Gemcitabine Inhibits Murine Head and Neck Squamous Cell Carcinoma Growth via Proteasome-Dependent Degradation of Chk1 Leading to Cell Cycle Arrest and Apoptosis

Anren Song, Jean Wu, Emily Whitaker, Nadarajah Vigneswaran

Department of Diagnostic and Biomedical Sciences, School of Dentistry, University of Texas Health Science Center at Houston, Houston, USA.
Medical Student, Washington University School of Medicine in St. Louis, St. Louis, USA.

DOI: 10.4236/jct.2012.35072                

Abstract

Head and neck squamous cell carcinoma (HNSCC) is a common tumor worldwide that often presents at an advanced stage with poor prognosis. Advanced HNSCC frequently exhibits resistance to chemotherapy limiting its efficacy. Gemcitabine is a pyrimidine-based analog that is currently used for the treatment of metastatic pancreatic cancer. In this study, we examined the anti-tumor effects of gemcitabine in a highly aggressive murine model of HNSCC (LY2). In vitro cell viability and in vivo tumor growth inhibitory assays were carried out to determine the sensitivity of LY2 cells to gemcitabine. Immunohistochemical, Western blotting, RT-PCR and RNAi-mediated silencing assays were used to characterize effects of gemcitabine cell proliferation, DNA synthesis, apoptosis, pro-survival and DNA damage response signaling pathways. LY2 cells treated with gemcitabine undergo apoptosis mediated by the activation of both caspase-3 and -9. Gemcitabine on treatment induces rapid phosphorylation of checkpoint kinase 1 (Chk1) in LY2 cells and subsequent degradation in a time and dose dependent manner. Proteasome inhibitor MG132 blocks Chk1 degradation and decreases LY2 cells susceptibility to gemcitabine. Inhibition of Chk1 function, either using inhibitor PD 407824 or small interfering RNA (siRNA), increases the sensitivity of LY2 cells to gemcitabine. Gemcitabine treatment resulted in significant reduction in tumor growth relative to saline-treated control in a syngeneic orthotopic murine model of HNSCC. Gemcitabine-induced DNA replication stress in LY2 cells activates Chk1 by phosphorylation and promotes Chk1 degradation via the ubiquitin-proteasome pathway. Depletion of Chk1 terminates S-phase check point in LY2 cells resulting in apoptotic cell death. Our data provides an important rationale for integrating gemcitabine to optimize chemotherapeutic efficacy in HNSCC.

Keywords

Chk1; Gemcitabine; Head and Neck Cancer; Apoptosis; siRNA

Conflicts of Interest

The authors declare no conflicts of interest.

