Functional Inactivation of Endogenous MDM2 and CHIP by HSP90 Causes Aberrant Stabilization of Mutant P53 in Human Cancer Cells

Published OnlineFirst April 8, 2011; DOI: 10.1158/1541-7786.MCR-10-0534

Molecular Cancer Cancer Genes and Genomics Research

Functional Inactivation of Endogenous MDM2 and CHIP by HSP90 Causes Aberrant Stabilization of Mutant p53 in Human Cancer Cells

Dun Li1, Natalia D. Marchenko1, Ramona Schulz3, Victoria Fischer2, Talia Velasco-Hernandez1, Flaminia Talos1, and Ute M. Moll1,3

Abstract The tight control of wild-type p53 by mainly MDM2 in normal cells is permanently lost in tumors harboring mutant p53, which exhibit dramatic constitutive p53 hyperstabilization that far exceeds that of wild-type p53 tumors. Importantly, mutant p53 hyperstabilization is critical for oncogenic gain of function of mutant p53 in vivo. Current insight into the mechanism of this dysregulation is fragmentary and largely derived from ectopically constructed cell systems. Importantly, mutant p53 knock-in mice established that normal mutant p53 tissues have sufficient enzymatic reserves in MDM2 and other E3 ligases to maintain full control of mutant p53. We find that in human cancer cells, endogenous mutant p53, despite its ability to interact with MDM2, suffers from a profound lack of ubiquitination as the root of its degradation defect. In contrast to wild-type p53, the many mutant p53 proteins which are conformationally aberrant are engaged in complexes with the HSP90 chaperone machinery to prevent its aggregation. In contrast to wild-type p53 cancer cells, we show that in mutant p53 cancer cells, this HSP90 interaction blocks the endogenous MDM2 and CHIP (carboxy-terminus of Hsp70-interacting protein) E3 ligase activity. Interference with HSP90 either by RNA interference against HSF1, the transcriptional regulator of the HSP90 pathway, or by direct knockdown of Hsp90 protein or by pharmacologic inhibition of Hsp90 activity with 17AAG (17-allylamino-17-demethoxygeldanamycin) destroys the complex, liberates mutant p53, and reactivates endogenous MDM2 and CHIP to degrade mutant p53. Of note, 17AAG induces a stronger viability loss in mutant p53 than in wild-type p53 cancer cells. Our data support the rationale that suppression of mutant p53 levels in vivo in established cancers might achieve clinically significant effects. Mol Cancer Res; 9(5); 577–88. 2011 AACR.

Introduction transactivation of MDM2, itself a wild-type p53 target gene that forms an autoregulatory loop (1). Surprisingly, however, Missense mutations in the p53 gene occur in more than recently generated knock-in (KI) mice expressing mutant 50% of human cancers. In normal unstressed cells, the level p53 R172H in all tissues clearly establish that mutant p53 is of wild-type p53 protein is very low due to rapid turnover by inherently unstable in normal cells. Despite impairment of its main physiologic E3 ligase MDM2, which is interrupted mutant p53 to transcriptionally induce MDM2, only only when needed in response to stress. This tight control is tumors but not normal tissues of these mice display con- permanently lost in tumors harboring mutant p53, which stitutive stabilization of mutant p53 (2–4). Expression of exhibits a dramatic constitutive p53 stabilization compared murine and human MDM2 is controlled by 2 different with wild-type p53 tumors. Mutant p53 hyperstability was promoters: the constitutive p53-independent P1 promoter always assumed to be entirely due to the loss of p53-mediated and the p53-responsive P2 promoter (5). Thus, in these KI mice, the constitutive p53-independent transcription of MDM2 from the P1 promoter alone is apparently sufficient Authors' Affiliations: Departments of 1Pathology and 2Pharmacology, to degrade mutant p53 in normal tissues. This finding Stony Brook University, Stony Brook, New York; and 3Department of eliminates the notion that transcriptional inability of mutant Molecular Oncology, Gottingen€ Center of Molecular Biosciences, Univer- sity of Gottingen,€ Gottingen,€ Germany p53 to induce sufficient levels of MDM2 is the sole or even primary cause for mutant p53 hyperstability. Rather, on Note: Supplementary data for this article are available at Molecular Cancer Research Online (http://mcr.aacrjournals.org/). malignant conversion, some undefined additional alteration Corresponding Author: Natalia Marchenko, Department of Pathology, (s) must occur that stabilize mutant p53. Stony Brook University, Stony Brook, NY 11794-8691. Phone: 631-444- Compared with p53 null mice, mutant p53 KI mice show 3520; Fax: 631-444-3424. E-mail: [email protected] an oncogenic gain of function (GOF) phenotype (2, 3). In doi: 10.1158/1541-7786.MCR-10-0534 agreement, depletion of mutant p53 by siRNA in human 2011 American Association for Cancer Research. tumor cells leads to suppressed tumor growth in culture and

