npgrj_nchembio_2007-10 1..10

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© 2007 Nature Publishing Group http://www.nature.com/naturechemicalbiology

Selective compounds define Hsp90 as a major inhibitor of apoptosis in small-cell lung cancer Anna Rodina1,9, Maria Vilenchik1,8,9, Kamalika Moulick1, Julia Aguirre1, Joungnam Kim1, Anne Chiang2, Julie Litz3, Cristina C Clement1, Yanlong Kang1, Yuhong She1, Nian Wu1, Sara Felts4, Peter Wipf 5, Joan Massague2, Xuejun Jiang6, Jeffrey L Brodsky7, Geoffrey W Krystal3 & Gabriela Chiosis1 The heat shock protein 90 (Hsp90) has a critical role in malignant transformation. Whereas its ability to maintain the functional conformations of mutant and aberrant oncoproteins is established, a transformation-specific regulation of the antiapoptotic phenotype by Hsp90 is poorly understood. By using selective compounds, we have discovered that small-cell lung carcinoma is a distinctive cellular system in which apoptosis is mainly regulated by Hsp90. Unlike the well-characterized antiapoptotic chaperone Hsp70, Hsp90 is not a general inhibitor of apoptosis, but it assumes this role in systems such as small-cell lung carcinoma, in which apoptosis is uniquely dependent on and effected through the intrinsic pathway, without involvement of caspase elements upstream of mitochondria or alternate pathways that are not apoptosome-channeled. These results provide important evidence for a transformation-specific interplay between chaperones in regulating apoptosis in malignant cells.

Molecular chaperone Hsp90 is a protein with important roles in the maintenance of malignant transformation and is required to keep the folded and functionally active conformation of several aberrantly functioning oncoproteins1–4. In this respect, Hsp90 regulates (in a transformation-specific manner) signaling pathways necessary for the growth, survival and limitless replicative potential of most tumors. Whereas in normal cells Hsp90 interacts in a low-affinity, dynamic fashion with a plethora of proteins to help them fold and mature, in malignant cells tight association of Hsp90 with oncoclient proteins maintains their ability to function in the dysregulated state and seems to be essential for their transforming, aberrant activity1–4. Historically, v-Src was the first oncoprotein shown to have ‘‘high-affinity’’ interactions with Hsp90, a property that is not shared with nononcogenic c-Src3,4. Moreover, in contrast with v-Src, c-Src requires only limited assistance by Hsp90 for its maturation and cellular function. Many examples of such behavior have now been identified in transformed cells, as many proteins involved in cell type–specific oncogenic processes have been shown to be tightly regulated by Hsp90. These include activated kinases such as Bcr-Abl in chronic myelogenous leukemia, nucleophosminanaplastic lymphoma kinase in non-Hodgkin’s lymphoma, mutated Flt3 in acute myeloid leukemia, and epidermal growth factor receptor harboring kinase mutations in non–small cell lung cancer. Because of such roles, researchers have proposed that

Hsp90 is ‘‘a buffer of mutations’’ that otherwise would be lethal to the cell4. In addition to increased proliferation, enhanced survival of cancer cells may result from effective shutdown of death pathways. Surprisingly, transformation-specific roles for Hsp90 in regulating the antiapoptotic phenotype of cancer cells are poorly defined. Here we use specific chemical tools to understand roles played by Hsp90 in regulating apoptosis in the specific setting of small-cell lung cancer (SCLC) and to define possible interplays between chaperones in inhibiting apoptosis in a transformation-specific manner. RESULTS Characterization of transformation in SCLC Hsp90 is co-opted to buffer the malignant phenotype in a cell-specific manner1–4. Therefore, to probe its role in regulating transformation in SCLC we first identified the aberrant elements leading to the antiapoptotic phenotype in this tumor. SCLC is a highly malignant tumor accounting for about 20% of all lung cancers5. In spite of several advances in cancer treatment, survival for individuals afflicted with SCLC remains poor, with a median survival time of 10–14 months. Whereas this disease often initially responds well to chemotherapy, relapses occur almost without exception, and these are usually resistant to cytotoxic treatment. The prognosis for people with SCLC that has progressed despite chemotherapy is exceedingly poor

1Program in Molecular Pharmacology and Chemistry and 2Program in Cancer Biology and Genetics, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, New York 10021, USA. 3Department of Medicine, Medical College of Virginia/Virginia Commonwealth University and McGuire Veterans Affairs Medical Center, 1201 Broad Rock Boulevard, Richmond, Virginia 23249, USA. 4Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine, 200 First Street, Rochester, Minnesota 55905, USA. 5Department of Chemistry, University of Pittsburgh, 274A Crawford Hall, Pittsburgh, Pennsylvania 15260, USA. 6Program in Cell Biology, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, New York 10021, USA. 7Department of Biological Sciences, University of Pittsburgh, 274A Crawford Hall, Pittsburgh, Pennsylvania 15260, USA. 8Present address: Hoffmann-La Roche Inc., 500 Kingsland St., Nutley, New Jersey 07110-1046, USA. 9These authors contributed equally to this work. Correspondence should be addressed to G.C. ([email protected]).

Received 16 March; accepted 8 June; published online 1 July 2007; doi:10.1038/nchembio.2007.10

