Hsp90 Inhibition Depletes Chk1 and Sensitizes Tumor Cells to Replication Stress

Abstract

DNA damage and replication stress activate the Chk1 signaling pathway, which blocks S phase progression, stabilizes stalled replication forks, and participates in G2 arrest. In this study, we show that Chk1 interacts with Hsp90, a molecular chaperone that participates in the folding, assembly, maturation, and stabilization of specific proteins known as clients. Consistent with Chk1 being an Hsp90 client, we also found that Chk1 but not Chk2 is destabilized in cells treated with the Hsp90 inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG). 17-AAG-mediated Chk1 loss blocked the ability of Chk1 to target Cdc25A for proteolytic destruction, demonstrating that the Chk1 signaling pathway was disrupted in the 17-AAG-treated cells. Finally, 17-AAG-mediated disruption of Chk1 activation dramatically sensitized various tumor cells to gemcitabine, an S phase-active chemotherapeutic agent. Collectively, our studies identify Chk1 as a novel Hsp90 client and suggest that pharmacologic inhibition of Hsp90 may sensitize tumor cells to chemotherapeutic agents by disrupting Chk1 function during replication stress. DNA damage and replication stress activate the Chk1 signaling pathway, which blocks S phase progression, stabilizes stalled replication forks, and participates in G2 arrest. In this study, we show that Chk1 interacts with Hsp90, a molecular chaperone that participates in the folding, assembly, maturation, and stabilization of specific proteins known as clients. Consistent with Chk1 being an Hsp90 client, we also found that Chk1 but not Chk2 is destabilized in cells treated with the Hsp90 inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG). 17-AAG-mediated Chk1 loss blocked the ability of Chk1 to target Cdc25A for proteolytic destruction, demonstrating that the Chk1 signaling pathway was disrupted in the 17-AAG-treated cells. Finally, 17-AAG-mediated disruption of Chk1 activation dramatically sensitized various tumor cells to gemcitabine, an S phase-active chemotherapeutic agent. Collectively, our studies identify Chk1 as a novel Hsp90 client and suggest that pharmacologic inhibition of Hsp90 may sensitize tumor cells to chemotherapeutic agents by disrupting Chk1 function during replication stress. Hsp90 is a ubiquitously expressed and abundant molecular chaperone that participates in the folding, assembly, maturation, and stabilization of specific proteins (known as clients). Hsp90 carries out these functions as an integral component of a multiprotein chaperone complex that contains additional chaperones and co-chaperones (reviewed in Refs. 1Pearl L.H. Prodromou C. Adv. Protein Chem. 2001; 59: 157-186Crossref PubMed Scopus (178) Google Scholar, 2Neckers L. Trends Mol. Med. 2002; 8: S55-S61Abstract Full Text Full Text PDF PubMed Scopus (622) Google Scholar, 3Pratt W. Toft D. Exp. Biol. Med. 2003; 228: 111-133Crossref PubMed Scopus (1276) Google Scholar). Hsp90 regulates client function by cycling between two physiologically important states. In its ATP-bound state, Hsp90 interacts with co-chaperones Cdc37, p23, and an assortment of immunophilin-like proteins, forming a complex that stabilizes and protects client proteins from proteasomal degradation (1Pearl L.H. Prodromou C. Adv. Protein Chem. 2001; 59: 157-186Crossref PubMed Scopus (178) Google Scholar, 2Neckers L. Trends Mol. Med. 2002; 8: S55-S61Abstract Full Text Full Text PDF PubMed Scopus (622) Google Scholar, 3Pratt W. Toft D. Exp. Biol. Med. 2003; 228: 111-133Crossref PubMed Scopus (1276) Google Scholar). In its ADP-bound form, Hsp90 recruits Hsp70 and p60/Hop, forming a complex that targets clients for proteasomal degradation. Many new Hsp90 clients have been discovered recently with the aid of the Hsp90 inhibitors