The importance of the PI3K/AKT/MTOR pathway in the progression of ovarian cancer

International Journal of Molecular Sciences

Volumn 14, Issue 4, 2013, Pages 8213-8227

  Dobbin, Zachary C ^a   Landen, Charles N ^a  

^a UNIVERSITY OF ALABAMA AT BIRMINGHAM   (United States)

Author keywords

AKT; mTOR; Ovarian cancer; PI3K

Indexed keywords

AKT1 PROTEIN; CELL PROTEIN; GELATINASE A; GELATINASE B; HYDROXYMETHYLGLUTARYL COENZYME A REDUCTASE KINASE; MAMMALIAN TARGET OF RAPAMYCIN; MAMMALIAN TARGET OF RAPAMYCIN COMPLEX 1; MAMMALIAN TARGET OF RAPAMYCIN COMPLEX 2; MAMMALIAN TARGET OF RAPAMYCIN INHIBITOR; MICRORNA; MICRORNA 152; MICRORNA 204; MICRORNA 93; ONCOPROTEIN; PHOSPHATIDYLINOSITOL 3 KINASE; PHOSPHATIDYLINOSITOL 3 KINASE INHIBITOR; PROTEIN KINASE B; PROTEIN KINASE B BETA; PROTEIN KINASE B INHIBITOR; PROTEIN PIK3CA; TISSUE INHIBITOR OF METALLOPROTEINASE 1; TISSUE INHIBITOR OF METALLOPROTEINASE 2; UNCLASSIFIED DRUG; MTOR PROTEIN, HUMAN; RNA; TARGET OF RAPAMYCIN KINASE;

BREAST CANCER; CANCER GRADING; CANCER GROWTH; CANCER PROGNOSIS; CANCER RESEARCH; CANCER RESISTANCE; CANCER SURVIVAL; CARCINOGENESIS; DRUG EFFICACY; ENDOMETRIUM CANCER; ENZYME ACTIVITY; HUMAN; MOLECULAR PATHOLOGY; NONHUMAN; OVARY CANCER; OVARY CARCINOMA; PROTEIN EXPRESSION; PROTEIN FUNCTION; PROTEIN TARGETING; REVIEW; ANIMAL; CELL PROLIFERATION; DISEASE COURSE; FEMALE; GENETICS; METABOLISM; MUTATION; NEOPLASMS, EXPERIMENTAL; OVARIAN NEOPLASMS; SIGNAL TRANSDUCTION; TUMOR CELL LINE;

ANIMALS; CELL LINE, TUMOR; CELL PROLIFERATION; DISEASE PROGRESSION; FEMALE; HUMANS; MICRORNAS; MUTATION; NEOPLASMS, EXPERIMENTAL; OVARIAN NEOPLASMS; PHOSPHATIDYLINOSITOL 3-KINASES; PROTO-ONCOGENE PROTEINS C-AKT; RNA, NEOPLASM; SIGNAL TRANSDUCTION; TOR SERINE-THREONINE KINASES;

EID: 84877063389     PISSN: 16616596     EISSN: 14220067     Source Type: Journal    
DOI: 10.3390/ijms14048213     Document Type: Review

References (76)

