Telling Cells How to Die Docetaxel Therapy in Cancer Cell Lines

Extra View Telling Cells How to Die Docetaxel Therapy in Cancer Cell Lines

Héctor Hernández-Vargas1 Abstract José Palacios2 Taxanes (paclitaxel and docetaxel) are antimicrotubule agents used in the clinical Gema Moreno-Bueno3,* practice for the treatment of solid tumors. The molecular mechanisms of taxane‑ dependent cytotoxicity are only partially known. In vitro, different modes of action have 1Spanish National Cancer Centre (CNIO); Madrid, Spain been established for paclitaxel according to its concentration. A similar phenomenon has been shown for other microtubule‑stabilizing drugs, including docetaxel. Recently, 2Servicio de Anatomía Patológica, HHUU Virgen del Rocío; Sevilla, Spain we described two different forms of mitotic exit in breast cancer cell lines exposed to 3Instituto de Investigaciones Biomedicas (IIB)-Alberto Sols; Unidad de Biología docetaxel. The causes and consequences of the dual mechanism of cytotoxicity of this Molecular y Celular del Cáncer; Madrid, Spain agent are discussed here. *Correspondence to: Dra. Gema Moreno-Bueno; Instituto de Investigaciones Biomedicas (IIB)-Alberto Sols.; Unidad de Biología Molecular y Celular del Cáncer; C/ Arturo Duperier 4; 28029 Madrid, Spain; Email: [email protected]

Original manuscript submitted: 02/14/07 Antimicrotubule Agents and Known Mechanisms of Cell Death Manuscript accepted: 02/22/07 Dynamic instability describes the process of slow growth and rapid shortening of Previously published online as a Cell Cycle E-publication: microtubules by continuous addition and loss of tubulin at their ends.1 The precise regu- http://www.landesbioscience.com/journals/cc/abstract.php?id=4050 lation of this process is fundamental to the multiple functions of microtubules, especially those related with cell division.2 Several agents affecting microtubule dynamics have been Key words shown to be effective against tumor cells. They are classified into those inducing either docetaxel, aberrant mitosis, mitotic slippage, microtubule depolymerization or their polymerization. The last group is represented by apoptosis taxanes, which include paclitaxel and docetaxel, both currently approved for clinical use.3,4 By binding to the b subunit of tubulin, these agents impair the dynamic of microtubules, Acknowledgements promote their polymerization, and arrest the cells in mitosis. As a consequence, cells die by apoptosis through pathways still not fully characterized. The authors thank P. Wachowicz, H. Peinado Most studies, especially with paclitaxel, have shown cell cycle arrest in mitosis as a and M. Hergueta for their critical feedback necessary step for cell death. However, it is also necessary that cells overcome this arrest, and thoughtful suggestions. This work was advancing to interphase without cytokinesis (a process called mitotic slippage), and supported by Ministerio de Educación y entering a tetraploid or “pseudo‑G ” phase followed by apoptotic cell death.5,6 Mitotic Ciencia, Grant Reference PTR1995-0753- 1 arrest induced by taxanes is dependent on the activation of the spindle‑assembly check- OP and Ministerio de Ciencia y Tecnologı’ point, which monitors any defects in microtubule‑kinetochore attachment or in the tension a SAF2001-0065 and SAF2004-08258-C02- of the mitotic spindles. This checkpoint is usually weakened—but not absent—in human 01. G.M.B. is a junior investigator of the tumor cells, with cells able to respond with an initial cell cycle arrest but less able to main- Ramón y Cajal Program (2004). tain it.7,8 Therefore, tumor cells usually arrest in response to taxanes, but this arrest is weak or transitory permitting the mitotic slippage required for cells to die. Several questions still remain to be answered regarding the mechanisms of taxane‑ dependent cytotoxicity in tumor cells, including the induction of cell death without mitotic arrest in some experimental conditions, the dependence of these processes on transcriptional mechanisms, the importance of a functional p53 protein, and the exact mechanism(s) by which tumor cells die.

