The class I PI3K/Akt pathway is critical for cancer cell survival in dogs and offers an opportunity for therapeutic intervention

BMC Veterinary Research

David J Argyle¹  Lisa Y Pang¹  Karen AL Tan¹  Yu-Ting Chen¹ 

[1] Royal (Dick) School of Veterinary Studies and the Roslin Institute, University of Edinburgh, Edinburgh, UK, EH25 9RG

关键词: Target;    Therapeutic;    MTOR;    AKT;    PI3;    Cancer;    Canine;   

Others  :  1119814
DOI  :  10.1186/1746-6148-8-73

 received in 2012-01-16, accepted in 2012-05-02,  发布年份 2012

【 摘 要 】

Background

Using novel small-molecular inhibitors, we explored the feasibility of the class I PI3K/Akt/mTORC1 signaling pathway as a therapeutic target in canine oncology either by using pathway inhibitors alone, in combination or combined with conventional chemotherapeutic drugs in vitro.

Results

We demonstrate that growth and survival of the cell lines tested are predominantly dependent on class I PI3K/Akt signaling rather than mTORC1 signaling. In addition, the newly developed inhibitors ZSTK474 and KP372-1 which selectively target pan-class I PI3K and Akt, respectively, and Rapamycin which has been well-established as highly specific mTOR inhibitor, decrease viability of canine cancer cell lines. All inhibitors demonstrated inhibition of phosphorylation of pathway members. Annexin V staining demonstrated that KP372-1 is a potent inducer of apoptosis whereas ZSTK474 and Rapamycin are weaker inducers of apoptosis. Simultaneous inhibition of class I PI3K and mTORC1 by ZSTK474 combined with Rapamycin additively or synergistically reduced cell viability whereas responses to the PI3K pathway inhibitors in combination with conventional drug Doxorubicin were cell line-dependent.

Conclusion

This study highlighted the importance of class I PI3K/Akt axis signaling in canine tumour cells and identifies it as a promising therapeutic target.