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in xenografts and to enhanced chemosensitivity (6, 7). tional), a specific inhibitor of deubiquitinases, was included Importantly, mutant p53 hyperstabilization is critical for in all buffers. Treatment with 5 mmol/L 17AAG (17-allyla- manifestation of its GOF in vivo. In support, constitutive mino-17-demethoxygeldanamycin; LC Laboratories) was MDM2 deficiency in p53 R172H/R172H mice (in short for 24 hours, 5 mmol/L camptothecin for 3 hours, and nutlin p53H/H mice) causes earlier tumor onset, increased tumor (20 mmol/L; Sigma) for 24 hours. Cell viability was deter- incidence and metastasis, and shortened survival compared mined by CellTiter-Blue Assay (Promega) ina96-well format with MDM2-proficient p53H/H mice, implying that GOF (10,000 cells/well, seeded 24 hours prior). Proliferation was depends on mutant p53 levels (4). Thus, tumor-specific measured by cell counts. Nude mice were injected subcuta- stabilization of mutant p53 is a critical determinant of its neously with MDA 231- or SW480 shp53 cells or vector GOF. controls (106 cells per injection site, 6 sites per mouse). Little conclusive insight currently exists about the precise Tumors were harvested after 12 or 20 days. mechanisms responsible for dysregulating mutant p53 protein levels in cancer cells. In fact, this important question Plasmids constitutes a major unexplored area in the p53 field, with the CMV-MDM2 plasmid carrying a neomycin resistance exciting prospect that advances have high translational gene (11) was transfected with Lipofectamine (Invitrogen). potential that might be exploited for a mutant p53–directed Stably transfected clones were selected in 700 mg/mL G418 cancer strategy. The existing studies provide only fragmen- (Gibco). The p53 R280K plasmid was subcloned into a tary insights mostly derived from ectopically overexpressed retroviral REBNA puro vector. Phoenix A cells were trans- mutant p53, analyzed in constructed nonphysiologic cell fected with mut p53R280K REBNA or empty vector. After systems of wild-type p53 or null p53 background. In contrast 48 hours, supernatants containing the retroviral particles to wild-type p53, many mutant p53 protein species are were collected and used to infect T47D cells overnight, conformationally aberrant. To prevent aggregation, this followed by puromycin (1 mg/mL) selection 48 hours later. dictates their engagement in stable complexes with Hsp90 and Hsp70, members of the HSP90 chaperone machinery RNA interference that is upregulated and activated in cancer (8–10). By Pools of 4 different siRNA duplexes specific for human analyzing the endogenous status, we show here that in MDM2 (Ambion) and CHIP (Dharmacon) were trans- human cancer cells harboring mutant p53, this HSP90 fected with Lipofectamine 2000. Cells were harvested 48 interaction, while, on the one hand, stabilizes the mutant hours later and analyzed. For Hsp90 silencing, 5637 and p53 conformation and, on the other hand, blocks its degra- MDA 231 cells were transfected with 10 pmol of Silencer dation by inhibiting constitutive MDM2 and CHIP (car- Select siRNAs (Ambion) and analyzed 3 days later. boxy-terminus of Hsp70-interacting protein) E3 ligase activity. Interference with the HSP90 pathway, using either Immunoblots and immunoprecipitations RNAi (RNA interference) against the upstream regulator or For immunoblots, equal total protein of crude cell lysates against Hsp90, or with a pharmacologic Hsp90 inhibitor, (2.5–5 mg) was loaded. When loading was normalized for destroys the complex, liberates mutant p53, and reactivates equal amounts of nonubiquitinated p53, a first quantitation endogenous MDM2 and CHIP for mutant p53 degradation. immunoblot was run prior to the second definitive immunoblot. For nuclear/cytoplasmic fractionations the Pierce kit Materials and Methods was used. Antibodies were FL393 and DO1 for p53, SMP14 for MDM2 (Santa Cruz), HAUSP (Calbiochem), p14Arf Human cancer cells (Abcam), p53 Ser15 (Cell Signaling), MDMX (Bethyl Lab), Cancer cell lines MCF7 (breast), RKO and HCT 116 Hsp70, Hsp90, and histone deacetylase (HDAC; all Affinity (colon), and U2OS (osteosarcoma), as well as immortalized Bioreagents), PCNA (proliferating cell nuclear antigen), MCF10A (breast) and MRC5 (diploid fibroblasts), contain tubulin, actin, and rabbit IgG (immunoglobulin G; all functional wild-type p53. Conversely, breast cancer MDA Sigma). For detecting endogenous complexes, crude lysates 231 (R280K), MDA 468 (R273H), T47D (L194F), and SK- were immunoprecipitated with 1 mg of antibody for 2 hours. BR3 (R175H), prostate cancer DU145 (P223L, V274F), Beads were washed 3 times with SNNTE plus 2 radio- pancreatic cancer PANC1, bladder cancer 5637 (R280T), immunoprecipitation assay buffer (50 mmol/L Tris, 150 and ovarian cancer EB2 cell lines all harbor mutant p53. mmol/L NaCl, 1% Triton X-100, 0.1% SDS, 1% Na Stable mutant SW480 cells (p53 R273H/P309S) inducibly deoxycholate, pH 7.4) before immunoblotting. Immuno- express shp53 under the control of a tetracycline-regulated fluorescence was done as described (12). promoter on adding tetracycline in culture (1.0 mg/mL) or feeding it to nude mice (2 g/L in drinking water; ref. 7). Stable Results and Discussion MDA 231-Luc (control) and MDA 231-shp53 cells were a gift from Dr. S. Deb. Cells were cultured in 10% fetal calf Tumor-derived endogenous mutant p53 shows serum/Dulbecco's modiﬁed Eagle's medium. Where indi- complete lack of ubiquitination, causing its profound cated, cells were treated with 25 mmol/L ALLN (Calbio- degradation defect chem) for 3 hours. CHX (cycloheximide; Sigma) was added Although stabilization of mutant p53 was noted pre- to the medium (final 50 mg/mL). UbAL (BioMol Interna- viously, the ubiquitination status of endogenous mutant