Mitotic Exit after Exposure to Taxanes Recently, we described a dual mechanism of action for docetaxel in breast cancer cell lines,9 that was suggested by a biphasic growth inhibition curve. The presence of at least ©2007 LANDES BIOSCIENCE.two mechanisms of action wasDO confirmed NOT by the DISTRIBUTE. observation of different cell cycle and cell death profiles in both cell lines, and further studied by gene expression microarrays performed after two concentrations of docetaxel. The most striking differences were found at the level of cell cycle. Low concentrations (2–4 nM) induced aberrant mitosis followed by aneuploidy, whereas higher concentrations (100 nM) induced sustained mitotic arrest followed by mitotic slippage (Fig. 1). Although mitotic arrest and mitotic slippage are well characterized responses to this agent, there are also some previous reports

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Figure 1. Exit from mitosis after exposure to docetaxel. The effect of docetaxel on cell cycle is represented here after a synchronization experiment. Cells normally replicating their DNA from G1 (2n) to G2 (4n) are then subjected to low and high concentrations of the drug. Cells not receiving treatment exit normal mitosis (M) to start a new cell cycle from G1 (1). In contrast, low and high concentrations of docetaxel induce transient (3) or prolonged (2) arrest in mitosis, respectively. The respective cell cycle profiles (obtained by DNA staining with propidium iodide) are shown. indicating that docetaxel is able to induce aberrant mitosis at lower concentrations.10,11 However, most studies indicating a dual mechanism of action of taxanes had been reported with paclitaxel.12‑15 In accordance to our findings with docetaxel, Ikui et al reported Figure 2. Transcriptional responses to docetaxel. Gene expression profiles that higher concentrations of paclitaxel induce a longer spindle‑as- of selected transcripts after treatment of MCF7 cells with 2 concentrations sembly checkpoint in HeLa cells. In contrast, low concentrations of docetaxel (4 nM and 100 nM), and different time points (8, 24 and of paclitaxel cause mitotic delay followed by premature dissociation 48 hours). Gene expression is represented as units relative to nontreated cells. of p55CDC/CDC20 from Mad2 and BubR1 and abrogation of the spindle‑assembly checkpoint, leading to aneuploidy.16 As represented in Figure 1, mitotic exit will be produced through at least two duplication during S phase, cells synchronized in G2 and treated with possible processes depending on drug concentration. Concentrations 2 nM docetaxel during 6 hours, were still able to show aneuploidy known to stabilize the microtubules, induce a prolonged activation after passing through mitosis. This suggests that mechanisms inde- of the spindle‑assembly checkpoint (the mitotic index was maximal pendent of centrosome duplication are related with the induction of after 24 hours). This is followed by mitotic slippage, and manifested aberrant mitosis in this context. by multinucleation. In contrast, low concentrations of docetaxel will Our gene expression data supports some of these previous find- affect the dynamics of microtubules, inducing a transient activation ings. Favoring the stronger mitotic arrest with higher concentrations of the spindle‑assembly checkpoint. In the breast cancer cells that of docetaxel, an induction of mitosis‑related genes (AURKA, PLK1) we evaluated, this checkpoint is not strong enough to stop the was observed in this condition (Fig. 2). Checkpoint‑related genes segregation of chromosomes, and this is manifested by aneuploidy.17 (BUB1, MAD2L1) showed a similar profile in the first time points Aneuploidy©2007 is known to be LANDESthe result of aberrant BIOSCIENCE. mitosis which can of treatment. DO However, NOT as can DISTRIBUTE. be seen in Figure 2, a downregulation be caused by divisions in the presence of a multipolar spindle, as of these transcripts was observed after 48 hours. This can be related has been described for docetaxel. However, it can also be the result to a release from the spindle checkpoint and the induction of mitotic of previous defects in cytokinesis or centrosome amplification, by slippage. Although exit from a normal mitosis is controlled by defects in chromosome cohesion, by spindle attachment defects, or ubiquitin‑dependent proteolysis and phosphorylation/dephosphory- by impairment of the mitotic checkpoint response.7 Interestingly, lation events, it is possible that transcriptional regulation participates although docetaxel has been described to interfere with centrosome in the prolonged activation of the spindle‑assembly checkpoint after www.landesbioscience.com Cell Cycle 781 Docetaxel Therapy in Cancer Cell Lines

Together, these data point to a mechanism regulating the different consequences of docetaxel exposure according to the duration of mitotic arrest. This mechanism can be partially regulated at the transcriptional level by changes both in p21 and checkpoint genes.