【 授权许可】

   
2012 Chen et al.; licensee BioMed Central Ltd.

【 图 表 】

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【 参考文献 】

• [1]Altomare DA, Testa JR: Perturbations of the AKT signaling pathway in human cancer. Oncogene 2005, 24:7455-7464.
• [2]Bjornsti MA, Houghton PJ: The TOR pathway: a target for cancer therapy. Nat Rev Cancer 2004, 4:335-348.
• [3]Jiang BH, Liu LZ: PI3K/PTEN signaling in angiogenesis and tumorigenesis. Adv Cancer Res 2009, 102:19-65.
• [4]Wymann MP, Pirola L: Structure and function of phosphoinositide 3-kinases. Biochim Biophys Acta 1998, 1436:127-150.
• [5]Schlessinger J: Cell signaling by receptor tyrosine kinases. Cell 2000, 103:211-225.
• [6]Kurig B, Shymanets A, Bohnacker T, Prajwal , Brock C, Ahmadian MR, et al.: Ras is an indispensable coregulator of the class IB phosphoinositide 3-kinase p87/p110gamma. Proc Natl Acad Sci U S A 2009, 106:20312-20317.
• [7]Rodriguez-Viciana P, Warne PH, Vanhaesebroeck B, Waterfield MD, Downward J: Activation of phosphoinositide 3-kinase by interaction with Ras and by point mutation. EMBO J 1996, 15:2442-2451.
• [8]Gupta S, Ramjaun AR, Haiko P, Wang Y, Warne PH, Nicke B, et al.: Binding of ras to phosphoinositide 3-kinase p110alpha is required for ras-driven tumorigenesis in mice. Cell 2007, 129:957-968.
• [9]Faivre S, Kroemer G, Raymond E: Current development of mTOR inhibitors as anticancer agents. Nat Rev Drug Discov 2006, 5:671-688.
• [10]Brunet A, Bonni A, Zigmond MJ, Lin MZ, Juo P, Hu LS, et al.: Akt promotes cell survival by phosphorylating and inhibiting a Forkhead transcription factor. Cell 1999, 96:857-868.
• [11]Tsuruta F, Masuyama N, Gotoh Y: The phosphatidylinositol 3-kinase (PI3K)-Akt pathway suppresses Bax translocation to mitochondria. J Biol Chem 2002, 277:14040-14047.
• [12]Datta SR, Dudek H, Tao X, Masters S, Fu H, Gotoh Y, et al.: Akt phosphorylation of BAD couples survival signals to the cell-intrinsic death machinery. Cell 1997, 91:231-241.
• [13]Cardone MH, Roy N, Stennicke HR, Salvesen GS, Franke TF, Stanbridge E, et al.: Regulation of cell death protease caspase-9 by phosphorylation. Science 1998, 282:1318-1321.
• [14]Mayo LD, Donner DB: The PTEN, Mdm2, p53 tumor suppressor-oncoprotein network. Trends Biochem Sci 2002, 27:462-467.
• [15]Ozes ON, Mayo LD, Gustin JA, Pfeffer SR, Pfeffer LM, Donner DB: NF-kappaB activation by tumour necrosis factor requires the Akt serine-threonine kinase. Nature 1999, 401:82-85.
• [16]Miyamoto S, Murphy AN, Brown JH: Akt mediates mitochondrial protection in cardiomyocytes through phosphorylation of mitochondrial hexokinase-II. Cell Death Differ 2008, 15:521-529.
• [17]Cross DA, Alessi DR, Cohen P, Andjelkovich M, Hemmings BA: Inhibition of glycogen synthase kinase-3 by insulin mediated by protein kinase B. Nature 1995, 378:785-789.
• [18]Zhou BP, Liao Y, Xia W, Spohn B, Lee MH, Hung MC: Cytoplasmic localization of p21Cip1/WAF1 by Akt-induced phosphorylation in HER-2/neu-overexpressing cells. Nat Cell Biol 2001, 3:245-252.
• [19]Liang J, Zubovitz J, Petrocelli T, Kotchetkov R, Connor MK, Han K, et al.: PKB/Akt phosphorylates p27, impairs nuclear import of p27 and opposes p27-mediated G1 arrest. Nat Med 2002, 8:1153-1160.
• [20]Manning BD, Tee AR, Logsdon MN, Blenis J, Cantley LC: Identification of the tuberous sclerosis complex-2 tumor suppressor gene product tuberin as a target of the phosphoinositide 3-kinase/akt pathway. Mol Cell 2002, 10:151-162.
• [21]Vander Haar E, Lee SI, Bandhakavi S, Griffin TJ, Kim DH: Insulin signalling to mTOR mediated by the Akt/PKB substrate PRAS40. Nat Cell Biol 2007, 9:316-323.