References

[1]	A. Aguilera and B. Gomez-Gonzalez, “Genome Instability: A Mechanistic View of Its Causes and Consequences,” Nature Reviews Genetics, Vol. 9, No. 5, 2008, pp. 204-217. doi:10.1038/nrg2268
[2]	J. A. Pietenpol and Z. A. Stewart, “Cell Cycle Checkpoint Signaling: Cell Cycle Arrest versus Apoptosis,” Toxicology, Vol. 181, 2002, pp. 475-481. doi:10.1016/S0300-483X(02)00460-2
[3]	S. Jalal, J. N. Earley and J. J. Turchi, “DNA Repair: From Genome Maintenance to Biomarker and Therapeutic Target,” Clinical Cancer Research, Vol. 17, No. 22, pp. 6973-6984. doi:10.1158/1078-0432.CCR-11-0761
[4]	R. T. Abraham, “Cell Cycle Checkpoint Signaling through the ATM and ATR Kinases,” Genes & Development, Vol. 15, No. 17, 2001, pp. 2177-2196. doi:10.1101/gad.914401
[5]	D. Branzei and M. Foiani, “Regulation of DNA Repair throughout the Cell Cycle,” Nature Reviews Molecular Cell Biology, Vol. 9, No. 4, 2008, pp. 297-308. doi:10.1038/nrm2351
[6]	B. B. Zhou and S. J. Elledge, “The DNA Damage Response: Putting Checkpoints in Perspective,” Nature, Vol. 408, No. 6811, 2000, pp. 433-439. doi:10.1038/35044005
[7]	H. Zhao and H. Piwnica-Worms, “ATR-Mediated Checkpoint Pathways Regulate Phosphorylation and Activation of Human Chk1,” Molecular and Cellular Biology, Vol. 21, No. 13, 2001, pp. 4129-4139. doi:10.1128/MCB.21.13.4129-4139.2001
[8]	S. Lapenna and A. Giordano, “Cell Cycle Kinases as Therapeutic Targets for Cancer,” Nature Reviews Drug Discovery, Vol. 8, No. 7, 2009, pp. 547-566. doi:10.1038/nrd2907
[9]	B. B. Zhou and J. Bartek, “Targeting the Checkpoint Kinases: Chemosensitization versus Chemoprotection,” Nature Reviews Cancer, Vol. 4, No. 3, 2004, pp. 216-225. doi:10.1038/nrc1296
[10]	N. Bucher and C. D. Britten, “G2 Checkpoint Abrogation and Checkpoint Kinase-1 Targeting in the Treatment of Cancer,” British Journal of Cancer, Vol. 98, No. 3, 2008, pp. 523-528. doi:10.1038/sj.bjc.6604208
[11]	C. Merry, K. Fu, J. Wang, I. J. Yeh and Y. Zhang, “Targeting the Checkpoint Kinase Chk1 in Cancer Therapy,” Cell Cycle, Vol. 9, No. 2, 2010, pp. 279-283. doi:10.4161/cc.9.2.10445
[12]	A. M. Bergman, V. W. Ruiz van Haperen, G. Veerman, C. M. Kuiper and G. J. Peters, “Synergistic Interaction between Cisplatin and Gemcitabine in Ovarian and Colon Cancer Cell Lines,” Advances in Experimental Medicine and Biology, Vol. 370, 1994, pp. 139-143.
[13]	P. Huang and W. Plunkett, “Induction of Apoptosis by Gemcitabine,” Seminars in Oncology, Vol. 22, No. 4, Suppl. 11, 1995, pp. 19-25.
[14]	M. A. Morgan, L. A. Parsels, J. Maybaum and T. S. Lawrence, “Improving Gemcitabine-Mediated Radiosensitization Using Molecularly Targeted Therapy: A Review,” Clinical Cancer Research, Vol. 14, No. 21, 2008, pp. 6744-6750. doi:10.1158/1078-0432.CCR-08-1032
[15]	J. D. Raguse, H. J. Gath, J. Bier, H. Riess and H. Oettle, “Gemcitabine in the Treatment of Advanced Head and Neck Cancer,” Clinical Oncology, Vol. 17, No. 6, 2005, pp. 425-429. doi:10.1016/j.clon.2005.05.006
[16]	P. M. Specenier, J. Weyler, C. Van Laer, D. Van den Weyngaert, J. Van den Brande, M. T. Huizing, et al., “A Non-Randomized Comparison of Gemcitabine-Based Chemoradiation with or without Induction Chemotherapy for Locally Advanced Squamous Cell Carcinoma of the Head and Neck,” BMC Cancer, Vol. 9, 2009, p. 273. doi:10.1186/1471-2407-9-273