A Dual Mechanism Implies Dual Outcomes Induction of apoptosis has been described as a main mechanism of cell death after antimicrotubule agents, implicating different downstream events including the dysregulation of cell cycle‑related proteins (particularly p34cdc‑2),21 the activation of the jun N‑terminal kinase (JNK)22 and Raf‑1 kinase23,24 ��pathways, and the final induction of a misbalance of the Bcl‑2 family of proteins.22,24‑27 However,�� two different effects on mitosis that we described were also followed by different types of cell death (Fig. 3). Although 100 nM docetaxel induced a clear pattern of early apoptosis by Annexin‑V staining at short time points (24 hours or less), cell death was lower with 2–4 nM concentrations, and it was mainly represented by necrosis.9 This is consistent with reports showing that cells undergoing aberrant mitosis eventually result in reduced clonogenic survival,28,29 usually due to nonapoptotic mechanisms.30 In this sense, some groups have described alternative caspase‑independent mechanisms of cell death after taxane therapy.31 More recently, it was shown that autophagy can be an important form of programmed cell death after treatment of MCF7 breast cancer cells with paclitaxel analogs and docetaxel, by using MAP I LC3 antibody as an specific marker of autopha- gosomes.32 Therefore, the most likely scenery is a combination of apoptotic and nonapoptotic mechanisms. In fact, a known mediator of apoptosis after taxane therapy is the cell death receptor CD95/ FAS. Our own gene expression data shows that the induction of FAS is a common response to docetaxel. Moreover, this response was dose‑ and cell line‑independent (Fig. 2). However, it is possible that nonapoptotic pathways override the induction of FAS after aberrant mitosis induced by low concentrations of docetaxel. One possible Figure 3. Model of cell outcome according to drug concentration. A model for the dual mechanism of action of docetaxel is represented in the diagram. mechanism triggering cell death in this context is the induction of The main effects and outcomes are shown in a color gradient, corresponding to DNA damage. It is likely that aberrant mitosis creates chromosomal the increasing concentrations of the drug. The hypothetic correlation between breakage, interchromosomal concatenation and a lethal genetic induction of the spindle‑assembly checkpoint (S‑A‑checkpoint) and accumula- imbalance that leads to cell death.5,33 In this sense, we have observed tion of p53 protein is shown, as well as the inverse correlation between p53, accumulation of phospho‑H2AX after docetaxel, especially in the and the induction of p53 targets. p53‑non functional MDA‑MB‑231 cells, at low concentrations of docetaxel. In addition, it was recently shown that DNA damage can taxane exposure. In support of this view, the results of Chen et al. be also involved in the activation of the G1 checkpoint after mitotic suggest that the mitotic slippage induced by microtubule‑stabilizing slippage induced by antimitotic agents.34 drugs may result from altered transcriptional regulation of G2/M and spindle checkpoint genes when higher concentrations of drug Concluding Remarks are used.15 Finally, we observed a different expression profile of known p53 targets when comparing low and high concentrations The impairment of microtubule dynamics results in cell cycle of docetaxel in the p53‑functional cell line MCF7. Specifically, alterations and cell death. Both effects can be the result of independent p21 and TP53INP1 were induced at earlier time points with lower activities of docetaxel. However, our observations point to the possi- concentrations. In addition, there was an inverse correlation between bility that the mechanism of cell death is the direct consequence of p53 protein accumulation and p21 transcript and protein expression the previous type of mitotic exit induced by this agent. Therefore, the (Figs. 2 and 3). This observation supports a model in which p53 understanding of the mechanisms underlying docetaxel cytotoxicity protein accumulation is a marker of the timing of mitotic arrest.18 will rest on a better knowledge of the link between checkpoint activa- In addition,©2007 the earlier induction LANDES of p21 after low BIOSCIENCE. concentrations of tion and DO cell cycle NOT arrest, and DISTRIBUTE. the subsequent induction of cell death. docetaxel may be related with the mechanism of aberrant mitosis, The elucidation of these mechanisms should consider the signaling as has been reported.19 In fact, previous studies have suggested pathways, and possible crosstalk among those pathways, activated in that p21 is not required for the mitotic arrest in response to pacli- a concentration‑dependent manner. taxel, but instead it could have a role in facilitating the exit from abnormal mitosis.20