NATURE CHEMICAL BIOLOGY

ADVANCE ONLINE PUBLICATION

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regardless of stage, and expected median survival is 2 to 3 months. It is therefore important to understand the elements that lead to a transformed phenotype in SCLC and to identify targets on which both chemotherapy-naı¨ve and chemotherapy-resistant tumors depend for their viability. One of the characteristics of SCLC tumors is substantial perturbations in apoptosis6. In many such cancers there is not only loss of proapoptotic proteins but also activation or overexpression of antiapoptotic molecules. Among these, several caspases, such as caspase-1, caspase-4, caspase-8 and caspase-10, are either absent or inactivated in SCLC cell lines and tumors7. In addition, SCLC is a human cancer of neural-neuroendocrin origin8 that is also characterized by a defective retinoblastoma gene product (Rb)9. pRb suppresses apoptosis by repressing a distinct proapoptotic set of E2F target genes that includes the apoptotic protease activating factor-1 (Apaf-1) and caspases10. Studies with Rb mutant mice have further shed light on the consequence of this cellular defect on apoptosis10. In Rb mutant embryos, Apaf-1 is absolutely required for apoptosis in the central nervous system and lens. As Apaf-1 is critical for apoptosome function, these data suggest that death in the Rb-null cells requires and is channeled through the intrinsic apoptotic machinery. Components of the intrinsic apoptotic pathway, such as Apaf-1 and caspase-9, are expressed and active in SCLC cells6. These data suggest that in order to remain viable, SCLC cells must tightly downregulate the activation of these apoptotic factors with an efficacy comparable to that of the Rb tumor suppressor. A defective phosphatidylinositol-3-OH kinase (PI(3)K)-Akt signal promoting SCLC growth, survival and chemotherapy resistance was further identified in SCLC11,12. First, several SCLC cells harbor PI(3)K or phosphatase and tensin homolog (PTEN) mutations, which leads

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to constitutive activation of PI(3)K that in turn promotes anchorage-independent proliferation11,12. Second, inhibition of Akt activity in these cells enhances the apoptotic effect of chemotherapy11. Third, laminin-mediated 10 100 activation of the PI(3)K-Akt-mTOR pathway is proposed as a mechanism of cellular survival and therapeutic resistance in SCLC cells, which suggests that inhibition of this pathway is one strategy for overcoming SCLC resistance12. In summary, deficiencies in apoptosis, combined with the increased survival conferred by the PI(3)K-Akt pathway, characterize transformation in SCLC and facilitate its resistance to routine chemotherapy. This suggests that components of both the intrinsic apoptotic pathway and the PI(3)K-Akt survival pathway may require Hsp90 assistance to tightly regulate their aberrant function, and to facilitate the viability of SCLC cells under the specific transforming pressure. Inhibition of Hsp90 leads to significant cell killing Because reduction of as much as 50% in Hsp90 expression in cancer cells has little phenotypic consequence, much knowledge of Hsp90 biology in cancer cells comes from probing its function with pharmacologic tools such as geldanamycin (1) and its derivative 17-allylamino-17-demethoxy-geldanamycin (17AAG; 2)2. However, there are several caveats with the use of these agents. The activity of ansamycins is modulated by a reductive environment in the cell, which results in effects that are not a direct consequence of Hsp90 inhibition13. Further, a lack or reduced activity of these agents in certain cells has been observed owing to drug efflux from cells by multidrugresistance elements1–4 or metabolic inactivation of these agents by nucleophiles14. Our laboratory has designed and developed chemical tools (the PU class of Hsp90 inhibitors) that bind specifically to the Hsp90 N-terminal site regulatory pocket15–18. Further work by us and others has demonstrated that PU derivatives are selective for Hsp90, and moreover they exert their biological effects via specific interference with tumor Hsp90 (refs. 15–18 and Supplementary Fig. 1 online). In addition to being selective for the high-affinity tumor Hsp90, these agents are not metabolically sensitive to the cellular environment, nor



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are they P-glycoprotein substrates (F. Burrows, Biogen Idec, personal communication). Such characteristics render them suitable chemical tools to specifically translate Hsp90 inhibition into biological activity in a particular transformed system. To probe the role of Hsp90 in regulating malignant perturbations in SCLC, we made use of three representative PU derivatives: 8-(2-chloro-3,4,5-trimethoxybenzyl)2-fluoro-9-(pent-4-ynyl)-9H-purin-6-amine (PU24FCl; 3), 8-(6-bromobenzo[d][1,3]dioxol-5-ylthio)-9-(pent-4-ynyl)-9H-purin-6-amine (PU-H58; 4) and 8-(6-iodobenzo[d][1,3]dioxol-5-ylthio)-9-(3-(isopropylamino)propyl)-9H-purin-6-amine (PU-H71; 5) (Fig. 1a). We compared these derivatives’ affinities for SCLC-specific Hsp90 with their cytotoxic activities in these cells (Fig. 1b–f). To probe binding, we used an assay that measures interactions of cellular Hsp90 with inhibitors of its ATPase activity17,18. Expression of Hsp90 was almost identical among the tested SCLC cell lines (Fig. 1b), and the PU derivatives bound similarly to each SCLC cell-specific Hsp90; only a 2.5- to 4-fold difference in affinity was observed among these cell lines for a particular inhibitor (Fig. 1c). The difference in affinity among the three inhibitors followed the trend previously recorded in other cancer cells: PU-H58 interacted B10 times more avidly than PU24FCl with Hsp90 (Fig. 1c), whereas PU-H71 was B5 times more potent than PU-H58 (Fig. 1e)16–18. In addition, we determined the cytotoxic effect of PU derivatives in these SCLC cells. Both PU24FCl and its higher-potency derivative, PU-H58, inhibited the growth and caused substantial death in all tested SCLC cells (Fig. 1d). At concentrations of PU24FCl, PU-H58 and PU-H71 in excess of 10–20 mM, 1–3 mM and 0.3–0.5 mM, respectively, 50–100% of the starting cell populations were dead at 72 h or 96 h post-treatment. Collectively, these data demonstrate that the Hsp90 binding affinity shown by these three drugs (Fig. 1c,e) correlates well with their death-inducing potencies (Fig. 1d,f), which suggests that their biological effect is a result of Hsp90 inhibition. To further demonstrate that significant cytotoxicity in these cells is due to Hsp90 inhibition and not to possible PU class off-target effects, we made use of several distinct Hsp90 inhibitor chemotypes (Supplementary Fig. 2 online). These are represented by radicicol (6) and