1. Siegel, R.; Ward, E.; Brawley, O.; Jemal, A. Cancer statistics, 2011. CA A Cancer J. Clin. 2011, 61, 212-236.
2. Armstrong, D.K. Relapsed ovarian cancer: Challenges and management strategies for a chronic disease. Oncologist 2002, 7, 20-28.
3. Badgwell, D.; Bast, R.C., Jr. Early detection of ovarian cancer. Dis. Markers 2007, 23, 397-410.
4. Bast, R.C., Jr.; Brewer, M.; Zou, C.; Hernandez, M.A.; Daley, M.; Ozols, R.; Lu, K.; Lu, Z.; Badgwell, D.; Mills, G.B. et al. Prevention and early detection of ovarian cancer: Mission impossible? In Cancer Prevention, Recent Results in Cancer Research; Springer: Berlin, Germany, 2007; Volume 174, pp. 91-100.
5. Kobayashi, E.; Ueda, Y.; Matsuzaki, S.; Yokoyama, T.; Kimura, T.; Yoshino, K.; Fujita, M.; Kimura, T.; Enomoto, T. Biomarkers for Screening, Diagnosis and Monitoring of Ovarian Cancer. Cancer Epidemiol. Biomarkers Prevent 2012, doi:0.1158/1055-9965.EPI-12-0646
6. Bast, R.C.; Mills, G.B. Dissecting "pi3kness": The complexity of personalized therapy for ovarian cancer. Cancer Discov. 2012, 2, 16-18.
7. The Cancer Genome Atlas Research Network. Integrated genomic analyses of ovarian carcinoma. Nature 2011, 474, 609-615.
8. Cantley, L.C. The phosphoinositide 3-kinase pathway. Science 2002, 296, 1655-1657.
9. Markman, B.; Dienstmann, R.; Tabernero, J. Targeting the pi3k/akt/mtor pathway-Beyond rapalogs. Oncotarget 2010, 7, 530-543.
10. Engelman, J.A.; Luo, J.; Cantley, L.C. The evolution of phosphatidylinositol 3-kinases as regulators of growth and metabolism. Nat. Rev. Genet. 2006, 7, 606-619.
11. Sarbassov, D.D.; Guertin, D.A.; Ali, S.M.; Sabatini, D.M. Phosphorylation and regulation of akt/pkb by the rictor-mtor complex. Science 2005, 307, 1098-1101.
12. Brunet, A.; Bonni, A.; Zigmond, M.J.; Lin, M.Z.; Juo, P.; Hu, L.S.; Anderson, M.J.; Arden, K.C.; Blenis, J.; Greenberg, M.E. Akt promotes cell survival by phosphorylating and inhibiting a forkhead transcription factor. Cell 1999, 96, 857-868.
13. Diehl, J.A.; Cheng, M.; Roussel, M.F.; Sherr, C.J. Glycogen synthase kinase-3β regulates cyclin d1 proteolysis and subcellular localization. Genes Dev. 1998, 12, 3499-3511.
14. Heitman, J.; Movva, N.R.; Hall, M.N. Targets for cell cycle arrest by the immunosuppressant rapamycin in yeast. Science 1991, 253, 905-909.
15. Sabatini, D.M.; Erdjument-Bromage, H.; Lui, M.; Tempst, P.; Snyder, S.H. Raft1: A mammalian protein that binds to fkbp12 in a rapamycin-dependent fashion and is homologous to yeast tors. Cell 1994, 78, 35-43.
16. Brown, E.J.; Albers, M.W.; Bum Shin, T.; ichikawa, K.; Keith, C.T.; Lane, W.S.; Schreiber, S.L. A mammalian protein targeted by g1-arresting rapamycin-receptor complex. Nature 1994, 369, 756-758.
17. Montero, J.C.; Chen, X.; Ocaña, A.; Pandiella, A. Predominance of mtorc1 over mtorc2 in the regulation of proliferation of ovarian cancer cells: Therapeutic implications. Mol. Cancer Ther. 2012, 11, 1342-1352.
18. Zoncu, R.; Efeyan, A.; Sabatini, D.M. Mtor: From growth signal integration to cancer, diabetes and ageing. Nat. Rev. Mol. Cell Biol. 2011, 12, 21-35.
19. Peterson, T.R.; Laplante, M.; Thoreen, C.C.; Sancak, Y.; Kang, S.A.; Kuehl, W.M.; Gray, N.S.; Sabatini, D.M. Deptor is an mtor inhibitor frequently overexpressed in multiple myeloma cells and required for their survival. Cell 2009, 137, 873-886.