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HSP90-Mediated MDM2 and CHIP Inactivation Causes Mutant p53 Stabilization in Cancer

AB wt p53 mutp53 Figure 1. Tumor-derived mutant wt p53 mutp53 p53 proteins show a complete lack of ubiquitination, causing T47D DU145 SK-BR3 MDA 435 MDA MCF7 MCF10 231 MDA their profound degradation defect. 468 MDA A, ubiquitination of endogenous MDA 231 MDA DU145 MCF7 p53 in a panel of wild-type and MCF10 468 MDA T47D mutant human cancer cell lines. ALLN + - + - + - + - + - + - p53 loading was normalized for p53-Ub p53-Ub comparable amounts of nonubiquitinated p53. DO1 p53 long exp immunoblot. B, proteasome p53 long exp inhibition (by ALLN) results in p53 short exp accumulation of ubiquitinated p53 only in wild-type p53 cells, p53 short exp whereas mutant p53 proteins remain completely nonubiquitinated. Normalized loading and blotting as in A. C, left, MCF7 MDA 231 ubiquitinated wild-type p53 is C wt p53 mut p53 mainly located in the cytoplasm. In wt p53 mut p53 contrast, both cytoplasm and ALLN - - + + - - + + nucleus are completely devoid of ubiquitinated mutant p53. Right, p53-Ub ubiquitinated wild-type p53 RKO HCT 116 resides mainly in the cytoplasm 231 MDA 468 MDA T47D and is stabilized by ALLN- p53 long exp mediated proteasome inhibition. p53-Ub In contrast, mutant p53 is HSP90 nonubiquitinated in both compartments and remains so p53 long exp HDAC even after ALLN treatment. N C N C N C N C Immunoblots normalized for p53 p53 short exp loading. HDAC and Hsp90 as nuclear and cytoplasmic markers. HSP90 Abbreviations: wt p53, wild-type HDAC p53; mut p53, mutant p53. N C N C N C N C N C left right

