and reduced phosphorylation of Cdc2 in p53 wild-type melanoma cells 24 h after genotoxic stress induced by doxorubicin pulse treatment was somewhat surprising, because Cdc2

Fig. 7. Cell cycle analysis of melanoma cells with knockdown of p53 or p21 under genotoxic stress and Chk1 inhibition. (A-C) Melanoma cell line SK-Mel-147 was transiently transfected with control siRNA (si Co), siRNA directed against p53(si p53) or siRNA against p21 (si p21). Subsequently, melanoma cells were pulse-treated with 250 nM doxorubicin for 1 h and with Chk1 inhibitor CHIR-124 for 48 h. Cell cycle analysis was performed by flow cytometry after staining of melanoma cells with propidium iodide (PI). Data are shown from one representative experiment of two independent experiments. *, P value ≤0.05; **, P value ≤0.01; ***, P value ≤0.001; n.s., not significant. Doxo, doxorubicin.

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Fig. 8. Cell cycle analysis of melanoma cells under genotoxic stress, Chk2 and ATM inhibition. (A/B) Melanoma cell line SK-Mel-147 was pulse-treated with 250 nM doxorubicin for 1 h and with Chk2 inhibitor II or ATM inhibitor KU55933 for 48 h. Cell cycle analysis was performed by flow cytometry after staining of melanoma cells with propidium iodide (PI). Data are given as mean +/− SD of three independent experiments; unpaired t-test was used to analyse statistical significance. *, P value ≤0.05; **, P value ≤0.01; ***, P value ≤0.001; n.s., not significant. Doxo, doxorubicin.

phosphorylation in benign human fibroblasts remained largely stable under these conditions. We reasoned that PLK1 might induce Wee1 degradation and subsequent reduced Cdc2 phosphorylation, as PLK1 plays an important role for re-entry of cell cycle after repair of DNA damage. PLK1 phosphorylates Wee1, targeting it for degradation. PLK1 is not only involved in Wee1 degradation after normal G2–M arrest, but under stress conditions [24,32]. Moreover, melanoma cells have been shown to express high levels PLK1 [33,34]. However, inhibition of PLK1 had no influence on Wee1 expression and activity in our experiments, and PLK1 expression was even downregulated under stress conditions. A reasonable explanation for these latter findings might be that melanoma cells expressed high levels of p53 after stress induction. p53, which is a well-known transcriptional repressor of PLK1, may thus downregulate PLK1 [25]. In line with our observations, activation of p53–p21 in murine T-cell lymphoma cells resulted in the downregulation of Weel expression, dephosphorylation of Cdc2 and enhanced apoptosis [19]. It was speculated that p53 might negatively impact on WEE1 gene transcription, although the precise mechanism remains to be defined. Thus, downmodulation of Wee1 in melanoma cells may be due to enhanced p53 activity. Wee1 degradation may also be activation-dependent as described recently [35]. Time course experiments in the present report showed that Cdc2 phosphorylation on Y15 started to decrease after 7 h in p53-proficient cells, while p53 expression was significantly enhanced. At the same time, induction of G2–M arrest was observed. Earlier reports showed that p53 and p21 support sustained G2–M arrest after DNA damage [36]. One feasible explanation for enhanced p53 activation under stress conditions and Wee1 inhibition might be, that in case of compromised G2–M arrest, DNA damage accumulates and finally leads to p53–p21 activation via DNA damage sensing Mre11–Rad50–Nb1 or ATR/ATRIP complex recruiting and activating ATM, ATR or both, which then activate p53–p21 pathway, similar to what was suggested by others for Chk1 inhibition [37–39]. By this means, ATM–Chk2–p53 may mount either enhanced G0–G1 or G2–M arrest. However, in our analyses inhibition of ATM and Chk2 under genotoxic stress inhibited G0–G1 arrest, but not G2–M arrest. The ATM–Chk2–p53–p21 and ATR–Chk1–Cdc25/Wee1–Cdc2 pathways are generally regarded as parallel and largely independent in the regulation of G2–M arrest, although both may converge on ATR kinase to activate Chk1 and p53 after UV irradiation [18]. Classically, p53 is directly activated via ATM and Chk2 induced by genotoxic stress. ATM activity mediates G0–G1 and G2–M arrest via Chk2 and p53 after DNA double-strand breaks [40]. ATR mediates G2–M arrest via Chk1 and Wee1 after single-strand breaks or unpaired single-stranded DNA, which may be part of stalled replication forks or appear during double