20. Borman, S.M.; Christian, P.J.; Sipes, I.G.; Hoyer, P.B. Ovotoxicity in female fischer rats and b6 mice induced by low-dose exposure to three polycyclic aromatic hydrocarbons: Comparison through calculation of an ovotoxic index. Toxicol. Appl. Pharmacol. 2000, 167, 191-198.
21. Shiromizu, K.; Mattison, D.R. Murine oocyte destruction following intraovarian treatment with 3-methylcholanthrene or 7,12-dimethylbenz(a)anthracene: Protection by alpha-naphthoflavone. Teratog. Carcinog. Mutagen. 1985, 5, 463-472.
22. Sobinoff, A.P.; Nixon, B.; Roman, S.D.; McLaughlin, E.A. Staying alive: Pi3k pathway promotes primordial follicle activation and survival in response to 3mc-induced ovotoxicity. Toxicol. Sci. 2012, 128, 258-271.
23. Kaku, T.; Ogawa, S.; Kawano, Y.; Ohishi, Y.; Kobayashi, H.; Hirakawa, T.; Nakano, H. Histological classification of ovarian cancer. Med. Electron Microsc. 2003, 36, 9-17.
24. McCluggage, W.G. Morphological subtypes of ovarian carcinoma: A review with emphasis on new developments and pathogenesis. Pathology 2011, 43, 420-432.
25. Dubeau, L. The cell of origin of ovarian epithelial tumours. Lancet Oncol. 2008, 9, 1191-1197.
26. Kim, J.; Coffey, D.M.; Creighton, C.J.; Yu, Z.; Hawkins, S.M.; Matzuk, M.M. High-grade serous ovarian cancer arises from fallopian tube in a mouse model. Proc. Natl. Acad. Sci. USA 2012, 109, 3921-3926.
27. Wu, R.; Hu, T.C.; Rehemtulla, A.; Fearon, E.R.; Cho, K.R. Preclinical testing of pi3k/akt/mtor signaling inhibitors in a mouse model of ovarian endometrioid adenocarcinoma. Clin. Cancer Res. 2011, 17, 7359-7372.
28. Kurman, R.J.; Shih, I.-M. Pathogenesis of ovarian cancer: Lessons from morphology and molecular biology and their clinical implications. Int. J. Gynecol. Pathol. 2008, 27, 151-160.
29. Shih Ie, M.; Kurman, R.J. Ovarian tumorigenesis: A proposed model based on morphological and molecular genetic analysis. Am. J. Pathol. 2004, 164, 1511-1518.
30. Kinross, K.M.; Montgomery, K.G.; Kleinschmidt, M.; Waring, P.; Ivetac, I.; Tikoo, A.; Saad, M.; Hare, L.; Roh, V.; Mantamadiotis, T. et al. An activating pik3ca mutation coupled with pten loss is sufficient to initiate ovarian tumorigenesis in mice. J. Clin. Investig. 2012, 122, 553-557.
31. Myers, M.P.; Pass, I.; Batty, I.H.; van der Kaay, J.; Stolarov, J.P.; Hemmings, B.A.; Wigler, M.H.; Downes, C.P.; Tonks, N.K. The lipid phosphatase activity of pten is critical for its tumor supressor function. Proc. Natl. Acad. Sci. USA 1998, 95, 13513-13518.
32. Markman, B.; Atzori, F.; Pérez-García, J.Tabernero, J.Baselga, J. Status of pi3k inhibition and biomarker development in cancer therapeutics. Ann. Oncol. 2010, 21, 683-691.
33. Ihle, N.T.; Lemos, R.; Wipf, P.; Yacoub, A.; Mitchell, C.; Siwak, D.; Mills, G.B.; Dent, P.; Kirkpatrick, D.L.; Powis, G. Mutations in the phosphatidylinositol-3-kinase pathway predict for antitumor activity of the inhibitor px-866 whereas oncogenic ras is a dominant predictor for resistance. Cancer Res. 2009, 69, 143-150.
34. Santiskulvong, C.; Konecny, G.E.; Fekete, M.; Chen, K.-Y.M.; Karam, A.; Mulholland, D.; Eng, C.; Wu, H.; Song, M.; Dorigo, O. Dual targeting of phosphoinositide 3-kinase and mammalian target of rapamycin using nvp-bez235 as a novel therapeutic approach in human ovarian carcinoma. Clin. Cancer Res. 2011, 17, 2373-2384.