geldanamycin, radicicol, and their derivatives. These agents occupy the Hsp90 ATP-binding site, mimicking the ADP-bound state and targeting clients to the proteasome for degradation (4Prodromou C. Roe S.M. O'Brien R. Ladbury J.E. Piper P.W. Pearl L.H. Cell. 1997; 90: 65-75Abstract Full Text Full Text PDF PubMed Scopus (1125) Google Scholar, 5Grenert J.P. Sullivan W.P. Fadden P. Haystead T.A. Clark J. Mimnaugh E. Krutzsch H. Ochel H.J. Schulte T.W. Sausville E. Neckers L.M. Toft D.O. J. Biol. Chem. 1997; 272: 23843-23850Abstract Full Text Full Text PDF PubMed Scopus (509) Google Scholar, 6Roe S.M. Prodromou C. O'Brien R. Ladbury J.E. Piper P.W. Pearl L.H. J. Med. Chem. 1999; 42: 260-266Crossref PubMed Scopus (896) Google Scholar, 7Schulte T.W. Akinaga S. Soga S. Sullivan W. Stensgard B. Toft D. Neckers L.M. Cell Stress Chaperones. 1998; 3: 100-108Crossref PubMed Scopus (365) Google Scholar). Several of these clients, such as Akt, Her2/Neu, Bcr-Abl, and Raf-1, are important participants in signaling pathways that drive tumor cell proliferation and survival (8Vivanco I. Sawyers C.L. Nat. Rev. Cancer. 2002; 2: 489-501Crossref PubMed Scopus (5171) Google Scholar, 9Neve R.M. Lane H.A. Hynes N.E. Ann. Oncol. 2001; 12: S9-S13Abstract Full Text PDF PubMed Scopus (131) Google Scholar, 10Raitano A.B. Whang Y.E. Sawyers C.L. Biochim. Biophys. Acta. 1997; 1333: F201-F216PubMed Google Scholar, 11Naumann U. Eisenmann-Tappe I. Rapp U.R. Recent Res. Cancer Res. 1997; 143: 237-244Crossref PubMed Scopus (41) Google Scholar). In addition to their role in proliferation, the constitutive activation of these signaling proteins also enhances the survival of tumor cells following DNA damage. Thus, Hsp90-directed therapy has been viewed as a mechanism to simultaneously target numerous oncogenic signaling pathways and sensitize cells to chemotherapeutic agents (2Neckers L. Trends Mol. Med. 2002; 8: S55-S61Abstract Full Text Full Text PDF PubMed Scopus (622) Google Scholar, 12Blagosklonny M.V. Leukemia. 2002; 16: 455-462Crossref PubMed Scopus (229) Google Scholar). Although the oncogenic signaling pathways enhance survival following DNA damage, DNA damage also activates checkpoint signaling pathways that play pivotal roles in the survival of genotoxin-treated cells. The DNA damage-activated checkpoint pathways are evolutionarily conserved signaling pathways that regulate cell cycle progression, programmed cell death, and DNA repair (reviewed in Refs. 13Abraham R.T. Genes Dev. 2001; 15: 2177-2196Crossref PubMed Scopus (1676) Google Scholar and 14Zhou B.B. Elledge S.J. Nature. 2000; 408: 433-439Crossref PubMed Scopus (2645) Google Scholar). One checkpoint signaling pathway that has emerged as a key regulator of cellular responses is the Chk1 signaling pathway, which is activated by replication stress and various types of DNA damage. Following replication fork stalling, single-stranded regions of DNA accumulate that bind the single-stranded DNA-binding protein RPA (15Walter J. Newport J. Mol. Cell. 2000; 5: 617-627Abstract Full Text Full Text PDF PubMed Scopus (324) Google Scholar, 16Lupardus P.J. Byun T. Yee M.C. Hekmat-Nejad M. Cimprich K.A. Genes Dev. 2002; 16: 2327-2332Crossref PubMed Scopus (144) Google Scholar, 17You Z. Kong L. Newport J. J. Biol. Chem. 2002; 277: 27088-27093Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar, 18Zou L. Elledge S.J. Science. 2003; 300: 1542-1548Crossref PubMed Scopus (2059) Google Scholar). RPA binding is then followed by the chromatin recruitment of the phosphatidylinositol 3-kinase-related kinase ATR and its binding partner ATRIP (18Zou L. Elledge S.J. Science. 