-strand break repair [39,41]. However, here we provided substantial evidence that ATM–Chk2–p53–p21 pathway is not involved in G2–M arrest in melanoma cells under genotoxic stress induced by doxorubicin, but G2–M arrest is mediated by ATR–Chk1 pathway. To further analyse the mechanisms underlying the establishment of G2–M arrest in melanoma cells, Wee1 upstream kinase Chk1 was inhibited by the chemical inhibitor CHIR-124. Chk1 inhibitor alone induced massive γ-H2AX expression, a surrogate marker for DNA damage. In line with this, one recent study showed that Chk1 inhibition under genotoxic stress induced strong γ-H2AX expression [21]. Earlier reports provided similar results showing that Chk1 inhibition in U2OS osteosarcoma cells led to phosphorylation of ATR targets H2AX, p53 and replication protein A (RPA) [37]. Authors further described inhibition of γ-H2AX expression after inhibition of Cdk2 and Cdc45, as markers for transcriptional initiation [35]. A model was proposed where Chk1 inhibition leads to Cdc25A stabilisation, accompanied by hyperactivation of Cdk2, loading of replication factor Cdc45, activation of replication origins, association of RPA and destabilisation of the genome with accumulation of DNA strand breaks with subsequent γ-H2AX expression [12,37]. Thus, Chk1 inhibition may induce γ-H2AX expression, but at least in melanoma cells, Chk1 inhibition alone is only a week activator of p53 expression. Chk1 inhibition alone induced dramatic reduction of G0–G1 arrest and slight induction of G2–M followed but high rates of apoptotic cells. In contrast, Chk1 inhibition in the presence of genotoxic stress induced by doxorubicin almost completely abrogated G2–M, while leaving G0–G1 arrest largely intact. Together, these findings suggest that Chk1 is the major regulator of G2–M under genotoxic stress, and doxorubicin induces significant G0–G1 arrest under Chk1 inhibition, likely due to enhanced p53 expression as compared with Chk1 inhibitor alone. In line with this, we observed an almost completely abolished G0– G1 arrest and significantly higher numbers of apoptotic cells in p53deficient (SK-Mel-28) cells than in p53 wild-type cells (SK-Mel-147) under genotoxic stress with doxorubicin after Chk1 inhibition. The relative apoptosis resistance of p53-proficient cells under these conditions might be explained by p53-induced G0–G1 arrest in melanoma cells under combined treatment, since p53-deficient cells were unable to mount a significant G0–G1 arrest under conditions of combined doxorubicin and Chk1 inhibitor treatment. Consequently, knockdown of p21, and to a lesser extend of p53, under combined treatment with doxorubicin and Chk1 inhibitor reduced G0–G1 arrest followed by dramatic induction of apoptosis, accompanied by high levels of γ-H2AX as indicator of massive DNA damage. Similar results were reported for PC3 prostate cancer cells by others [42]. Interestingly, effects of p53 knockdown were less striking than of p21 knockdown, a finding that cannot