[17]	L. Milas, T. Fujii, N. Hunter, M. Elshaikh, K. Mason, W. Plunkett, et al., “Enhancement of Tumor Radioresponse in Vivo by Gemcitabine,” Cancer Research, Vol. 59, No. 1, 1999, pp. 107-114.
[18]	B. Pauwels, J. B. Vermorken, A. Wouters, J. Ides, S. Van Laere, H. A. Lambrechts, et al., “The Role of Apoptotic Cell Death in the Radiosensitising Effect of Gemcitabine,” British Journal of Cancer, Vol. 101, No. 4, 2009, pp. 628-636. doi:10.1038/sj.bjc.6605145
[19]	H. Ueno, K. Kiyosawa and N. Kaniwa, “Pharmacogenomics of Gemcitabine: Can Genetic Studies Lead to Tailor-Made Therapy?” British Journal of Cancer, Vol. 97, No. 2, 2007, pp. 145-151. doi:10.1038/sj.bjc.6603860
[20]	E. Mini, S. Nobili, B. Caciagli, I. Landini and T. Mazzei, “Cellular Pharmacology of Gemcitabine,” Annals of Oncology, Vol. 17, Suppl. 5, 2006, pp. 7-12. doi:10.1093/annonc/mdj941
[21]	C. Nabhan, D. Gajria, N. L. Krett, V. Gandhi, K. Ghias and S. T. Rosen, “Caspase Activation Is Required for Gemcitabine Activity in Multiple Myeloma Cell Lines,” Molecular Cancer Therapeutics, Vol. 1, No. 13, 2002, pp. 1221-1227.
[22]	L. M. Karnitz, K. S. Flatten, J. M. Wagner, D. Loegering, J. S. Hackbarth, S. J. Arlander, et al., “Gemcitabine Induced Activation of Checkpoint Signaling Pathways that Affect Tumor Cell Survival,” Molecular Pharmacology, Vol. 68, No. 6, 2005, pp. 1636-1644.
[23]	N. Vigneswaran, J. Wu, A. Song, A. Annapragada and W. Zacharias, “Hypoxia-Induced Autophagic Response Is Associated with Aggressive Phenotype and Elevated Incidence of Metastasis in Orthotopic Immunocompetent Murine Models of Head and Neck Squamous Cell Carcinomas (HNSCC),” Experimental and Molecular Pathology, Vol. 90, No. 2, 2011, pp. 215-225. doi:10.1016/j.yexmp.2010.11.011
[24]	Z. Chen, C. W. Smith, D. Kiel and C. Van Waes, “Metastatic Variants Derived Following in Vivo Tumor Progression of an in Vitro Transformed Squamous Cell Carcinoma Line Acquire a Differential Growth Advantage Requiring Tumor-Host Interaction,” Clinical and Experimental Metastasis, Vol. 15, No. 5, 1997, pp. 527-537. doi:10.1023/A:1018474910432
[25]	P. Huang, S. Chubb, L. W. Hertel, G. B. Grindey and W. Plunkett, “Action of 2',2'-Difluorodeoxycytidine on DNA Synthesis,” Cancer Research, Vol. 51, No. 22, 1991, pp. 6110-6117.
[26]	M. Hochstrasser, “Ubiquitin, Proteasomes, and the Regulation of Intracellular Protein Degradation,” Current Opinion in Cell Biology, Vol. 7, No. 2, 1995, pp. 215-223. doi:10.1016/0955-0674(95)80031-X
[27]	K. L. Rock, C. Gramm, L. Rothstein, K. Clark, R. Stein, L. Dick, et al., “Inhibitors of the Proteasome Block the Degradation of Most Cell Proteins and the Generation of Peptides Presented on MHC Class I Molecules,” Cell, Vol. 78, No. 5, 1994, pp. 761-771. doi:10.1016/S0092-8674(94)90462-6
[28]	J. H. Lorch, M. R. Posner, L. J. Wirth and R. I. Haddad, “Induction Chemotherapy in Locally Advanced Head and Neck Cancer: A New Standard of Care?” Hematology/ Oncology Clinics of North America, Vol. 22, No. 6, 2008, pp. 1155-1163. doi:10.1016/j.hoc.2008.08.004
[29]	R. I. Haddad and D. M. Shin, “Recent Advances in Head and Neck Cancer,” New England Journal of Medicine, Vol. 359, No. 11, 2008, pp. 1143-1154. doi:10.1056/NEJMra0707975