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References 32. Górka M, Daniewski WM, Gajkowska B, Lusakowska E, Godlewski MM, Motyl T. 1. Mitchinson T, Kirschner M. Dynamic instability of microtubule growth. Nature 1984; Autophagy is the dominant type of programmed cell death in breast cancer MCF‑7 cells 312:237‑42. exposed to AGS 115 and EFDAC, new sequiterpene analogs of paclitaxel. Anti‑Cancer 2. Jordan MA, Wilson L. Microtubules as a target for anticancer drugs. Nat Rev Cancer 2004; Drugs 2005; 16:777‑88. 4:253‑65. 33. Fujikawa‑Yamamoto K, Ohodoi C, Yamagishi H, Zong ZP, Murakami M, Yamaguchi N. 3. Montero A, Fosella F, Hortobagyi GN, Valero V. Docetaxel for treatment of solid tumours: Lack of synchrony among multiple nuclei induces partial DNA fragmentation in V79 cells A systematic review of clinical data. The Lancet Oncology 2005; 6:229‑39. polyploidized by demecolcine. Cell Proliferation 1999; 32:337‑49. 4. Eniu A, Palmieri FM, Perez EA. Weekly administration of docetaxel and paclitaxel in meta- 34. Quignon F, Rozier L, Lachages AM, Bieth A, Simili M, Debatisse M. Sustained mitotic static or advanced breast cancer. Oncologist 2005; 10:665‑85. block elicits DNA breaks: One‑step alteration of ploidy and chromosome integrity in mam- 5. Blajeski AL, Kottke TJ, Kaufmann SH. A multistep model for paclitaxel‑induced apoptosis malian cells. Oncogene 2006; 26:165‑72. in human breast cancer cell lines. Experimental Cell Research 2001; 270:277‑88. 6. Tao W. The mitotic checkpoint in cancer therapy. Cell Cycle 2005; 4:1495-9. 7. Kops GJPL, Weaver BAA, Cleveland DW. On the road to cancer: Aneuploidy and the mitotic checkpoint. Nat Rev Cancer 2005; 5:773‑85. 8. Weaber BAA, Cleveland DW. Decoding the links between mitosis, cancer, and chemotherapy: The mitotic checkpoint, adaptation, and cell death. Cancer Cell 2005; 8:7‑12. 9. Hernández‑Vargas H, Palacios J, Moreno‑Bueno G. Molecular profiling of docetaxel cytotoxicity in breast cancer cells: Uncoupling of aberrant mitosis and apoptosis. Oncogene 2007; 26:2901-13. 10. Paoletti A, Giocanti N, Favaudon V, Bornens M. Pulse treatment of interphasic HeLa cells with nanomolar doses of docetaxel affects centrosome organization and leads to catastrophic exit of mitosis. J Cell Sci 1997; 110:2403‑15. 11. Morse DL, Gray H, Payne CM, Gillies RJ. Docetaxel induces cell death through mitotic catastrophe in human breast cancer cells. Mol Cancer Ther 2005; 4:1495‑504. 12. Torres K, Horwitz S. Mechanisms of Taxol‑induced cell death are concentration dependent. Cancer Res 1998; 58:3620‑6. 13. Giannakakou P, Robey R, Fojo T, Blagosklonny M. Low concentrations of paclitaxel induce cell type‑dependent p53, p21 and G1/G2 arrest instead of mitotic arrest: Molecular deter- minants of paclitaxel‑induced cytotoxicity. Oncogene 2001; 20:3806‑13. 14. Chen Z, Xiao Z, Chen J, Ng SC, Sowin T, Sham H, Rosenberg S, Fesik S, Zhang H. Human chk1 expression is dispensable for somatic cell death and critical for sustaining G2 DNA damage checkpoint. Mol Cancer Ther 2003; 2:543‑8. 15. Chen JG, Yang CPH, Cammer M, Band Horwitz S. Gene expression and mitotic exit induced by microtubule‑stabilizing drugs. Cancer Res 2003; 63:7891‑9. 16. Ikui AE, Yang CH, Matsumoto T, Horwitz SB. Low concentrations of Taxol cause mitotic delay followed by premature dissociation of p55CDC from Mad2 and BubR1 and abrogation of the spindle checkpoint, leading to aneuploidy. Cell Cycle 2005; 4:1385‑8. 17. Yoon DS, Wersto RP, Zhou W, Chrest FJ, Garrett ES, Kwon TK, Gabrielson E. Variable levels of chromosomal instability and mitotic spindle checkpoint defects in breast cancer. Am J Pathol 2002; 161:391‑7. 18. Blagosklonny M. Prolonged mitosis versus tetraploid checkpoint: How p53 measures the duration of mitosis. Cell Cycle 2006; 5:971‑5. 19. Chang BD, Broude E, Fang J, Kalinichenko TV, Abdryashitov R, Poole JC, Roninson IB. p21Waf1/Cip1/Sdi1‑induced growth arrest is associated with depletion of mitosis‑control proteins and leads to abnormal mitosis and endoreduplication in recovering cells. Oncogene 2000; 19:2165‑70. 20. Barboule N, Chadebech P, Baldin V, Vidal S, Valette A. Involvement of p21 in mitotic exit after paclitaxel treatment in MCF‑7 breast adenocarcinoma cell line. Oncogene 1997; 15:2867‑75.