35. Meng, Q.; Xia, C.; Fang, J.; Rojanasakul, Y.; Jiang, B.-H., Role of pi3k and akt specific isoforms in ovarian cancer cell migration, invasion and proliferation through the p70s6k1 pathway. Cell. Signal. 2006, 18, 2262-2271.
36. Huang, J.; Zhang, L.; Greshock, J.; Colligon, T.A.; Wang, Y.; Ward, R.; Katsaros, D.; Lassus, H.; Butzow, R.; Godwin, A.K.; et al. Frequent genetic abnormalities of the pi3k/akt pathway in primary ovarian cancer predict patient outcome. Genes Chromosom. Cancer 2011, 50, 606-618.
37. Altomare, D.A.; Wang, H.Q.; Skele, K.L.; de Rienzo, A.; Klein-Szanto, A.J.; Godwin, A.K.; Testa, J.R. Akt and mtor phosphorylation is frequently detected in ovarian cancer and can be targeted to disrupt ovarian tumor cell growth. Oncogene 2004, 23, 5853-5857.
38. Itamochi, H.; Oishi, T.; Shimada, M.; Sato, S.; Uegaki, K.; Naniwa, J.; Sato, S.; Nonaka, M.; Terakawa, N.; Kigawa, J. et al. Inhibiting the mtor pathway synergistically enhances cytotoxicity in ovarian cancer cells induced by etoposide through upregulation of c-jun. Clin. Cancer Res. 2011, 17, 4742-4750.
39. Curran, S.; Murray, G.I. Matrix metalloproteinases: Molecular aspects of their roles in tumour invasion and metastasis. Eur. J. Cancer 2000, 36, 1621-1630.
40. Stamenkovic, I. Matrix metalloproteinases in tumor invasion and metastasis. Semin. Cancer Biol. 2000, 10, 415-433.
41. Brew, K.; Dinakarpandian, D.; Nagase, H. Tissue inhibitors of metalloproteinases: Evolution, structure and function. Biochim. Biophys. Acta 2000, 1477, 267-283.
42. Karam, A.K.; Santiskulvong, C.; Fekete, M.; Zabih, S.; Eng, C.; Dorigo, O. Cisplatin and pi3kinase inhibition decrease invasion and migration of human ovarian carcinoma cells and regulate matrix-metalloproteinase expression. Cytoskeleton 2010, 67, 535-544.
43. Vasko, V.; Saji, M.; Hardy, E.; Kruhlak, M.; Larin, A.; Savchenko, V.; Miyakawa, M.; Isozaki, O.; Murakami, H.; Tsushima, T. et al. Akt activation and localisation correlate with tumour invasion and oncogene expression in thyroid cancer. J. Med. Genet. 2004, 41, 161-170.
44. Furuya, F.; Hanover, J.A.; Cheng, S.-Y. Activation of phosphatidylinositol 3-kinase signaling by a mutant thyroid hormone β receptor. Proc. Natl. Acad. Sci. USA 2006, 103, 1780-1785.
45. Fang, J.; Zhou, Q.; Liu, L.-Z.; Xia, C.; Hu, X.; Shi, X.; Jiang, B.-H. Apigenin inhibits tumor angiogenesis through decreasing hif-1α and vegf expression. Carcinogenesis 2006, 28, 858-864.
46. He, J.; Xu, Q.; Wang, M.; Li, C.; Qian, X.; Shi, Z.; Liu, L.-Z.; Jiang, B.-H. Oral administration of apigenin inhibits metastasis through akt/p70s6k1/mmp-9 pathway in orthotopic ovarian tumor model. Int. J. Mol. Sci. 2012, 13, 7271-7282.
47. Vakana, E.; Altman, J.K.; Platanias, L.C. Targeting ampk in the treatment of malignancies. J. Cell. Biochem. 2012, 113, 404-409.
48. Rattan, R.; Graham, R.P.; Maguire, J.L.; Giri, S.; Shridhar, V. Metformin suppresses ovarian cancer growth and metastasis with enhancement of cisplatin cytotoxicity in vivo. Neoplasia 2011, 13, 483-491.
49. Byun, H.J.; Kim, B.R.; Yoo, R.; Park, S.Y.; Rho, S.B. Smek1 enhances gemcitabine anti-cancer activity through inhibition of phosphorylation of akt/mtor. Apoptosis 2012, 17, 1095-1103.