cycloproparadicicol (7), members of the macrolactone-Hsp90 inhibitor class, and 17-dimethylaminoethylamino-17-demethoxygeldanamycin (17DMAG; 8) and 17AAG, members of the ansamycin class19. These agents bind effectively to Hsp90 in NCI-H526 cells (Supplementary Fig. 2) and result in across-the-board in substantial cell killing (Supplementary Fig. 2), which suggests that demise of SCLC cells is a result of Hsp90 inhibition. Hsp90 inhibits the intrinsic apoptotic pathway To determine whether cell death was attributable to apoptosis, we treated cells with the Hsp90 inhibitors and analyzed their effects on several effectors and mediators of apoptosis (Fig. 2a–c and Supplementary Fig. 3 online). Given the absence of a functional death receptor pathway, apoptosis in SCLC is probably mitochondrialmediated6. Apoptotic signals to the mitochondria lead to membrane depolarization, resulting in activation of the effector caspase-3, poly(ADP-ribose) polymerase (PARP) cleavage, and DNA fragmentation20,21. Notably, we observed a substantial increase in caspase-3 and caspase-7 activity upon addition of these agents to SCLC cells: a two- to ten-fold boost in the apoptotic cell population was seen at 3 to 12 mM concentrations of PU24FCl (Fig. 2a) and 0.2 to 1.0 mM concentrations of PU-H58 (Supplementary Fig. 3), respectively (Supplementary Methods online). Several caspases may be responsible for caspase-3 activation. Caspase-3 activation by PU24FCl was associated with cleavage of both caspase-9 and caspase-2, and further paralleled by PARP cleavage (Fig. 2b and Supplementary Fig. 3). Cleavage of caspases occurred at doses of PU24FCl that correlate with the Hsp90 inactivation potency of this agent (Fig. 1c); cleavage was evident at doses above 5 mM. Similar activation of the intrinsic apoptotic pathway, as demonstrated by cleavage of both caspase-3 and PARP, was seen in these cells with all tested Hsp90 inhibitor chemotypes (Supplementary Fig. 2). To extend these results, we analyzed the effects of Hsp90 inhibitors on the mitochondrial membrane potential (MMP) using the dye JC-1. We observed in a panel of NCI-H526 (Fig. 2d), NCI-N417, NCI-H187 and NCI-H69 SCLC cells (Supplementary Fig. 3) that a total shift toward cells that emit only green fluorescence, indicating membrane depolarization, was evident above 5 mM PU24FCl. We also evaluated the DNA-fragmenting effects of PU24FCl on a panel of asynchronously growing SCLC cells, and we observed a considerable accumulation of cells containing sub-G1 DNA content, which is characteristic of apoptosis, with increasing doses of PU24FCl. When treated with 0, 2.5, 5, 15 and 25 mM of PU24FCl, the sub-G1 population increased

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respectively from 10 to 18, 40, 60 and 80% in NCI-N526 cells (Fig. 2e), from 13 to 38, 48, 66 and 89% in NCI-N417 cells (data not shown), and from 11 to 36, 71, 82 and 83% in NCI-H187 cells (data not shown). Together, the massive apoptotic response and significant cell population demise upon Hsp90 inhibition suggest that the chaperone is an important regulator of the intrinsic apoptotic pathway in SCLC cells. They also demonstrate that death induced by pharmacologic Hsp90 inhibition in SCLC cells is mainly channeled through the intrinsic apoptotic pathway. Mechanism of apoptosis inhibition by Hsp90 To elucidate the mechanism by which Hsp90 regulates apoptosis in SCLC cells, we made use of both the broad-spectrum caspase inhibitor Boc-D-FMK and the irreversible caspase-9 inhibitor Z-LEHD-FMK. First, we analyzed SCLC cells pretreated with Boc-D-FMK for 3 h before PU24FCl addition for changes in MMP and cell death by JC-1 and propidium iodide staining, respectively. The broad-spectrum caspase inhibitor had no statistically significant influence on the mitochondrial depolarizing activity of PU24FCl, which indicates that PU24FCl acts independently of upstream caspase activation (Fig. 3a). Boc-D-FMK, however, completely blocked death in NCIH526 cells, which suggests that death in these cells occurred mainly via caspase-executed apoptosis (Fig. 3b). Next, NCI-H526 cells were pretreated with Z-LEHD-FMK. The caspase-9 inhibitor inhibited the activation of caspase-3 (Fig. 3c) and prevented its full cleavage (Fig. 3d) by PU24FCl, which suggests that Hsp90-regulated apoptosis in SCLC is channeled through the caspase-9–caspase-3 pathway. In contrast, we failed to note increased proteolytic activation of Bid, which occurs when apoptosis is triggered either by the death receptor pathway21 or by caspase-2 when activated upstream of the mitochondria22 (Fig. 2c). These data suggest that the observed activation of caspase-2 by Hsp90 inhibition in SCLC cells (Fig. 2b) must occur downstream of the mitochondria. Release of caspase-2 from mitochondria is documented upon membrane depolarization23, and our data may identify SCLC cells as an endogenous system in which this phenomenon occurs upon Hsp90 inactivation. It was reported that high levels of expression of both apoptosis inhibitory proteins, such as Bcl-2, and inhibitors of apoptosis proteins, such as survivin, are characteristic of many SCLC tumors, likely raising the apoptotic threshold in these tumors6. In some systems,

