Elsevier

Cellular Signalling

Volume 27, Issue 5, May 2015, Pages 951-960
Cellular Signalling

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

https://doi.org/10.1016/j.cellsig.2015.01.020Get rights and content

Highlights

  • Treatment of p53-proficient melanoma cells with Chk1 inhibitor blocks G2-M arrest and induces massive apoptosis.

  • Combined treatment with Chk1 inhibitor and doxorubicin has an apoptosis-protective effect via induction of a G0-G1 arrest.

  • Combined treatment with ATM/Chk2 inhibitors and doxorubicin abolishes G0-G1 arrest and induces massive apoptosis.

  • Combinations of chemotherapeutic agents with cell cycle inhibitors may produce apoptosis-protective effects.

Abstract

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.

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 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], [8], [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, 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 stress-induced 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.

Section snippets

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

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 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

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.

Interestingly, enhanced induction of p53 was

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

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 (47)

  • P. Hersey et al.

    Ann. Oncol.

    (2009)
  • C.J. Sherr

    Cell

    (2004)
  • S.L. Harvey et al.

    Cell

    (2005)
  • S.E. Mir et al.

    Cancer Cell

    (2010)
  • C.X. Ma et al.

    Trends Mol. Med.

    (2011)
  • A. Saalbach et al.

    J. Invest. Dermatol.

    (2010)
  • M.A. van Vugt et al.

    Mol. Cell

    (2004)
  • I. Masgras et al.

    J. Biol. Chem.

    (2012)
  • A. Jalili et al.

    J. Invest. Dermatol.

    (2011)
  • T.L. Schmit et al.

    J. Invest. Dermatol.

    (2009)
  • L. Owens et al.

    J. Biol. Chem.

    (2010)
  • H.C. Reinhardt et al.

    Curr. Opin. Cell Biol.

    (2009)
  • B. Shiotani et al.

    Mol. Cell

    (2009)
  • A.J. Miller et al.

    N. Engl. J. Med.

    (2006)
  • C. Garbe et al.

    Oncologist

    (2011)
  • A.K. Salama et al.

    Clin. Cancer Res.

    (2013)
  • A.E. Siroy et al.

    J. Invest. Dermatol.

    (Aug 22 2014)
  • R. Nazarian et al.

    Nature

    (2010)
  • M.B. Kastan et al.

    Nature

    (2004)
  • A.J. Levine et al.

    Nat. Rev. Cancer

    (2009)
  • D.R. Kellogg

    J. Cell Sci.

    (2003)
  • C.S. Sørensen et al.

    Nucleic Acids Res.

    (2012)
  • J.P. Mak et al.

    Oncotarget

    (2014)
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    Both authors contributed equally to this work.

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