[30]	S. L. Koolen, P. O. Witteveen, R. S. Jansen, M. H. Langenberg, R. H. Kronemeijer, A. Nol, et al., “Phase I Study of Oral Gemcitabine Prodrug (LY2334737) Alone and in Combination with Erlotinib in Patients with Advanced Solid Tumors,” Clinical Cancer Research, Vol. 17, No. 18, 2011, pp. 6071-6082. doi:10.1158/1078-0432.CCR-11-0353
[31]	P. Pourquier, C. Gioffre, G. Kohlhagen, Y. Urasaki, F. Goldwasser, L. W. Hertel, et al., “Gemcitabine (2’,2’- Difluoro-2’-deoxycytidine), an Antimetabolite That Poisons Topoisomerase I,” Clinical Cancer Research, Vol. 8, No. 8, 2002, pp. 2499-2504.
[32]	E. Petermann, A. Maya-Mendoza, G. Zachos, D. A. Gillespie, D. A. Jackson and K. W. Caldecott, “Chk1 Requirement for High Global Rates of Replication Fork Progression during Normal Vertebrate S Phase,” Molecular and Cellular Biology, Vol. 26, No. 8, 2006, pp. 3319-3326. doi:10.1128/MCB.26.8.3319-3326.2006
[33]	G. Zachos, E. J. Black, M. Walker, M. T. Scott, P. Vagnarelli, W. C. Earnshaw, et al., “Chk1 Is Required for Spindle Checkpoint Function,” Developmental Cell, Vol. 12, No. 2, 2007, pp. 247-260. doi:10.1016/j.devcel.2007.01.003
[34]	A. Hoglund, L. M. Nilsson, S. V. Muralidharan, L. A. Hasvold, P. Merta, M. Rudelius, et al., “Therapeutic Implications for the Induced Levels of Chk1 in Myc-Expressing Cancer Cells,” Clinical Cancer Research, Vol. 17, No. 22, 2011, pp. 7067-7079. doi:10.1158/1078-0432.CCR-11-1198
[35]	K. Myers, M. E. Gagou, P. Zuazua-Villar, R. Rodriguez and M. Meuth, “ATR and Chk1 Suppress a Caspase- 3-Dependent Apoptotic Response Following DNA Replication Stress,” PLoS Genetics, Vol. 5, No. 1, 2009, p. e1000324. doi:10.1371/journal.pgen.1000324
[36]	R. Rodriguez and M. Meuth, “Chk1 and p21 Cooperate to Prevent Apoptosis during DNA Replication Fork Stress,” Molecular Biology of the Cell, Vol. 17, No. 1, 2006, pp. 402-412. doi:10.1091/mbc.E05-07-0594
[37]	A. Loercher, T. L. Lee, J. L. Ricker, A. Howard, J. Geoghegen, Z. Chen, et al., “Nuclear Factor-{kappa}B Is an Important Modulator of the Altered Gene Expression Profile and Malignant Phenotype in Squamous Cell Carcinoma,” Cancer Research, Vol. 64, No. 18, 2004, pp. 6511-6523. doi:10.1158/0008-5472.CAN-04-0852
[38]	T. P. Heffernan, M. Kawasumi, A. Blasina, K. Anderes, A. H. Conney and P. Nghiem, “ATR-Chk1 Pathway Inhibition Promotes Apoptosis after UV Treatment in Primary Human Keratinocytes: Potential Basis for the UV Protective Effects of Caffeine,” Journal of Investigative Dermatology, Vol. 129, No. 7, 2009, pp. 1805-1815. doi:10.1038/jid.2008.435
[39]	V. W. Ruiz van Haperen, G. Veerman, J. B. Vermorken and G. J. Peters, “2’,2’-Difluoro-Deoxycytidine (Gemcitabine) Incorporation into RNA and DNA of Tumour Cell Lines,” Biochemical Pharmacology, Vol. 46, No. 4, 1993, pp. 762-766. doi:10.1016/0006-2952(93)90566-F
[40]	F. Y. Feng, S. Varambally, S. A. Tomlins, P. Y. Chun, C. A. Lopez, X. Li, et al., “Role of Epidermal Growth Factor Receptor Degradation in Gemcitabine-Mediated Cytotoxicity,” Oncogene, Vol. 26, No. 23, 2007, pp. 3431-3439. doi:10.1038/sj.onc.1210129
[41]	C. C. Chen, R. D. Kennedy, S. Sidi, A. T. Look and A. D’Andrea, “CHK1 Inhibition as a Strategy for Targeting Fanconi Anemia (FA) DNA Repair Pathway Deficient Tumors,” Molecular Cancer, Vol. 8, 2009, p. 24. doi:10.1186/1476-4598-8-24
[42]	Z. F. Tao and N. H. Lin, “Chk1 Inhibitors for Novel Cancer Treatment,” Anti-Cancer Agents in Medicinal Chemistry-Anti-Cancer Agents, Vol. 6, No. 4, 2006, pp. 377-388. doi:10.2174/187152006777698132