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Hsp90 chaperones survivin function24. In SCLC cells, however, Hsp90 does not seem to tightly regulate the stability of these proteins, as evidenced by the lack of an effect of PU24FCl on the steady state levels of Bcl-2, Bcl-xL and survivin (Fig. 2c). Apaf-1 is necessary to promote activation of both caspase-9 and caspase-9–mediated activation of caspase-3 (ref. 21). We found that Apaf-1 forms a complex with Hsp90 in SCLC cells. Inhibition of Hsp90 by PU24FCl led to a time-dependent release of Apaf-1 from the chaperone complex, which correlated with increased association between caspase-9 and Apaf-1 (Fig. 4a). Whereas a gradual shift of Apaf-1 from the Hsp90 complex to the caspase-9 complex was observed at as early as 4 h (Fig. 4a), a substantial activation of the apoptotic processes in these cells was delayed to over 10 h (Fig. 4b), which suggests that Apaf-1 release is necessary but not sufficient to trigger massive apoptosis in SCLC by Hsp90 inhibition. Activation of caspase-9 requires release of cytochrome c from the mitochondria, which indicates that at 4 h, apoptotic stimuli leading to mitochondrial depolarization may not yet be sufficiently activated by the Hsp90 inhibitor. Another important determinant involved in promoting survival and resistance to chemotherapy in SCLC is the PI(3)K-Akt pathway11,12. The principal mediator of this pathway is Akt, a PI(3)Kactivated protein kinase25. Akt has direct effects on the apoptosis machinery; for example, it phosphorylates the proapoptotic Bcl-2– related protein BAD on Ser136 (ref. 25). The function and stability of Akt is regulated by Hsp9026, and because inhibition of Hsp90 results in the ubiquitination and proteasome degradation of Akt, we investigated whether Akt and its downstream client BAD are affected by the Hsp90 inhibitors and whether they have a role in triggering massive apoptosis in SCLC cells. Treatment of SCLC cells with concentrations of PU24FCl that inhibit Hsp90 (Fig. 1c) and induce apoptosis (Fig. 2) led to Akt degradation and to a reduction in BAD phosphorylation (Fig. 2b). The kinetics of Akt inactivation by the Hsp90 inhibitor further correlate with induction of apoptosis (Fig. 4b and Supplementary Fig. 3), which suggests that BAD dephosphorylation leads to subsequent cytochrome c release from the mitochondria and initiates apoptosis. To test this hypothesis, we treated SCLC cells with a quinoxaline-scaffold small-molecule Akt inhibitor (9) (Fig. 4c). Inactivation of Akt by this agent, as suggested by reduction of Akt phosphorylation on Ser473 and BAD dephosphorylation on Ser136, resulted in disruption of the Hsp90–Apaf-1 complex (Fig. 4d) and



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Figure 4 Mechanism of apoptotic regulation by Hsp90 in SCLC. (a,b) NCI-H526 cells were treated for the indicated times with PU24FCl (20 mM), and protein complexes were isolated with antibodies specific for Apaf-1 (top panel) or caspase-9 (lower panel) or with a control IgG (a). Indicated proteins were identified by western blot analysis. Whole lysate protein content was analyzed by western blot (b). (c) Western blot analysis of NCI-H526 (top panel) and SKBr3 (lower panel) cells treated for the indicated times with the Akt inhibitor (9) (50 mM). b-actin was used as protein quantification control. (d) NCI-H526 cells were treated for 24 h with either PU24FCl (20 mM), vehicle, Akt inhibitor (50 mM) or MAL3-101 (50 mM). Proteins isolated with an Apaf-1–specific antibody (top panel) and in the whole lysate (lower panel) were analyzed by western blot. (e) SKBr3 cells were treated for 24 h with vehicle, PU24FCl (20 mM), KNK437 (100 mM), MAL3-101 (50 mM) or Akt inhibitor (50 mM), and cellular fractions were prepared as described in Methods. The presence of cytochrome c in these fractions was determined by western blot analysis. The mitochondrial-specific protein Hsp60 was used as a control. The data are consistent with those obtained from multiple repeat experiments (n Z 3).

activation of the intrinsic apoptotic pathway, as demonstrated by cleavage of caspase-3, caspase-9 and PARP (Fig. 4c,d) in a fashion similar to that observed during Hsp90 inhibition (Fig. 4d). These results suggest that in SCLC cells, cytochrome c released from the mitochondria upon BAD dephosphorylation may compete with Hsp90 for Apaf-1 binding, thereby leading to the formation of an active apoptosome. Collectively, these findings imply that pharmacological inhibition of Hsp90 function in SCLC cells frees Apaf-1 to form an Apaf-1–caspase-9 complex, but this becomes activated to effect substantial apoptosis only upon cytochrome c release triggered by Akt inactivation and consequent BAD dephosphorylation. These data portray a dual role of Hsp90 in regulating apoptosis in SCLC by acting as a negative regulator of Apaf-1 and by controlling the PI(3) K-Akt survival pathway. Role of Hsp90 is cell- and transformation-specific Hsp90 interacts with several client substrates (such as kinases, hormone receptors and transcription factors) that are directly involved in driving multistep malignancy, and also with mutated oncogenic proteins required for the transformed phenotype1–4. Maintenance of these oncogenic proteins and thus of the transformed phenotype by Hsp90 occurs in a cell- and transformation-specific manner. To investigate whether this specificity extends to maintaining the antiapoptotic phenotype in SCLC cells, we analyzed the effects of the



Hsp90 inhibitors against the breast carcinoma cell lines MCF-7 and SKBr3, and also against the normal diploid pulmonary fibroblast cell line MRC-5 (Figs. 4 and 5; Supplementary Fig. 4 online). Transformation in these two breast cancer cells is driven by distinct aberrant events. In SKBr3 cells, overexpression of the receptor tyrosine kinase Her2 leads to Akt activation, which in turn promotes cell survival27. MCF-7 cells seem to be driven by an activation of the Raf-1 kinase and by defects in the intrinsic caspase pathway28,29. However, we found that Hsp90 inactivation arrests the growth of MCF-7 and SKBr3 cells (Fig. 5a) and leads to Akt degradation (Supplementary Fig. 4 and ref. 16) and the release of Apaf-1 from the Hsp90 complex (Fig. 5b) at doses similar to those that were effective in SCLC cells (Fig. 4a). In contrast, there was little or no detectable apoptosis at 24 h, as measured by caspase-3, caspase-9 and PARP cleavage (Fig. 5c and Supplementary Fig. 4). Similar effects of PU24FCl and other Hsp90 inhibitors are documented in several other cancer cells, in which differentiation rather than spontaneous apoptosis is observed16,30. The Akt inhibitor also failed to induce apoptosis in the SKBr3 breast cancer cells at doses at which it was active in the NCI-H526 cells (Fig. 4c). Apoptosis induced by other interventions in these breast cancer cells is documented, which suggests that whereas the apoptotic pathway is functional, it is likely not regulated by Hsp90; nor is it mainly dependent on an Akt triggered-apoptosome–mediated mechanism as in the case of SCLC cells.