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be explained at the moment. It might be due to p53-independent signalling mechanisms that activate p21 under genotoxic stress such as ATR– Chk1 [43]. In line with our findings, a recent study on the role of Chk1 in genotoxic stress-induced cell cycle arrest in U2OS osteosarcoma cells showed that knockdown of Chk1 in the presence of different chemotherapeutic agents such as doxorubicin, irinotecan, cisplatin and taxol significantly reduced cell viability in both p53-proficient and p53deficient cells [21]. Thus, Chk1 appears to be involved in G2–M arrest and apoptosis protection in both p53-proficient and p53-deficient tumour cells. Similar findings were reported in an earlier study, which described that temozolomide-induced G2–M checkpoint abrogation and toxicity in U87MG (glioblastoma) cells are independent of p53 status after abrogation of the G2–M checkpoint by a non-specific Chk1 inhibitor [44]. Moreover, treatment of HCT116 colon carcinoma cells with selective Chk1 inhibitor CHIR-124 abrogated the ionising-radiation induced G2–M arrest and reduced clonogenic survival of cells [45]. This radiosensitising effect was similar in p53-deficient and p53-proficient HCT116 cells. Overall, p53-proficient cells seem to be not more viable than p53-depleted cells when Chk1 is inhibited under conditions of DNA damage. However, a higher percentage of p53−/− HCT116 entered mitotic catastrophe compared with p53 wild-type cells which may lead to subsequent apoptosis and thus be in line with our findings of enhanced apoptosis in p53-deficient melanoma cells. Taken together, p53 wild-type and p53-mutant melanoma cells are sensitive to apoptosis induction by genotoxic stress and Chk1 inhibition, but Chk1 inhibition alone may even be more effective. Future treatment options should take into account that chemotherapeutic agents such as doxorubicin might even have some apoptosis protective effects in the presence of wild-type p53. Interestingly, in a recent report on non-transformed human fibroblasts and epithelial cells, p21, but not Chk1 or Chk2, were required for G2–M arrest [46]. However, in the same study, p21 was not involved in G2–M arrest of U2OS osteosarcoma cells under treatment with bleomycin and ICRF-193, a topoisomerase II inhibitor that induces G2– M arrest. Authors found evidence for a negative feedback loop between p21 and ATR, i.e., in the absence of p21, Chk1 is activated via phosphorylation at Ser 317. It was further shown that p21 induction was significantly impaired in U2OS cells and these cells completely relied on Chk1 pathway for G2–M arrest. These data are partly in contrast to our experiments, which show strong induction of p21 in melanoma cells, and p21 appears to be active in these cells. But similar to U2OS, p21 did not play a role in G2–M arrest in melanoma cells, as G2–M arrest was blocked by Chk1 inhibition even in the presence of high p21 levels and p21 knockdown did not affect G2–M arrest. Interestingly, treatment of HCT116 colon cancer cell with thymidine, which inhibits ribonucleotide reductase and induces S phase arrest, did not lead to apoptosis [47]. However, apoptosis was readily induced after additional Chk1 inhibition. Apoptosis was even further enhanced in p21-deficient HCT116 cells under these conditions, but not in p53deficient cells [47]. G1 cells were retained after thymidine treatment of Chk1-depleted p21+/+ cells, but G1 was lost in p21−/− cells, which is in agreement with reduced G1 arrest of p21 knockdown melanoma cells under combined doxorubicin Chk1 inhibitor treatment. Authors proposed that p21 retards cell death in the absence of Chk1 through its role as a cyclin-dependent kinase (CDK) inhibitor by slowing Sphase entry where cell death is induced. Similar observations regarding apoptosis induction under Chk1 inhibition were made for hydroxurea, which induces S phase arrest and camptothecin, which induces G2–M arrest [47]. Taken together, we show that ATR–Chk1–Wee1–Cdc2 and ATM– Chk2–p53–p21 pathways are both active in stress-induced cell cycle arrest in melanoma cells. We provide strong evidence that G2–M arrest relies on ATR–Chk1–Wee1–Cdc2 while both pathways are active in G0–G1 arrest. A combination of classical chemotherapeutic agents such as doxorubicin and chemical inhibition of ATR–Chk1–Wee1–