50. Loganathan, S.; Kandala, P.K.; Gupta, P.; Srivastava, S.K. Inhibition of egfr-akt axis results in the suppression of ovarian tumors in vitro and in preclinical mouse model. PLoS One 2012, 7, e43577.
51. Prossnitz, E.R.; Barton, M. The g-protein-coupled estrogen receptor gper in health and disease. Nat. Rev. Endocrinol. 2011, 7, 715-726.
52. Smith, H.O.; Leslie, K.K.; Singh, M.; Qualls, C.R.; Revankar, C.M.; Joste, N.E.; Prossnitz, E.R. Gpr30: A novel indicator of poor survival for endometrial carcinoma. Am. J. Obstet. Gynecol. 2007, 196, 386.e1-386.e11.
53. Albanito, L.; Madeo, A.; Lappano, R.; Vivacqua, A.; Rago, V.; Carpino, A.; Oprea, T.I.; Prossnitz, E.R.; Musti, A.M.; Andò, S. et al. G protein-coupled receptor 30 (gpr30) mediates gene expression changes and growth response to 17β-estradiol and selective gpr30 ligand g-1 in ovarian cancer cells. Cancer Res. 2007, 67, 1859-1866.
54. He, Y.-Y.; Cai, B.; Yang, Y.-X.; Liu, X.-L.; Wan, X.-P. Estrogenic g protein-coupled receptor 30 signaling is involved in regulation of endometrial carcinoma by promoting proliferation, invasion potential, and interleukin-6 secretion via the mek/erk mitogen-activated protein kinase pathway. Cancer Sci. 2009, 100, 1051-1061.
55. Pandey, D.P.; Lappano, R.; Albanito, L.; Madeo, A.; Maggiolini, M.; Picard, D. Estrogenic gpr30 signalling induces proliferation and migration of breast cancer cells through ctgf. EMBO J. 2009, 28, 523-532.
56. Fujiwara, S.; Terai, Y.; Kawaguchi, H.; Takai, M.; Yoo, S.; Tanaka, Y.; Tanaka, T.; Tsunetoh, S.; Sasaki, H.; Kanemura, M.; et al. Gpr30 regulates the egfr-akt cascade and predicts lower survival in patients with ovarian cancer. J. Ovarian Res. 2012, 5, 35.
57. Smith, H.O.; Arias-Pulido, H.; Kuo, D.Y.; Howard, T.; Qualls, C.R.; Lee, S.-J.; Verschraegen, C.F.; Hathaway, H.J.; Joste, N.E.; Prossnitz, E.R. Gpr30 predicts poor survival for ovarian cancer. Gynecol. Oncol. 2009, 114, 465-471.
58. Alo, P.L.; Visca, P.; Framarino, M.L.; Botti, C.; Monaco, S.; Sebastiani, V.; Serpieri, D.E.; di Tondo, U. Immunohistochemical study of fatty acid synthase in ovarian neoplasms. Oncol. Rep. 2000, 7, 1383-1388.
59. Gansler, T.S.; Hardman, W., III; Hunt, D.A.; Schaffel, S.; Hennigar, R.A. Increased expression of fatty acid synthase (oa-519) in ovarian neoplasms predicts shorter survival. Hum. Pathol. 1997, 28, 686-692.
60. Tomek, K.; Wagner, R.; Varga, F.; Singer, C.F.; Karlic, H.; Grunt, T.W. Blockade of fatty acid synthase induces ubiquitination and degradation of phosphoinositide-3-kinase signaling proteins in ovarian cancer. Mol. Cancer Res. 2011, 9, 1767-1779.
61. Lee, R.C.; Feinbaum, R.L.; Ambros, V. The C. elegans heterochronic gene lin-4 encodes small rnas with antisense complementarity to lin-14. Cell 1993, 75, 843-854.
62. Calin, G.A.; Dumitru, C.D.; Shimizu, M.; Bichi, R.; Zupo, S.; Noch, E.; Aldler, H.; Rattan, S.; Keating, M.; Rai, K. et al. Frequent deletions and down-regulation of micro- rna genes mir15 and mir16 at 13q14 in chronic lymphocytic leukemia. Proc. Natl. Acad. Sci. USA 2002, 99, 15524-15529.
63. Zhang, L.; Volinia, S.; Bonome, T.; Calin, G.A.; Greshock, J.; Yang, N.; Liu, C.-G.; Giannakakis, A.; Alexiou, P.; Hasegawa, K. et al. Genomic and epigenetic alterations deregulate microrna expression in human epithelial ovarian cancer. Proc. Natl. Acad. Sci. USA 2008, 105, 7004-7009.