21. Stewart ZA, Mays D, Pietenpol JA. Defective G1‑S cell cycle checkpoint function sensitizes cells to microtubule inhibitor‑induced apoptosis. Cancer Res 1999; 59:3831‑7. 22. Wang Q, Wieder R. All‑trans retinoic acid potentiates Taxotere-induced cell death mediated by Jun N‑terminal kinase in breast cancer cells. Oncogene 2004; 23:426‑33. 23. Caraglia M, Giuberti G, Marra M, Di Gennaro E, Facchini G, Caponigro F, Iaffaioli R, Budillon A, Abbruzzese A. Docetaxel induces p53‑dependent apoptosis and synergizes with farnesyl transferase inhibitor r115777 in human epithelial cancer cells. Front Biosci 2005; 10:2566‑75. 24. Blagosklonny M, Schulte T, Nguyen P, Trepel J, Neckers L. Taxol‑induced apoptosis and phosphorylation of Bcl‑2 protein involves c‑Raf‑1 and represents a novel c‑Raf‑1 signal transduction pathway. Cancer Res 1996; 56:1851‑4. 25. Berchem G, Bosseler M, Mine N, Avalosse B. Nanomolar range docetaxel treatment sensitizes MCF‑7 cells to chemotherapy induced apoptosis, induces G2M arrest and phosphory- lates bcl‑2. Anticancer Res 1999; 19:535‑40. 26. Boudny V, Nakano S. Src tyrosine kinase augments taxotere-induced apoptosis through enhanced expression and phosphorylation of Bcl‑2. Br J Cancer 2002; 86:463‑9. 27. Haldar S, Basu A, Croce C. Bcl2 is the guardian of microtubule integrity. Cancer Res 1997; 57:229‑33. 28. Blagosklonny MV, Robey R, Sackett DL, Du L, Traganos F, Darzynkiewicz Z, Fojo T, Bates SE. Histone deacetylase inhibitors all induce p21 but differentially cause tubulin acetylation, mitotic arrest, and cytotoxicity. Mol Cancer Ther 2002; 1:937‑41. 29. Roninson©2007 IB, Broude EV, Chang LANDES BD. If not apoptosis, then BIOSCIENCE. what? Treatment��‑induced DO NOT DISTRIBUTE. senescence and mitotic catastrophe in tumor cells. Drug Resistance Updates 2001; 4:303‑13. 30. Mansilla S, Priebe W, Portugal J. Mitotic catastrophe results in cell death by caspase‑dependent and caspase‑independent mechanisms. Cell Cycle 2006; 5:53‑60. 31. Huisman C, Ferreira CG, Broker LE, Rodriguez JA, Smit EF, Postmus PE, Kruyt FAE, Giaccone G. Paclitaxel triggers cell death primarily via caspase‑independent routes in the non-small cell lung cancer cell line NCI‑H460. Clinical Cancer Research 2002; 8:596‑606. www.landesbioscience.com Cell Cycle 783