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Cdc2 pathway might open interesting perspectives for new therapeutic approaches in malignant melanoma. Chemical inhibitors for Chk1 and Wee1 have been tested in clinical trials, but up to now not in malignant melanoma [18,27]. 5. Conclusions Metastatic melanoma poorly responds to chemotherapeutic agents, and even new small molecule inhibitors prolong overall survival of patients for only a few months. Here we provide substantial evidence that targeting intracellular pathways controlling cell cycle checkpoints might be a promising strategy for future treatment approaches in melanoma. However, treatment combinations of chemotherapeutic agents with cell cycle inhibitors such as Chk1 inhibitors may produce counterintuitive effects as re-establishment of p53-dependent G0–G1 arrest in melanoma cells, which are in their majority p53-proficient, may hamper treatment success. This must be kept in mind when designing combination treatment strategies. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.cellsig.2015.01.020. Conflict of interest All authors declare no conflict on interest. Acknowledgements The initial idea for the investigation was suggested by M.K, J.V. and OW. Experiments were designed and performed by Y.R., J.V., O.W., M.K, T.K., A.B. and J.C.S. All the authors drafted the manuscript. This work was supported in part by the German Federal Ministry of Education and Research (BMBF), GerontoSys project ROSAge, grant number PTJ/0315892C of M.K., O.W. and J.V. References [1] A.J. Miller, M.C. Mihm Jr., N. Engl. J. Med. 355 (2006) 51–65 (PMID:16822996). [2] C. Garbe, T.K. Eigentler, U. Keilholz, A. Hauschild, J.M. Kirkwood, Oncologist 16 (1) (2011) 5–24. http://dx.doi.org/10.1634/theoncologist. 2010-0190 (PMID:21212434). [3] P. Hersey, L. Bastholt, V. Chiarion-Sileni, G. Cinat, R. Dummer, A.M. Eggermont, E. Espinosa, A. Hauschild, I. Quirt, C. Robert, D. Schadendorf, Ann. Oncol. 20 (Suppl. 6) (2009) vi35–vi40. http://dx.doi.org/10.1093/annonc/mdp254 (PMID:19617296). [4] A.K. Salama, K.T. Flaherty, Clin. Cancer Res. 19 (16) (2013) 4326–4334. http://dx.doi. org/10.1158/1078-0432.CCR-13-0779 (PMID:23770823). [5] A.E. Siroy, G.M. Boland, D.R. Milton, J. Roszik, S. Frankian, J. Malke, L. Haydu, V.G. Prieto, M. Tetzlaff, D. Ivan, et al., J. Invest. Dermatol. (Aug 22 2014)http://dx.doi. org/10.1038/jid.2014.366 (PMID:25148578, Epub ahead of print). [6] R. Nazarian, H. Shi, Q. Wang, X. Kong, R.C. Koya, H. Lee, Z. Chen, M.K. Lee, N. Attar, H. Sazegar, et al., Nature 468 (7326) (2010) 973–977. http://dx.doi.org/10.1038/ nature09626 (PMID:21107323). [7] C.J. Sherr, Cell 116 (2004) 235–246 (PMID:14744434). [8] M.B. Kastan, J. Bartek, Nature 432 (7015) (2004) 316–323 (PMID:15549093). [9] A.J. Levine, M. Oren, Nat. Rev. Cancer 9 (10) (2009) 749–758. http://dx.doi.org/10. 1038/nrc2723 (PMID:19776744). [10] D.R. Kellogg, J. Cell Sci. 116 (Pt 24) (2003) 4883–4890 (PMID:14625382). [11] S.L. Harvey, A. Charlet, W. Haas, S.P. Gygi, D.R. Kellogg, Cell 122 (3) (2005) 407–420 (PMID:16096060). [12] C.S. Sørensen, R.G. Syljuåsen, Nucleic Acids Res. 40 (2) (2012) 477–486. http://dx. doi.org/10.1093/nar/gkr697 PMID:21937510. [13] J.P. Mak, W.Y. Man, H.T. Ma, R.Y. Poon, Oncotarget 5 (21) (2014) 10546–10557 (PMID:25301733). [14] S.E. Mir, P.C. De Witt Hamer, P.M. Krawczyk, L. Balaj, A. Claes, J.M. Niers, A.A. Van Tilborg, A.H. Zwinderman, D. Geerts, G.J. Kaspers, et al., Cancer Cell 18 (3) (2010) 244–257. http://dx.doi.org/10.1016/j.ccr.2010.08.011 (PMID:20832752). [15] Y. Wang, S.J. Decker, J. Sebolt-Leopold, Cancer Biol. Ther. 3 (3) (2004) 305–313 (PMID:14726685). [16] A.N. Tse, T.N. Sheikh, H. Alan, T.C. Chou, G.K. Schwartz, Mol. Pharmacol. 75 (1) (2009) 124–133. http://dx.doi.org/10.1124/mol.108.050807 (PMID:18820127). [17] F. Wang, Y. Zhu, Y. Huang, S. McAvoy, W.B. Johnson, T.H. Cheung, T.K. Chung, K.W. Lo, S.F. Yim, M.M. Yu, et al., Oncogene 24 (24) (2005) 3875–3885 (PMID:15735666). [18] C.X. Ma, J.W. Janetka, H. Piwnica-Worms, Trends Mol. Med. 17 (2) (2011) 88–96. http://dx.doi.org/10.1016/j.molmed.2010.10.009 (PMID:21087899). [19] S.D. Leach, C.D. Scatena, C.J. Keefer, H.A. Goodman, S.Y. Song, L. Yang, J.A. Pietenpol, Cancer Res. 58 (15) (1998) 3231–3236 (PMID:9699647).