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Another chaperone that has critical roles in cell survival and whose inhibition can trigger cancer cell apoptosis is Hsp7031,32. Indeed, treatment of SKBr3 cells with MAL3-101 (10), an agent that interferes with Hsp70 activation by J-proteins33, or with quercetin (11) and KNK437 (12), both Hsp70 expression inhibitors34,35, resulted in substantial cell death (Fig. 5a and Supplementary Fig. 4). Similar massive death of human breast cancer cells36 as well as glioblastoma and colon cells37 was demonstrated following inhibition of Hsp70 synthesis by adenoviral transfer or classical transfection of antisense Hsp70 complementary DNA. Cell death induced by Hsp70 inhibition in SKBr3 cells was apoptotic, as indicated by caspase-3 and PARP cleavage and substantial caspase-3 activation (Fig. 5c–d). However, Apaf-1 was still regulated by Hsp90 and not Hsp70 (Fig. 5b), which suggests that apoptosis in these cells may not be apoptosomemediated. Indeed, in SKBr3 cells these agents induced caspase-3 cleavage while failing to release cytochrome c from the mitochondria (Fig. 4e) and induce caspase-9 cleavage (Fig. 5c), which suggests that in these cells apoptosis effected by caspase-3 may be initiated and channeled through other mechanisms, one of which may be the death receptor pathway. In contrast to the data described above, the three Hsp70 inhibitors failed both to activate an apoptotic response in the NCI-H526 cells (Fig. 5c) and to modulate the Apaf-1–Hsp90 complex (Fig. 4d). The insensitivity of these cells to Hsp70 inhibition is notable considering the important roles attributed to Hsp70 in inhibiting key effectors of the apoptotic machinery31,32. Specifically, Hsp70 has been suggested to block the release of cytochrome c and prevent Apaf-1 oligomerization and procaspase-9 recruitment. Considering its suggested substantial antiapoptotic capabilities and the specific apoptotic wiring of SCLC cells, induction of Hsp70 could potentially inhibit apoptosis and overcome the release by Hsp90 inhibitors of apoptotic elements from under Hsp90 control. In fact, upregulation of Hsp70 by Hsp90 inhibitors has been suggested to lessen the apoptotic effects of these inhibitors1–4,38. It is therefore noteworthy that in spite of substantial Hsp70 upregulation (Figs. 2b and 5c,e), this chaperone cannot significantly compensate for Hsp90 inhibition in SCLC. We observed that PU24FCl addition to NCI-H526 cells, which led to a

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Figure 5 Inhibition of apoptosis by Hsp90 is celland transformation-specific. (a) Cell growth over 72 h in the presence of indicated doses of inhibitors was assessed in SKBr3 and MCF-7 cells as indicated in Figure 1d,f. (b) SKBr3 cells were treated for the indicated times with PU24FCl (20 mM), or for 24 h with either vehicle, MAL3101 (50 mM) or KNK437 (100 mM), and protein complexes were isolated with an Apaf-1–specific antibody or a control IgG. Indicated proteins were identified by western blot analysis. (c) Western blot analyses of SKBr3 (left panel) and NCI-H526 (right panel) cells treated for 24 h with either PU24FCl (20 mM), MAL3-101 (50 mM), KNK437 (400 mM) or quercetin (200 mM). (d) SKBr3 cells were treated for 24 h with the indicated doses of MAL3-101. Effects on caspase-3 activation and cleavage were analyzed as indicated in Methods. (e) Western blot analyses of NCI-H526 cells treated for 24 h with vehicle only (1), PU24FCl (20 mM) (2) or MAL3-101 (25 mM (3) or 75 mM (5)). Alternatively, cells were pretreated for 4 h with MAL3-101 (25 mM (4) or 75 mM (6)) before addition of PU24FCl (20 mM). The data are consistent with those obtained from multiple repeat experiments (n Z 3). Points, mean; bars, s.d.

three- to ten-fold increase in Hsp70 expression, did not reduce its apoptotic effect at 24 h (Figs. 2b and 5c). Further, inhibition of Hsp70 activity by MAL3-101 did not modulate the apoptotic cascade unleashed by PU24FCl in these cells (Fig. 5e). Collectively, these data suggest that, at least in the above-presented transformed cells, Hsp70 does not regulate the intrinsic apoptotic pathway, and it may not interfere with cytochrome c release or inhibit the recruitment of Apaf-1 to caspase-9. As evidenced in the SKBr3 cells, Hsp70 regulates apoptotic pathways that, although caspase-3-effected, are not exclusively apoptosome-channeled. Normal cells are reported to be less susceptible to Hsp90 inhibition1–4. We observed that MRC-5 cells were insensitive to Hsp90 inactivation even at 72 h in the presence of PU24FCl (Supplementary Fig. 4). Doses of agent that induced massive death in SCLC cell lines had no significant effect on the growth of these cells, nor did the agent activate the apoptotic pathway. Hsp90 in chemotherapy-resistant SCLC SCLC tumors are initially sensitive to treatment with anticancer therapies, but the small population that remains resistant is characterized by fast dissemination and a more aggressive clinical evolution5,39,40. The development of drug resistance is the main limiting factor influencing the survival of individuals with SCLC. To assess whether Hsp90 remains an important antiapoptotic molecule in such cells, we made use of several multidrug-resistant cell lines, including H69AR, SKI-AC3 and WBA, in which the mechanisms of drug resistance are distinct (Fig. 6 and Supplementary Fig. 5 online). The H69AR line was established from NCI-H69 cells that were grown in the presence of increasing concentrations of adriamycin (doxorubicin) (13) over a total of 14 months41. Its resistance to chemotherapy seems to result from multiple mechanisms, including an increased concentration of the multidrug-resistance protein. The cell line is cross-resistant to anthracycline analogs, including daunomycin (14), epirubicin (15), menogaril (16) and mitoxantrone (17); it is also cross-resistant to acivicin (18), etoposide (19), colchicines (20) and the Vinca alkaloids vincristine (21) and vinblastine (22). SKI-AC3 is a



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marrow involvement, liver and adrenal metastasis, and extensive mediastinal and cervical adenopathy42. Resistance to chemotherapy in this cell is partly due to a constitutively active PI(3)K11. Regardless of these distinctions, however, PU24FCl and its higher potency derivatives, PU-H58 and PU-H71, inhibited Hsp90 in these cells (Fig. 6a) and caused substantial death at doses that were effective in all other tested SCLC lines (Fig. 6c and Supplementary Fig. 5). Cell

primary SCLC cell line harvested from the malignant pleural effusion of a person who had recurrent disease after several chemotherapy options (cisplatin (23), etoposide, topotecan (24), gemcitabine (25) and whole-brain radiation therapy) and had extensive metastasis to lung, liver, lymph nodes, subcutaneous tissue, left adrenal gland and brain. The mechanism of resistance in this cell line is not yet known. WBA cells were derived from a person with extensive SCLC with bone