2003; 300: 1542-1548Crossref PubMed Scopus (2059) Google Scholar). The stalled fork also recruits DNA polymerase α (16Lupardus P.J. Byun T. Yee M.C. Hekmat-Nejad M. Cimprich K.A. Genes Dev. 2002; 16: 2327-2332Crossref PubMed Scopus (144) Google Scholar, 17You Z. Kong L. Newport J. J. Biol. Chem. 2002; 277: 27088-27093Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar), which then participates in the Rad17-dependent recruitment of the PCNA-like Rad9-Hus1-Rad1 (9-1-1) clamp complex to chromatin (17You Z. Kong L. Newport J. J. Biol. Chem. 2002; 277: 27088-27093Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar, 19Zou L. Cortez D. Elledge S.J. Genes Dev. 2002; 16: 198-208Crossref PubMed Scopus (437) Google Scholar). Once bound to chromatin, the 9-1-1 complex facilitates the ATR-mediated phosphorylation and activation of Chk1 (reviewed in Ref. 20Melo J. Toczyski D. Curr. Opin. Cell Biol. 2002; 14: 237-245Crossref PubMed Scopus (399) Google Scholar). Activated Chk1 performs several functions that promote cell survival. First, Chk1 increases the time available for DNA repair by arresting cells in G2. This arrest occurs when Chk1 phosphorylates Cdc25C and Cdc25A, two cell cycle phosphatases that activate the Cdk1-cyclin B complex (reviewed in Ref. 21Rhind N. Russell P. J. Cell Sci. 2000; 113: 3889-3896Crossref PubMed Google Scholar). Second, activated Chk1 slows progression through S phase by blocking the firing of unfired origins of the replication (22Guo N. Faller D.V. Vaziri C. Cell Growth Differ. 2002; 13: 77-86PubMed Google Scholar, 23Heffernan T.P. Simpson D.A. Frank A.R. Heinloth A.N. Paules R.S. Cordeiro-Stone M. Kaufmann W.K. Mol. Cell Biol. 2002; 22: 8552-8561Crossref PubMed Scopus (208) Google Scholar, 24Zhao H. Watkins J.L. Piwnica-Worms H. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 14795-14800Crossref PubMed Scopus (422) Google Scholar, 25Zachos G. Rainey M.D. Gillespie D.A. EMBO J. 2003; 22: 713-723Crossref PubMed Scopus (223) Google Scholar). In this checkpoint, Chk1 phosphorylates Cdc25A, leading to Cdc25A degradation (24Zhao H. Watkins J.L. Piwnica-Worms H. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 14795-14800Crossref PubMed Scopus (422) Google Scholar, 26Shimuta K. Nakajo N. Uto K. Hayano Y. Okazaki K. Sagata N. EMBO J. 2002; 21: 3694-3703Crossref PubMed Scopus (111) Google Scholar, 27Mailand N. Falck J. Lukas C. Syljuasen R.G. Welcker M. Bartek J. Lukas J. Science. 2000; 288: 1425-1429Crossref PubMed Scopus (656) Google Scholar). Because Cdc25A is required for activation of Cdk2 complexes, which then control the firing of origins of replication, activation of this pathway blocks S phase progression (28Sexl V. Diehl J.A. Sherr C.J. Ashmun R. Beach D. Roussel M.F. Oncogene. 1999; 18: 573-582Crossref PubMed Scopus (87) Google Scholar, 29Blomberg I. Hoffman I. Mol. Cell Biol. 1999; 19: 6183-6194Crossref PubMed Scopus (255) Google Scholar, 30Hoffmann I. Draetta G. Karsenti E. EMBO J. 1994; 13: 4302-4310Crossref PubMed Scopus (424) Google Scholar, 31Jinno S. Suto K. Nagata A. Igarashi M. Kanaoka Y. Nojima H. Okayama H. EMBO J. 1994; 13: 1549-1556Crossref PubMed Scopus (400) Google Scholar). Third, Chk1 stabilizes stalled replication forks. Although the relevant substrate is not known, in the absence of Chk1 function, the stalled forks irreversibly collapse. Correspondingly, disruption of the Chk1 signaling pathway is associated with increased sensitivity to genotoxins. For example, cells lacking the upstream regulators Hus1 and Rad9 are highly sensitive to ultraviolet radiation and the replication inhibitor hydroxyurea (32Weiss R.S. Enoch T. Leder P. Genes Dev. 2000; 14: 1886-1898PubMed Google Scholar, 33Roos-Mattjus P. Hopkins K.M. Oestreich A.J. Vroman B.T. Johnson S. L.M. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). cells in which Chk1 has been by targeting are sensitive to the replication inhibitor and radiation G. Rainey M.D. Gillespie D.A. EMBO J. 2003; 22: 713-723Crossref PubMed Scopus (223) Google Scholar). Consistent with this a pharmacologic Chk1 inhibitor L. 