Please cite this article as: J. Vera, et al., Cell. Signal. (2015), http://dx.doi.org/10.1016/j.cellsig.2015.01.020

10

J. Vera et al. / Cellular Signalling xxx (2015) xxx–xxx

[20] V. Gottifredi, O. Karni-Schmidt, S.S. Shieh, C. Prives, Mol. Cell. Biol. 21 (4) (2001) 1066–1076 (PMID:11158294). [21] S. Zenvirt, N. Kravchenko-Balasha, A. Levitzki, Oncogene 29 (46) (2010) 6149–6159. http://dx.doi.org/10.1038/onc.2010.343 (PMID:20729914). [22] M.S. Soengas, P. Capodieci, D. Polsky, J. Mora, M. Esteller, X. Opitz-Araya, R. McCombie, J.G. Herman, W.L. Gerald, Y.A. Lazebnik, et al., Nature 409 (2001) 207–211 (PMID:11196646). [23] A. Saalbach, C. Klein, C. Schirmer, W. Briest, U. Anderegg, J.C. Simon, J. Invest. Dermatol. 130 (2) (2010) 444–454. http://dx.doi.org/10.1038/jid.2009.253 (PMID: 19710690). [24] M.A. van Vugt, A. Brás, R.H. Medema, Mol. Cell 15 (5) (2004) 799–811 (PMID: 15350223). [25] L. McKenzie, S. King, L. Marcar, S. Nicol, S.S. Dias, K. Schumm, P. Robertson, J.C. Bourdon, N. Perkins, F. Fuller-Pace, D.W. Meek, Cell Cycle 9 (20) (2010) 4200–4212 (PMID:20962589). [26] H.B. Lieberman, Curr. Med. Chem. 15 (4) (2008) 360–367 (PMID:18288990). [27] B.B. Zhou, J. Bartek, Nat. Rev. Cancer 4 (3) (2004) 216–225 (PMID:14993903). [28] C. Guillouf, F. Rosselli, K. Krishnaraju, E. Moustacchi, B. Hoffman, D.A. Liebermann, Oncogene 10 (11) (1995) 2263–2270 (PMID:7784074). [29] S. Macip, M. Igarashi, P. Berggren, J. Yu, S.W. Lee, S.A. Aaronson, Mol. Cell. Biol. 23 (23) (2003) 8576–8585 (PMID:14612402). [30] T. Abbas, A. Dutta, Nat. Rev. Cancer 9 (6) (2009) 400–414. http://dx.doi.org/10. 1038/nrc2657 (PMID:19440234). [31] I. Masgras, S. Carrera, P.J. de Verdier, P. Brennan, A. Majid, W. Makhtar, E. Tulchinsky, G.D. Jones, I.B. Roninson, S. Macip, J. Biol. Chem. 287 (13) (2012) 9845–9854. http:// dx.doi.org/10.1074/jbc.M111.250357 (PMID:22311974). [32] M.A. van Vugt, A.K. Gardino, R. Linding, G.J. Ostheimer, H.C. Reinhardt, S.E. Ong, C.S. Tan, H. Miao, S.M. Keezer, J. Li, et al., PLoS Biol. 8 (1) (2010) e1000287. http://dx.doi. org/10.1371/journal.pbio.1000287 (PMID:20126263). [33] A. Jalili, A. Moser, M. Pashenkov, C. Wagner, G. Pathria, V. Borgdorff, M. Gschaider, G. Stingl, S. Ramaswamy, S.N. Wagner, J. Invest. Dermatol. 131 (9) (2011) 1886–1895. http://dx.doi.org/10.1038/jid.2011.136 (PMID:21654832). [34] T.L. Schmit, W. Zhong, V. Setaluri, V.S. Spiegelman, N. Ahmad, J. Invest. Dermatol. 129 (12) (2009) 2843–2853. http://dx.doi.org/10.1038/jid.2009.172 (PMID: 19554017).