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Figure 7 Pharmacologic inactivation of Hsp90 in in vivo SCLC models results in tumor cell death through apoptosis. (a) Immunohistochemical analysis of representative NCI-N417 tumors (n ¼ 2) treated with five consecutive daily doses of PU24FCl (100 mg kg–1 administered i.p.) (lower panels) or vehicle (top panels) and killed at 12 h following the last administration. Scale bars, 150 mm. (b) Western blot analysis of representative NCI-H526 tumors treated i.p. with five consecutive indicated daily doses of PU-H71. Mice (n ¼ 2) were killed at 12 h following the last drug administration. (c,d) Western blot (c) and HPLC/MS/MS (d) analyses of NCI-N417 tumors from mice administered i.p. one dose of 75 mg kg–1 PU-H71. Animals (n ¼ 3) were killed at the indicated times and tumors and brains harvested and halved for analyses. PI(3)K levels were used as a loading control as its levels do not change with Hsp90 inhibition. C1 and C2 are representative untreated mice. (e) Inhibition of Hsp90 leads to tumor growth inhibition. Nude athymic mice (n ¼ 4) bearing NCI-N417 tumors (V ¼ 50–90 mm3) were administered i.p. either PU-H71 or 17AAG and tumor growth was monitored as indicated in Methods. Points, mean; bars, s.d. ***P o 0.001.



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ARTICLES death induced by the Hsp90 inhibitors was apoptotic, as demonstrated by both activation and cleavage of caspase-3 and by cleavage of PARP (Fig. 6b and Supplementary Fig. 5). These events were paralleled by Akt inactivation (Fig. 6b and Supplementary Fig. 5). We obtained comparable results with 17AAG in the WBA and SKI-AC3 cells (Fig. 6b). This drug, however, performed less effectively in the multidrug-resistance protein–expressing H69AR cells (Fig. 6b and Supplementary Fig. 5), which suggests that, as observed in other systems, resistance to 17AAG is mediated by drug efflux or metabolism and not by a change in Hsp90 biology1–3,13,14,19. Collectively, these observations suggest that irrespective of the mechanism of chemotherapy resistance, Hsp90 retains its role as a major inhibitor of apoptosis in SCLC. Inhibition of Hsp90 in SCLC tumors results in apoptosis To exclude the possibility that these effects may be a peculiarity of propagating cells in culture, we translated our findings to SCLC animal models (Fig. 7). Xenograft tumors were established by injecting cells with reconstituted basement membrane (Matrigel) in nude athymic (nu/nu) mice. Mice with tumors reaching 7–9 mm in diameter were treated intraperitoneally (i.p.) with five consecutive daily doses of PU24FCl (100 mg kg–1) (Fig. 7a) or with the indicated doses of PU-H71 (Fig. 7b), and they were killed 12 h after the last administration. NCI-N417 tumors treated with PU24FCl were prepared for immunohistochemistry and induction of apoptosis (caspase-3 cleavage), and changes in the proliferative index (Ki-67) and Akt phosphorylation were studied in these tissues. We observed that PU24FCl induced apoptosis in this tumor and reduced Akt activity (Fig. 7a). The presence of nonproliferating and necrotic pockets in the PU24FCl-treated tumors was also observed. The effects of Hsp90 inhibition were dose dependent, and for PU-H71 the effect was maximal in the NCI-H526 tumors at 75 mg kg–1 (Fig. 7b). In the NCI-N417 tumors, Akt inactivation and cleavage of PARP was detected as early as 6 h after PU-H71 administration, and the effects were still evident at 36 h, irrespective of significant Hsp70 upregulation (Fig. 7c). These effects occurred at pharmacologically relevant doses of PU-H71 that were retained in tumors (38.5 mM at 3 h, 19.8 mM at 6 h, 12.2 mM at 8 h, 12.7 mM at 12 h, 3.6 mM at 24 h and 5.2 mM at 36 h) (Fig. 7d). At the termination of treatment, we observed significant tumor growth inhibition with administration of nontoxic doses of PU-H71 (P o 0.0001) or 17AAG (P ¼ 0.0003) to mice bearing NCI-N417 tumors (Fig. 7e). To our knowledge these data portray the first documented induction of apoptosis by an Hsp90 inhibitor in vivo, thereby highlighting the unique dependency of apoptotic elements on Hsp90 regulation in SCLC. DISCUSSION In spite of the seemingly vital involvement of Hsp90 in maintaining the transformed phenotype, inhibition of its function results in little apoptosis and is mostly cytostatic in the majority of cancer cell lines30. Our data define SCLC as the first documented biological system in which Hsp90 is a major inhibitor of apoptosis, and they indicate that its pharmacologic inactivation is a particularly effective means to induce apoptosis in this tumor. Our observations suggest that Hsp90 restrains apoptosis in SCLC via the intrinsic apoptotic pathway and the survival pathway regulated by Akt. Apoptosis following Hsp90 inhibition in SCLC is likely triggered by inactivation of Akt. This leads to consequent reduction in BAD phosphorylation, releasing the protein from 14-3-3 so that it is free to heterodimerize with antiapoptotic members of the Bcl-2 family of proteins and/or to activate the proapoptotic proteins Bax and Bak in the mitochondrial