• [22]Kang SS, Kwon T, Kwon DY, Do SI: Akt protein kinase enhances human telomerase activity through phosphorylation of telomerase reverse transcriptase subunit. J Biol Chem 1999, 274:13085-13090.
• [23]Sabatini DM: mTOR and cancer: insights into a complex relationship. Nat Rev Cancer 2006, 6:729-734.
• [24]Jefferies HB, Fumagalli S, Dennis PB, Reinhard C, Pearson RB, Thomas G: Rapamycin suppresses 5'TOP mRNA translation through inhibition of p70s6k. EMBO J 1997, 16:3693-3704.
• [25]Iadevaia V, Caldarola S, Tino E, Amaldi F, Loreni F: All translation elongation factors and the e, f, and h subunits of translation initiation factor 3 are encoded by 5'-terminal oligopyrimidine (TOP) mRNAs. RNA 2008, 14:1730-1736.
• [26]Terada N, Patel HR, Takase K, Kohno K, Nairn AC, Gelfand EW: Rapamycin selectively inhibits translation of mRNAs encoding elongation factors and ribosomal proteins. Proc Natl Acad Sci U S A 1994, 91:11477-11481.
• [27]Bianchini A, Loiarro M, Bielli P, Busa R, Paronetto MP, Loreni F, et al.: Phosphorylation of eIF4E by MNKs supports protein synthesis, cell cycle progression and proliferation in prostate cancer cells. Carcinogenesis 2008, 29:2279-2288.
• [28]Sun SY, Rosenberg LM, Wang X, Zhou Z, Yue P, Fu H, et al.: Activation of Akt and eIF4E survival pathways by rapamycin-mediated mammalian target of rapamycin inhibition. Cancer Res 2005, 65:7052-7058.
• [29]Waskiewicz AJ, Flynn A, Proud CG, Cooper JA: Mitogen-activated protein kinases activate the serine/threonine kinases Mnk1 and Mnk2. EMBO J 1997, 16:1909-1920.
• [30]Raught B, Gingras AC: eIF4E activity is regulated at multiple levels. Int J Biochem Cell Biol 1999, 31:43-57.
• [31]De Benedetti A, Graff JR: eIF-4E expression and its role in malignancies and metastases. Oncogene 2004, 23:3189-3199.
• [32]Mamane Y, Petroulakis E, Rong L, Yoshida K, Ler LW, Sonenberg N: eIF4E–from translation to transformation. Oncogene 2004, 23:3172-3179.
• [33]Gleixner KV, Rebuzzi L, Mayerhofer M, Gruze A, Hadzijusufovic E, Sonneck K, et al.: Synergistic antiproliferative effects of KIT tyrosine kinase inhibitors on neoplastic canine mast cells. Exp Hematol 2007, 35:1510-1521.
• [34]Akin C: Molecular diagnosis of mast cell disorders: a paper from the 2005 William Beaumont Hospital Symposium on Molecular Pathology. J Mol Diagn 2006, 8:412-419.
• [35]Meshinchi S, Appelbaum FR: Structural and functional alterations of FLT3 in acute myeloid leukemia. Clin Cancer Res 2009, 15:4263-4269.
• [36]Richter A, Murua Escobar H, Gunther K, Soller JT, Winkler S, Nolte I, et al.: RAS gene hot-spot mutations in canine neoplasias. J Hered 2005, 96:764-765.
• [37]Philp AJ, Campbell IG, Leet C, Vincan E, Rockman SP, Whitehead RH, et al.: The phosphatidylinositol 3'-kinase p85alpha gene is an oncogene in human ovarian and colon tumors. Cancer Res 2001, 61:7426-7429.
• [38]Samuels Y, Wang Z, Bardelli A, Silliman N, Ptak J, Szabo S, et al.: High frequency of mutations of the PIK3CA gene in human cancers. Science 2004, 304:554.
• [39]Wang SI, Puc J, Li J, Bruce JN, Cairns P, Sidransky D, et al.: Somatic mutations of PTEN in glioblastoma multiforme. Cancer Res 1997, 57:4183-4186.
• [40]Silva A, Yunes JA, Cardoso BA, Martins LR, Jotta PY, Abecasis M, et al.: PTEN posttranslational inactivation and hyperactivation of the PI3K/Akt pathway sustain primary T cell leukemia viability. J Clin Invest 2008, 118:3762-3774.