p53 remains controversial. Although early reports noted p53 only in wild-type cancer cells, whereas mutant p53 higher stability of mutant p53 (13), recent studies using remained nonubiquitinated (Figs. 1B and C, right). Puta- ectopic expression suggest that mutant p53 is more ubiqui- tive mutations in the RING domain of MDM2 were tinated than wild-type p53 in cancer cells (4, 14, 15). excluded in all 6 mutant p53 lines, eliminating mutational Moreover, until now, studies on the regulation of mutant inactivation of MDM2 as possible explanation. Ubiquiti- p53 stability were mostly limited to genetic analysis of KI nated wild-type p53 mainly localizes to the cytoplasm, mice (2–4) or ectopic overexpression of mutant p53 in whereas the nucleus preferentially harbors nonubiquitinated tumor cells (1, 13, 14). Thus, to characterize the degrada- p53 (refs. 12, 16; Fig. 1C, left). Mutant p53 accumulates tion of endogenous mutant p53, we probed a panel of mainly in the nucleus. To further exclude that the dramatic randomly chosen human cancer cell lines expressing either accumulation of nonubiquitinated mutant p53 in the wild-type p53 or mutant p53. Mutant p53 tumor cell lines nucleus might mask a putative ubiquitinated pool in the exhibit a dramatic constitutive p53 stabilization ranging cytoplasm, we conducted fractionations. Again, ubiquiti- from 10- to 20-fold above wild-type p53 cancer lines nated wild-type p53 was mainly located in the cytoplasm (Supplementary Fig. S1A). To compare their p53 ubiqui- where it was further stabilized by ALLN (Fig. 1C left; ref. tination status side by side, immunoblots from total cell 12). In contrast, cytoplasm and nucleus were completely lysates were normalized for comparable amounts of non- devoid of ubiquitinated mutant p53 and remained unre- ubiquitinated p53. Although wild-type p53 ubiquitination sponsive to ALLN (Fig. 1C, right). Taken together, this was readily detected, mutant p53 ubiquitination remained indicates that the severely impaired degradation of mutant undetectable in all lines, even after prolonged exposure p53 is due to grossly defective ubiquitination by MDM2 (Fig. 1A). Moreover, proteasome inhibition by ALLN and possibly other related E3 ligases, thereby causing its treatment led to marked accumulation of ubiquitinated aberrant stabilization.

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A B wt p53 mut p53

Figure 2. Neither HAUSP or p14Arf expression nor p53 modification can explain mutant MCF7 MDA 468 MDA 231 DU145 T47D p53 hyperstability. A, all mutant ALLN - + - + - + - + - + wt p53 mut p53 p53 cell lines have lower (about 2- fold) but readily detectable levels HAUSP of MDM2. B, top, HAUSP expression is similar in wild-type Tubulin and mutant p53 lines. Bottom, SW480 T47D HCT 116 U2OS MCF7 MDA 231 RKO DU145 wt p53 mut p53 p14Arf is undetectable in all wild- MDM2 type p53 cell lines and upregulated in some but not all Actin mutant p53 cell lines. C, constitutive DNA damage does MCF7 RKO MCF10 HCT 116 SK-BR3 DU145 T47D MDA 231 U2OS EB2 MDA 435 MDA 468 not contribute to mutant p53 p14Arf stabilization. Ser15 PCNA phosphorylation is undetectable in the majority of mutant p53 cell lines. D, mutant p53 cancer cells C D express lower amounts of wt p53 mut p53 wt p53 mut p53 acetylated p53 than wild-type p53 cells. Loading normalized for comparable amounts of nonubiquitinated p53 and blotted with p53 acetyl 373/382 antibody. U2OS HCT 116 T47D MCF7 DU145 EB2 MDA 231 MDA 468 RKO RKO MDA 231 MDA 468 DU145 SW480 U2OS HCT 116 MCF7 T47D A to D, immunoblots, actin, tubulin p53 p53 and PCNA as loading controls. Ser 15 p53 Abbreviations: wt p53, wild-type Ac-p53 p53; mut p53, mutant p53. PCNA