[35] L. Owens, S. Simanski, C. Squire, A. Smith, J. Cartzendafner, V. Cavett, J. Caldwell Busby, T. Sato, N.G. Ayad, J. Biol. Chem. 285 (9) (2010) 6761–6769. http://dx.doi. org/10.1074/jbc.M109.093237 (PMID:20038582). [36] F. Bunz, A. Dutriaux, C. Lengauer, T. Waldman, S. Zhou, J.P. Brown, J.M. Sedivy, K.W. Kinzler, B. Vogelstein, Science 282 (5393) (1998) 1497–1501 (PMID:9822382). [37] R.G. Syljuåsen, C.S. Sørensen, L.T. Hansen, K. Fugger, C. Lundin, F. Johansson, T. Helleday, M. Sehested, J. Lukas, J. Bartek, Mol. Cell. Biol. 25 (9) (2005) 3553–3562 (PMID:15831461). [38] E. Berkovich, R.J. Monnat Jr., M.B. Kastan, Nat. Cell Biol. 9 (6) (2007) 683–690 (PMID: 17486112). [39] A. Jazayeri, J. Falck, C. Lukas, J. Bartek, G.C. Smith, J. Lukas, S.P. Jackson, Nat. Cell Biol. 8 (1) (2006) 37–45 (PMID:16327781). [40] H.C. Reinhardt, M.B. Yaffe, Curr. Opin. Cell Biol. 21 (2) (2009) 245–255. http://dx.doi. org/10.1016/j.ceb.2009.01.018 (PMID:19230643). [41] B. Shiotani, L. Zou, Mol. Cell 33 (5) (2009) 547–558. http://dx.doi.org/10.1016/j. molcel.2009.01.024 (PMID:19285939). [42] U.K. Mukhopadhyay, A.M. Senderowicz, G. Ferbeyre, Cancer Res. 65 (7) (2005) 2872–2881 PMID:15805289. [43] R. Beckerman, A.J. Donner, M. Mattia, M.J. Peart, J.L. Manley, J.M. Espinosa, C. Prives, Genes Dev. 23 (11) (2009) 1364–1377. http://dx.doi.org/10.1101/gad.1795709 (PMID:19487575). [44] Y. Hirose, M.S. Berger, R.O. Pieper, Cancer Res. 61 (15) (2001) 5843–5849 (PMID: 11479224). [45] Y. Tao, C. Leteur, C. Yang, P. Zhang, M. Castedo, A. Pierré, R.M. Golsteyn, J. Bourhis, G. Kroemer, E. Deutsch, Cell Cycle 8 (8) (2009) 1196–1205 (PMID:19305158). [46] G. Lossaint, E. Besnard, D. Fisher, J. Piette, V. Dulić, Oncogene 30 (41) (2011) 4261–4274. http://dx.doi.org/10.1038/onc.2011.135 (PMID:21532626). [47] R. Rodriguez, M. Meuth, Mol. Biol. Cell 17 (1) (2006) 402–412 (PMID:16280359).

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