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membrane. Abrogation of Bcl-2 antiapoptotic function leads to mitochondrial depolarization and cytochrome c release from the mitochondria. Concomitantly, inhibition of Hsp90 also releases Apaf-1 from the Hsp90 complex, thereby freeing it to interact with caspase-9 and subsequently induce the apoptotic cascade by activation of procaspase-3 (ref. 43). Whereas most Hsp90 client proteins are trapped in the chaperone complex after Hsp90 inhibition, which leads to the client protein’s ubiquitination and subsequent degradation by the proteasome1–4, Apaf-1 is released from the Hsp90 complex. This suggests that in contrast to oncoprotein regulation, in which Hsp90 binding has a positive regulatory role1–4, for Apaf-1, the chaperone plays a negative regulatory role. Although regulation of both Akt and Apaf-1 by Hsp90 has been previously reported26,43, to our knowledge our findings are the first to implicate this regulation in a cellular setting in which it has a major antiapoptotic consequence. The role played by Hsp90 as a key inhibitor of apoptosis in SCLC does not extend to all biological systems but is limited to systems in which apoptotic pathways otherwise under tight Hsp70 control are nonfunctional. Hsp90 assumes this role in systems in which apoptosis is uniquely dependent on and solely effected through the intrinsic pathway, without involvement of caspase apoptotic elements upstream of mitochondria or alternative pathways that are not apoptosomechanneled. These findings delineate the specificity of the Hsp90 chaperone in regulating the antiapoptotic phenotype in a transformation-specific manner that is dependent on the cell’s wiring and functionality of apoptotic pathways. Although both Hsp90 and Hsp70 have roles in suppressing apoptotic elements, the magnitude of the apoptotic outcome resulting from their inhibition is dictated by transformation itself. To gain a translational significance, it is therefore important to study and validate the roles of chaperones not in artificial in vitro systems, but in the endogenous system they regulate. In SCLC, the specific cellular wiring and functionality of apoptotic pathways may be a consequence of a defective Rb (ref. 10)—a lesion that has been identified in most of these tumors9. Retinoblastoma and large-cell neuroendocrine carcinoma are human cancers of neural/neuroendocrin origin that share this characteristic8,9, and in light of our data, it will be important to examine the effects of Hsp90 inhibitors on these cancers. Our data demonstrate that contrary to previous findings44, at least in the analyzed malignant systems, Hsp70 does not regulate the intrinsic apoptotic pathway, and it may not interfere with cytochrome c release or inhibit the recruitment of Apaf-1 to caspase-9, which suggests that Hsp70 regulates apoptosis in systems in which cell death is not exclusively apoptosome-mediated. In support of our observations are previous studies in which Hsp70 depletion in a panel of breast cancer cells led to apoptosis that was not mitochondria-mediated36. The significant induction of apoptosis in both cell line and xenograft models of SCLC after Hsp90 inactivation suggests the chaperone as a valid target in this disease. Further, the retained massive apoptotic effects in multidrug-resistant SCLC cell lines upon treatment with the Hsp90 inhibitors suggest both that Hsp90 remains a major regulator of apoptosis in these cells and that Hsp90 inhibitors have the potential to overcome resistance mechanisms to standard chemotherapeutic agents. We therefore propose that Hsp90 inhibitors, alone or in combination with standard chemotherapy, are a promising strategy for the treatment of SCLC, especially in chemoresistant recurrent disease for which viable therapeutic options are currently unavailable. With 17AAG and 17DMAG now in phase 1 and 2 trials, the first purine-scaffold Hsp90 inhibitor (CNF2024) now in phase 1 trials, and other chemotypes in late-stage preclinical



ARTICLES development19, this work further establishes a rationale for the translation of Hsp90 inhibitors to the treatment of people with SCLC, including those with recurrent disease.


METHODS Reagents. We synthesized and characterized PU24FCl, PU-H58 and PU-H71 as previously described16,18. MAL3-101 is a specific inhibitor of J-chaperone– stimulated Hsp70 ATPase activity, and its synthesis is described elsewhere33. The two ansamycin Hsp90 inhibitors, 17AAG and 17DMAG, were synthesized as reported45. We purchased quercetin from Sigma; we purchased KNK437, radicicol, the pan-caspase inhibitor Boc-D-FMK and the quinoxaline scaffold Akt inhibitor VIII, isozyme-selective Akti-1/2, from Calbiochem. We purchased propidium iodide from BD Biosciences, the caspase-9 inhibitor Z-LEHD-FMK from R&D Systems, and JC-1 (5,5¢,6,6¢-tetrachloro-1,1¢,3,3¢tetraethylbenzimidazolylcarbocyanine chloride) and the mitochondrial uncoupler CCCP (carbonyl cyanide m-chlorophenyl hydrazone) from Molecular Probes. Cell lines. We obtained the human SCLC cells NCI-H69, NCI-H146, NCIH187, NCI-H209, NCI-N417, NCI-H510, NCI-H526 and H69AR, the breast cancer cells MCF-7 and SKBr3, and the normal lung fibroblast cells MRC-5 from the American Type Culture Collection. After informed consent was obtained using a Memorial Sloan-Kettering Cancer Center Institutional Review Board (IRB)–approved tissue collection protocol (MSKCC IRB #92-055), pleural fluid was collected via chest tube placement from a person with SCLC widely metastatic to the lungs, liver, bone and subcutaneous tissue. After centrifugation and lysis of the red blood cells, the tumor cells were cultured in RPMI with 10% FBS (FBS) and antibiotics. We confirmed the cytology of the SCLC tumor cells by pathology. WBA cells were reported previously42. We cultured the SCLC cells in RPMI supplemented with 10 mM HEPES, 1 mM sodium pyruvate, 1.5 g l–1 sodium bicarbonate, 4.5 g l–1 glucose, 10% FBS, 1% L-glutamine, and 1% penicillin and streptomycin. We cultured the MRC-5 cells in minimum essential medium adjusted to contain 2 mM L-glutamine, 1.5 g l–1 sodium bicarbonate, 0.1 mM nonessential amino acids, 1 mM sodium pyruvate, 10% FBS, and 1% penicillin and streptomycin. Hsp90 binding assay. We performed measurements in black 96-well microtiter plates (Corning #3650) as previously described17,18. In short, cell lysates were prepared by rupturing cellular membranes via freezing at –70 1C and dissolving the cellular extract in HFB (20 mM HEPES (K) pH 7.3, 50 mM KCl, 5 mM MgCl2, 20 mM Na2MoO4, 0.01% Nonidet P-40) with added protease and phosphatase inhibitors. Saturation curves were recorded in which fluorescently labeled geldanamycin (Cy3B-GM)46 (3 nM) was treated with increasing amounts of cellular lysates. The amount of lysate that resulted in polarization (mP) readings corresponding to 90–99% bound ligand was chosen for the competition study. Here, each 96-well plate contained 3 nM Cy3B-GM, cellular lysate (amounts as determined above and normalized to total Hsp90 as determined by western blot analysis using Hsp90 purified from HeLa cells as standard (Stressgen #SPP-770)) and tested inhibitor (initial stock in DMSO) in a final volume of 100 ml. The plate was left on a shaker at 4 1C for 24 h and the fluorescence polarization (FP) values in mP were recorded. EC50 values were determined as the competitor concentrations at which 50% of the Cy3B-GM was displaced. FP measurements were performed on an Analyst GT instrument (Molecular Devices). Growth inhibition assay. We evaluated the antiproliferative effects of inhibitors using the CellTiter-Glo Luminescent Cell Viability Assay kit (Promega Corporation #G7571). Briefly, exponentially growing cancer cells were seeded into 96-well plates (Corning #3603) and incubated in medium containing either vehicle control (DMSO) or compounds for the indicated time at 37 1C. Plates containing four replicate wells per assay condition were seeded at a density of 15,000 cells for NCI-N417, NCI-H526, SKI-AC3 and WBA; 20,000 cells for NCI-H69, H69AR, NCI-H146, NCI-H187, NCI-H510 and NCI-H209; 1,000 cells for MCF-7 and 3,000 cells for SKBr3 in 100 ml of medium. After exposure of cells to inhibitors, plates were equilibrated to room temperature (20–25 1C) for approximately 30 min, and 100 ml of CellTiter-Glo reagent were added to each well. Plates were mixed for 2 min on an orbital shaker and then incubated