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Cell Biol. 15: PubMed Scopus Google Scholar). Hsp90 inhibition the ability of Chk1 to to upstream and to activated by we cells with for and Chk1 was by its upstream kinase in not Chk1 that when Hsp90 is this client to upstream these also that Hsp90 inhibition not of the upstream required for Chk1 the of Hsp90 inhibition Chk1 we a Chk1 expressed Chk1 in cells. to Chk1 in cell we not of cells with activated the of Chk1 and this activation was not blocked by a with these suggest that Hsp90 is not required to Chk1 in a state that is to and to upstream 17-AAG-mediated Chk1 Cdc25A Cdc25A phosphorylation to Cdc25A proteolytic destruction, blocking cell cycle progression (24Zhao H. Watkins J.L. Piwnica-Worms H. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 14795-14800Crossref PubMed Scopus (422) Google Scholar, 27Mailand N. Falck J. Lukas C. Syljuasen R.G. Welcker M. Bartek J. Lukas J. Science. 2000; 288: 1425-1429Crossref PubMed Scopus (656) Google Scholar, I. R. I. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar, Z. Z. S. H. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar, Syljuasen R.G. Falck J. T. L. B.B. Bartek J. Lukas J. Cancer Cell. 2003; 3: Full Text Full Text PDF PubMed Scopus Google Scholar). Chk1 loss Cdc25A destruction, we treated cells with for and then with for an additional and Cdc25A this the of Chk1 in cells is to that found in cells Consistent with our Chk1 kinase Chk1 was not in cells treated with for this time and not the Cdc25A destruction, demonstrating that Chk1 Chk1 loss Cdc25A we treated cells with for to S phase arrest and activate then for an additional to and Cdc25A Cdc25A was in the demonstrating that Cdc25A of to In the addition of during the of this the Cdc25A Thus, these show that to Chk1 loss and the loss of cellular responses to stress. of Chk1 to Cell an integral and role in activation of the S phase checkpoint (22Guo N. Faller D.V. Vaziri C. Cell Growth Differ. 2002; 13: 77-86PubMed Google Scholar, 23Heffernan T.P. Simpson D.A. Frank A.R. Heinloth A.N. Paules R.S. Cordeiro-Stone M. Kaufmann W.K. Mol. Cell Biol. 2002; 22: 8552-8561Crossref PubMed Scopus (208) Google Scholar, 24Zhao H. Watkins J.L. Piwnica-Worms H. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 14795-14800Crossref PubMed Scopus (422) Google Scholar, 25Zachos G. Rainey M.D. Gillespie D.A. EMBO J. 2003; 22: 713-723Crossref PubMed Scopus (223) Google Scholar), which stabilizes stalled DNA replication forks and blocks the firing of origins of has that cells in S phase with of are when the Chk1 inhibitor was to the cells Z. A. D. P. W. Cancer Res. 2001; Google Scholar). Because Chk1 is following we that also sensitize tumor cells to the S phase arrest by we the of to sensitize cells. Because is in S phase and cells in and we cells in S phase by for with then and the cells for an additional and Cell survival was by cells with and and cells and the addition of of the cells that cells the time In the of and cells this time the time cell when the was and as these studies to tumor cell we also the of these and cells. cell with of for and then treated with for an additional and the cell was with was with the and in cell in the and cells In with to these studies that dramatically tumor cells. In this we that a key component of the checkpoint activated by replication is an Hsp90 Hsp90 inhibitors to the loss of Chk1 and a disruption of Cdc25A and tumor cells in S phase by Collectively, these new the of the Chk1 signaling pathway and suggest a novel mechanism to sensitize tumor cells to the that Hsp90 function, numerous Hsp90 clients have been and our of the role of Hsp90 in cellular has a These studies have that the and and the and Akt, of which regulate tumor cell proliferation and are Hsp90 clients (reviewed in Refs. 2Neckers L. Trends Mol. Med. 2002; 8: S55-S61Abstract Full Text Full Text PDF PubMed Scopus (622) Google Scholar and 12Blagosklonny M.V. Leukemia. 