• [41]Knobbe CB, Reifenberger G: Genetic alterations and aberrant expression of genes related to the phosphatidyl-inositol-3'-kinase/protein kinase B (Akt) signal transduction pathway in glioblastomas. Brain Pathol 2003, 13:507-518.
• [42]Carpten JD, Faber AL, Horn C, Donoho GP, Briggs SL, Robbins CM, et al.: A transforming mutation in the pleckstrin homology domain of AKT1 in cancer. Nature 2007, 448:439-444.
• [43]Kim MS, Jeong EG, Yoo NJ, Lee SH: Mutational analysis of oncogenic AKT E17K mutation in common solid cancers and acute leukaemias. Br J Cancer 2008, 98:1533-1535.
• [44]Bellacosa A, de Feo D, Godwin AK, Bell DW, Cheng JQ, Altomare DA, et al.: Molecular alterations of the AKT2 oncogene in ovarian and breast carcinomas. Int J Cancer 1995, 64:280-285.
• [45]Nakatani K, Thompson DA, Barthel A, Sakaue H, Liu W, Weigel RJ, et al.: Up-regulation of Akt3 in estrogen receptor-deficient breast cancers and androgen-independent prostate cancer lines. J Biol Chem 1999, 274:21528-21532.
• [46]Davies MA, Stemke-Hale K, Tellez C, Calderone TL, Deng W, Prieto VG, et al.: A novel AKT3 mutation in melanoma tumours and cell lines. Br J Cancer 2008, 99:1265-1268.
• [47]Shan X, Czar MJ, Bunnell SC, Liu P, Liu Y, Schwartzberg PL, et al.: Deficiency of PTEN in Jurkat T cells causes constitutive localization of Itk to the plasma membrane and hyperresponsiveness to CD3 stimulation. Mol Cell Biol 2000, 20:6945-6957.
• [48]Carracedo A, Pandolfi PP: The PTEN-PI3K pathway: of feedbacks and cross-talks. Oncogene 2008, 27:5527-5541.
• [49]Kwitkowski VE, Prowell TM, Ibrahim A, Farrell AT, Justice R, Mitchell SS, et al.: FDA approval summary: temsirolimus as treatment for advanced renal cell carcinoma. Oncologist 2010, 15:428-435.
• [50]Atkins MB, Hidalgo M, Stadler WM, Logan TF, Dutcher JP, Hudes GR, et al.: Randomized phase II study of multiple dose levels of CCI-779, a novel mammalian target of rapamycin kinase inhibitor, in patients with advanced refractory renal cell carcinoma. J Clin Oncol 2004, 22:909-918.
• [51]Paoloni MC, Mazcko C, Fox E, Fan T, Lana S, Kisseberth W, et al.: Rapamycin pharmacokinetic and pharmacodynamic relationships in osteosarcoma: a comparative oncology study in dogs. PLoS One 2010, 5:e11013.
• [52]Buck E, Eyzaguirre A, Brown E, Petti F, McCormack S, Haley JD, et al.: Rapamycin synergizes with the epidermal growth factor receptor inhibitor erlotinib in non-small-cell lung, pancreatic, colon, and breast tumors. Mol Cancer Ther 2006, 5:2676-2684.
• [53]Dal Col J, Zancai P, Terrin L, Guidoboni M, Ponzoni M, Pavan A, et al.: Distinct functional significance of Akt and mTOR constitutive activation in mantle cell lymphoma. Blood 2008, 111:5142-5151.
• [54]Sarbassov DD, Ali SM, Sengupta S, Sheen JH, Hsu PP, Bagley AF, et al.: Prolonged rapamycin treatment inhibits mTORC2 assembly and Akt/PKB. Mol Cell 2006, 22:159-168.
• [55]Kong D, Dan S, Yamazaki K, Yamori T: Inhibition profiles of phosphatidylinositol 3-kinase inhibitors against PI3K superfamily and human cancer cell line panel JFCR39. Eur J Cancer 2010, 46:1111-1121.
• [56]Yaguchi S, Fukui Y, Koshimizu I, Yoshimi H, Matsuno T, Gouda H, et al.: Antitumor activity of ZSTK474, a new phosphatidylinositol 3-kinase inhibitor. J Natl Cancer Inst 2006, 98:545-556.
• [57]Kong D, Yaguchi S, Yamori T: Effect of ZSTK474, a novel phosphatidylinositol 3-kinase inhibitor, on DNA-dependent protein kinase. Biol Pharm Bull 2009, 32:297-300.
• [58]Kong D, Yamori T: ZSTK474 is an ATP-competitive inhibitor of class I phosphatidylinositol 3 kinase isoforms. Cancer Sci 2007, 98:1638-1642.