for 20 min to 5 h at room temperature (optimal incubation time was determined for each cell line). The luminescence of each well was measured in an Analyst GT microplate reader (Molecular Devices) with an integration time of 0.5 s well–1. The percentage cell growth inhibition was calculated by comparison of the luminescence reading obtained from treated versus control cells, accounting for initial cell population. For the normal lung fibroblast cells line MRC-5, effects on growth were measured using the sulforhodamine B assay. In summary, experimental cultures were plated in microtiter plates (Nunc). One column of wells lacked cells and served as the blank. Otherwise, cells were allowed to attach overnight. The following day, growth medium having either drug or DMSO at twice the desired initial concentration was added to the plate in triplicate and was serially diluted at a 1:1 ratio in the microtiter plate. After 96 h of growth, the cell number in treated versus control wells was estimated after treatment with 10% trichloroacetic acid and staining with 0.4% sulforhodamine B in 1% acetic acid. The IC50 was calculated as the drug concentration that inhibits cell growth by 50% compared with the control. Because DMSO was toxic toward MRC-5 cells at the tested concentrations, drug stocks were made in ethanol. Protein assays. Cells were grown to 60–70% confluence and exposed to drugs or DMSO vehicle for the indicated time periods. Lysates were prepared using 50 mM Tris pH 7.4, 150 mM NaCl and 1% NP-40 lysis buffer. Protein concentrations were determined using the BCA kit (Pierce) according to the manufacturer’s instructions. Protein lysates (20–100 mg) were electrophoretically resolved by SDS-PAGE, transferred to nitrocellulose membrane, and probed with the indicated primary antibodies. Membranes were then incubated with a 1:5,000 dilution of a peroxidase-conjugated corresponding secondary antibody. Equal loading of the protein samples was confirmed by parallel western blots for b-actin or PI(3)K (p85 subunit). Enhanced chemiluminescence (Amersham) was performed according to the manufacturer’s instructions. Blots were visualized by autoradiography. Note: Supplementary information and chemical compound information is available on the Nature Chemical Biology website. ACKNOWLEDGMENTS This work was supported by SynCure Cancer Research Foundation (G.C.), Susan G. Komen Breast Cancer Foundation (G.C. and Y.K.), AACR-Cancer Research and Prevention Foundation (G.C.), W.H. Goodwin and A. Goodwin and the Commonwealth Cancer Foundation for Research, The Experimental Therapeutics Center of Memorial Sloan-Kettering Cancer Center (MSKCC) (G.C.), Steps for breath (G.C.), the Translational and Integrative Medicine Research Fund of MSKCC (G.C.), Partnership for Cure and the Goldman Philanthropic Partnerships (J.L.B.), the Tri-institutional Program in Chemical Biology (K.M), and the University of Pittsburgh Combinatorial Chemistry Center (P.W., P50-GM067082). We thank D.M. Turner and S. Werner (University of Pittsburgh) for the preparation of MAL3-101, D. Zatorska and H. He (MSKCC) for the preparation of PU24FCl, PU-H58, PU-H71, 17AAG and 17DMAG, S. Danishefsky (MSKCC, New York) for providing a sample of cycloproparadicicol, the MSKCC Thoracic Service for providing the clinical sample used to establish the SKI-AC3 cell line, and Y. Lazebnik (Cold Spring Harbor Laboratory, New York) and D. Toft (Mayo Clinic) for the generous gifts of antibodies. We thank Y. Lazebnik and N. Rosen for useful discussions in the preparation of this manuscript. AUTHOR CONTRIBUTIONS A.R., M.V. and C.C.C. designed, performed and analyzed experiments and helped write the paper; K.M., J.A., J.K., A.C., J.L. and Y.K. performed and analyzed experiments; Y.S., S.F. and N.W. designed and analyzed experiments; G.C., X.J., J.M., P.W., G.W.K. and J.L.B. designed and analyzed experiments and helped write the paper. COMPETING INTERESTS STATEMENT The authors declare competing financial interests: details accompany the full-text HTML version of the paper at http://www.nature.com/naturechemicalbiology/. Published online at http://www.nature.com/naturechemicalbiology Reprints and permissions information is available online at http://npg.nature.com/ reprintsandpermissions 1. Workman, P. & Maloney, A. HSP90 as a new therapeutic target for cancer therapy: the story unfolds. Expert Opin. Biol. Ther. 2, 3–24 (2002).

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