64. Fu, X.; Tian, J.; Zhang, L.; Chen, Y.; Hao, Q. Involvement of microrna-93, a new regulator of pten/akt signaling pathway, in regulation of chemotherapeutic drug cisplatin chemosensitivity in ovarian cancer cells. FEBS Lett. 2012, 586, 1279-1286.
65. Polytarchou, C.; Iliopoulos, D.; Hatziapostolou, M.; Kottakis, F.; Maroulakou, I.; Struhl, K.; Tsichlis, P.N. Akt2 regulates all akt isoforms and promotes resistance to hypoxia through induction of mir-21 upon oxygen deprivation. Cancer Res. 2011, 71, 4720-4731.
66. Lou, Y.; Yang, X.; Wang, F.; Cui, Z.; Huang, Y. Microrna-21 promotes the cell proliferation, invasion and migration abilities in ovarian epithelial carcinomas through inhibiting the expression of pten protein. Int. J. Mol. Med. 2010, 26, 819-827.
67. Wang, Y.-Q.; Guo, R.-D.; Guo, R.-M.; Sheng, W.; Yin, L.-R. Microrna-182 promotes cell growth, invasion and chemoresistance by targeting programmed cell death 4 (pdcd4) in human ovarian carcinomas. J. Cell. Biochem. 2012, doi:10.1002/jcb.24488
68. Xiang, Y.; Ma, N.; Wang, D.; Zhang, Y.; Zhou, J.; Wu, G.; Zhao, R.; Huang, H.; Wang, X.; Qiao, Y. et al. Mir-152 and mir-185 co-contribute to ovarian cancer cells cisplatin sensitivity by targeting dnmt1 directly: A novel epigenetic therapy independent of decitabine. Oncogene 2013, doi:10.1038/onc.2012.575
69. Gong, M.; Ma, J.; Li, M.; Zhou, M.; Hock, J.M.; Yu, X. Microrna-204 critically regulates carcinogenesis in malignant peripheral nerve sheath tumors. Neuro-Oncology 2012, 14, 1007-1017.
70. Chung, T.K.H.; Lau, T.S.; Cheung, T.H.; Yim, S.F.; Lo, K.W.K.; Siu, N.S.S.; Chan, L.K.Y.; Yu, M.Y.; Kwong, J.; Doran,G. et al. Dysregulation of microrna-204 mediates migration and invasion of endometrial cancer by regulating foxc1. Int. J. Cancer 2012, 130, 1036-1045.
71. Imam, J.S.; Plyler, J.R.; Bansal, H.; Prajapati, S.; Bansal, S.; Rebeles, J.; Chen, H.-I.H.; Chang, Y.-F.; Panneerdoss, S.; Zoghi, B. et al. Genomic loss of tumor suppressor mirna-204 promotes cancer cell migration and invasion by activating akt/mtor/rac1 signaling and actin reorganization. PLoS One 2012, 7, e52397.
72. Bellacosa, A.; de Feo, D.; Godwin, A.K.; Bell, D.W.; Cheng, J.Q.; Altomare, D.A.; Wan, M.; Dubeau, L.; Scambia, G.Masciullo, V.et al; Molecular alterations of the akt2 oncogene in ovarian and breast carcinomas. Int. J. Cancer 1995, 64, 280-285.
73. Carden, C.P.; Stewart, A.; Thavasu, P.; Kipps, E.; Pope, L.; Crespo, M.; Miranda, S.; Attard, G.; Garrett, M.D.; Clarke, P.A. et al. The association of pi3 kinase signaling and chemoresistance in advanced ovarian cancer. Mol. Cancer Ther. 2012, 11, 1609-1617.
74. Shimizu, T.; Tolcher, A.W.; Papadopoulos, K.P.; Beeram, M.; Rasco, D.W.; Smith, L.S.; Gunn, S.; Smetzer, L.; Mays, T.A.; Kaiser, B. et al. The clinical effect of the dual-targeting strategy involving pi3k/akt/mtor and ras/mek/erk pathways in patients with advanced cancer. Clin. Cancer Res. 2012, 18, 2316-2325.
75. Carracedo, A.; Pandolfi, P.P. The pten-pi3k pathway: Of feedbacks and cross-talks. Oncogene 2008, 27, 5527-5541.
76. Hoeflich, K.P.; O'Brien, C.; Boyd, Z.; Cavet, G.; Guerrero, S.; Jung, K.; Januario, T.; Savage, H.; Punnoose, E.Truong, T.et al; In vivo antitumor activity of mek and phosphatidylinositol 3-kinase inhibitors in basal-like breast cancer models. Clin. Cancer Res. 2009, 15, 4649-4664.