• [59]Zeng Z, Samudio IJ, Zhang W, Estrov Z, Pelicano H, Harris D, et al.: Simultaneous inhibition of PDK1/AKT and Fms-like tyrosine kinase 3 signaling by a small-molecule KP372-1 induces mitochondrial dysfunction and apoptosis in acute myelogenous leukemia. Cancer Res 2006, 66:3737-3746.
• [60]Chen XG, Liu F, Song XF, Wang ZH, Dong ZQ, Hu ZQ, et al.: Rapamycin regulates Akt and ERK phosphorylation through mTORC1 and mTORC2 signaling pathways. Mol Carcinog 2010, 49:603-610.
• [61]Shor B, Zhang WG, Toral-Barza L, Lucas J, Abraham RT, Gibbons JJ, et al.: A new pharmacologic action of CCI-779 involves FKBP12-independent inhibition of mTOR kinase activity and profound repression of global protein synthesis. Cancer Res 2008, 68:2934-2943.
• [62]Carracedo A, Ma L, Teruya-Feldstein J, Rojo F, Salmena L, Alimonti A, et al.: Inhibition of mTORC1 leads to MAPK pathway activation through a PI3K-dependent feedback loop in human cancer. J Clin Invest 2008, 118:3065-3074.
• [63]Wang X, Yue P, Chan CB, Ye K, Ueda T, Watanabe-Fukunaga R, et al.: Inhibition of mammalian target of rapamycin induces phosphatidylinositol 3-kinase-dependent and Mnk-mediated eukaryotic translation initiation factor 4E phosphorylation. Mol Cell Biol 2007, 27:7405-7413.
• [64]Harrington LS, Findlay GM, Gray A, Tolkacheva T, Wigfield S, Rebholz H, et al.: The TSC1-2 tumor suppressor controls insulin-PI3K signaling via regulation of IRS proteins. J Cell Biol 2004, 166:213-223.
• [65]Tamburini J, Chapuis N, Bardet V, Park S, Sujobert P, Willems L, et al.: Mammalian target of rapamycin (mTOR) inhibition activates phosphatidylinositol 3-kinase/Akt by up-regulating insulin-like growth factor-1 receptor signaling in acute myeloid leukemia: rationale for therapeutic inhibition of both pathways. Blood 2008, 111:379-382.
• [66]Chaturvedi D, Gao X, Cohen MS, Taunton J, Patel TB: Rapamycin induces transactivation of the EGFR and increases cell survival. Oncogene 2009, 28:1187-1196.
• [67]Lin YW, Beharry ZM, Hill EG, Song JH, Wang W, Xia Z, et al.: A small molecule inhibitor of Pim protein kinases blocks the growth of precursor T-cell lymphoblastic leukemia/lymphoma. Blood 2010, 115:824-833.
• [68]Sarbassov DD, Guertin DA, Ali SM, Sabatini DM: Phosphorylation and regulation of Akt/PKB by the rictor-mTOR complex. Science 2005, 307:1098-1101.
• [69]Ikenoue T, Inoki K, Yang Q, Zhou X, Guan KL: Essential function of TORC2 in PKC and Akt turn motif phosphorylation, maturation and signalling. EMBO J 2008, 27:1919-1931.
• [70]Garcia-Martinez JM, Alessi DR: mTOR complex 2 (mTORC2) controls hydrophobic motif phosphorylation and activation of serum- and glucocorticoid-induced protein kinase 1 (SGK1). Biochem J 2008, 416:375-385.
• [71]Pullen N, Dennis PB, Andjelkovic M, Dufner A, Kozma SC, Hemmings BA, et al.: Phosphorylation and activation of p70s6k by PDK1. Science 1998, 279:707-710.
• [72]Roux PP, Shahbazian D, Vu H, Holz MK, Cohen MS, Taunton J, et al.: RAS/ERK signaling promotes site-specific ribosomal protein S6 phosphorylation via RSK and stimulates cap-dependent translation. J Biol Chem 2007, 282:14056-14064.
• [73]Burnett PE, Barrow RK, Cohen NA, Snyder SH, Sabatini DM: RAFT1 phosphorylation of the translational regulators p70 S6 kinase and 4E-BP1. Proc Natl Acad Sci U S A 1998, 95:1432-1437.
• [74]Downward J: Targeting RAS and PI3K in lung cancer. Nat Med 2008, 14:1315-1316.
• [75]Castellano E, Downward J: RAS Interaction with PI3K: More Than Just Another Effector Pathway. Genes Cancer 2010, 2:261-274.