2002; 16: 455-462Crossref PubMed Scopus (229) Google Scholar). Because these signaling pathways are activated in tumor cells and Hsp90 is in tumor Hsp90 inhibitors may agents that simultaneously signaling pathways that drive tumor cell proliferation and survival. In several studies have that and its also sensitize tumor cells to agents (2Neckers L. Trends Mol. Med. 2002; 8: S55-S61Abstract Full Text Full Text PDF PubMed Scopus (622) Google Scholar). One for the is that proliferation and survival signaling such as the and are by Hsp90 the sensitivity of cells to genotoxins. the Chk1 signaling pathway also a role in the survival of genotoxin-treated cells. Because the suggest that Hsp90 inhibition also tumor cells to by disrupting Chk1 function, mechanism by which Hsp90 inhibitors may sensitize tumor cells to genotoxins. Chk1 an role in cell in genotoxin-treated cells (22Guo N. Faller D.V. Vaziri C. Cell Growth Differ. 2002; 13: 77-86PubMed Google Scholar, 23Heffernan T.P. Simpson D.A. Frank A.R. Heinloth A.N. Paules R.S. Cordeiro-Stone M. Kaufmann W.K. Mol. Cell Biol. 2002; 22: 8552-8561Crossref PubMed Scopus (208) Google Scholar, 24Zhao H. Watkins J.L. Piwnica-Worms H. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 14795-14800Crossref PubMed Scopus (422) Google Scholar, 25Zachos G. Rainey M.D. Gillespie D.A. EMBO J. 2003; 22: 713-723Crossref PubMed Scopus (223) Google Scholar, Nat. Cell Biol. 2000; 2: PubMed Scopus Google Scholar). Following replication fork and the of single-stranded the of the Chk1 signaling pathway are to Chk1 activation J. Toczyski D. Curr. Opin. Cell Biol. 2002; 14: 237-245Crossref PubMed Scopus (399) Google Scholar). Once Chk1 blocks the firing of unfired origins by targeting Cdc25A for degradation (24Zhao H. Watkins J.L. Piwnica-Worms H. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 14795-14800Crossref PubMed Scopus (422) Google Scholar, 26Shimuta K. Nakajo N. Uto K. Hayano Y. Okazaki K. Sagata N. EMBO J. 2002; 21: 3694-3703Crossref PubMed Scopus (111) Google Scholar, 27Mailand N. Falck J. Lukas C. Syljuasen R.G. Welcker M. Bartek J. Lukas J. Science. 2000; 288: 1425-1429Crossref PubMed Scopus (656) Google Scholar). This protects the by additional DNA replication when damage is The Chk1 signaling pathway also stabilizes stalled replication forks G. Rainey M.D. Gillespie D.A. EMBO J. 2003; 22: 713-723Crossref PubMed Scopus (223) Google Scholar, Nat. Cell Biol. 2000; 2: PubMed Scopus Google Scholar, C. C. R. D. J. C. J. Cell Biol. 2001; PubMed Scopus Google Scholar). Although the relevant Chk1 is not known, studies have that in the absence of Chk1 function, stalled forks G. Rainey M.D. Gillespie D.A. EMBO J. 2003; 22: 713-723Crossref PubMed Scopus (223) Google Scholar, C. C. R. D. J. C. J. Cell Biol. 2001; PubMed Scopus Google Scholar). the of the replication cells not proliferation, and the cells not G. Rainey M.D. Gillespie D.A. EMBO J. 2003; 22: 713-723Crossref PubMed Scopus (223) Google Scholar). Consistent with this of S cells with of the S phase cells cell cycle progression D. Z. W. Mol. 2002; PubMed Scopus Google Scholar, Z. A. D. P. W. Cancer Res. 2001; Google Scholar). 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Biol. 2002; 2: PubMed Scopus Google Scholar), may a pharmacologic to Chk1 function in treated with S phase-active In may have the of blocking additional signaling pathways that also to the suggest that of replication inhibitors and may to tumor In a in which are treated with replication inhibitors to arrest tumor cells in S phase followed by to Chk1 signaling may a to such J. for the and also S. Kaufmann for and of the and for