Neither HAUSP or p14Arf expression nor p53 the possibility that the MDM2 antagonist Arf could con- modification can explain mutant p53 hyperstability tribute to hyperstability of mutant p53 (4). We therefore To gain more insight into the mechanism of mutant p53 examined Arf levels in mutant and wild-type p53 cells. As stabilization in human cancer, we analyzed MDM2, expected, Arf was undetectable in all wild-type p53 cells HAUSP, and p14Arf, key molecules in the regulation of (Fig. 2B, bottom). Although Arf was upregulated in mutant p53 stability. MDM2 is a classic target gene of wild-type EB2, MDA 435, and DU145 cells, it remained undetect- p53. As expected, because of the lack of transcriptional able in T47D, MDA 231, and SK-BR3 cells. This lack of activity of mutant p53, levels of MDM2 mRNA and correlation between Arf expression and mutant p53 status protein were downregulated in mutant p53 cancer cell lines indicates that Arf upregulation is not a generic mechanism compared with wild-type p53 cancer lines but only by about for hyperstabilization of mutant p53, although it might 2- to 3-fold (Fig. 2A and Supplementary Fig. S1B). Impor- contribute in individual cancers when deregulated. tantly and parallel to normal tissues of mutant p53 knock-in Alternatively, it was suggested that constitutively acti- mice which express wild-type–like low levels of mutant p53 vated DNA damage signaling in mutant p53-harboring (4), although downregulated, all mutant human cancer lines tumors could account for stabilization of mutant p53 via express MDM2 protein constitutively. This renders the chronic Ser15 phosphorylation (19). We therefore assessed complete lack of mutant p53 ubiquitination disproportion- Ser15 status in unstressed cell panels. However, the majority ally severe and leaves it unexplained. of mutant p53 lines and all wild-type p53 lines lacked Because hyperstability of mutant p53 might not only be constitutive Ser15 phosphorylation. Although 2 of the 6 caused by reduced ubiquitination but also by increased mutant lines did show variably increased phosphorylation deubiquitination, we analyzed HAUSP, the major p53 (Fig. 2C), genotoxic stress (camptothecin) induced Ser15 deubiquitinase. However, levels of HAUSP and its inter- phosphorylation similarly in both wild-type p53 and action with p53 were similar in wild-type and mutant p53 mutant p53 cells (Supplementary Fig. S2A). Thus, this cells and thus did not contribute to mutant p53 hyperst- modification does not contribute to generic mutant p53 ability (Fig. 2B and Supplementary Fig. S1C). stabilization. Acetylation of the C-terminal lysines of wild- p14Arf protein levels, normally undetectable, are elevated type p53 via competitive inhibition of their ubiquitination in cells with a perturbed p53 signaling axis (17, 18), raising was proposed as an important mechanism for wild-type p53

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HSP90-Mediated MDM2 and CHIP Inactivation Causes Mutant p53 Stabilization in Cancer

stabilization in response to DNA damage. Moreover, acet- and wild-type p53 tumor cells. First, MDM2 levels in wild- ylation of wild-type p53 interferes with its interaction with type p53 breast cancer cells (MCF7) were downregulated by MDM2 (20). To test whether putative hyperacetylation of siRNA to match those of mutant p53 breast cancer cells mutant p53 may cause its ubiquitination defect, we ana- (MDA 231 and MDA 468; Fig. 4A). If MDM2 levels were lyzed the acetylation status of normalized amounts of the sole determinant as was previously assumed, one would mutant and wild-type p53. However, mutant p53 cells now expect wild-type p53 to hyperstabilize to levels matching generally express lower amounts of acetylated p53 than those of mutant p53. Surprisingly, however, wild-type p53 wild-type p53 cells (Fig. 2D), excluding hyperacetylation as stabilized by less than 2-fold in MCF7 cells, far below the contributor to the specific hyperstability of mutant p53. approximately 20-fold constitutive stabilization of mutant p53 levels in MDA 231 and MDA 468 cells (Fig. 4A). Similar Selective impairment of MDM2 E3 ligase activity in results were obtained for other wild-type p53 cells (RKO and mutant p53 but not wild-type p53 cancer cells HCT 116; Supplementary Fig. S1D). Conversely, we cor- Consistent with the lack of ubiquitination, the half-life of rected the lower MDM2 levels in mutant p53–harboring mutant p53 is dramatically increased compared with wild- MDA 231 back to those of wild-type p53–harboring MCF7 type p53 (Fig. 3A). Moreover, other bona fide substrates of cells by generating stable MDM2 clones that express about 2- MDM2, that is, MDMX and MDM2 itself, are also more fold higher MDM2 levels (Fig. 4B). MDA 231 cells do not stable in mutant compared with wild-type p53 cancer cells have elevated p14Arf as shown in Figure 2B, thereby exclud- (Fig. 3A) and are insensitive to proteasome inhibition in ing a possible negative effect on MDM2 activity. However, in mutant but not in wild-type p53 cancer cells (Fig. 3B). all successfully established MDA 231 clones, mutant p53 Thus, major physiologic substrates of MDM2 exhibit levels remained unaffected and ubiquitination nondetect- degradation deficiencies in mutant p53 cells. During stress, able, even after challenge with ALLN (Fig. 4B). This is DNA damage induces autoubiquitination and self-degrada- despite the fact that ectopic MDM2 undergoes effective tion of MDM2 as part of the stabilization mechanism of complex formation with endogenous mutant p53 wild-type p53 (21). However, although camptothecin (Fig. 4C). Likewise, ectopic MDM2 and endogenous destabilized MDM2 in wild-type p53 cells, this was not MDMX again display (self)-degradation defects in all mutant the case in mutant p53 cancer cells, again supporting their p53 MDM2 clones, judged by the poor (for MDM2) or selectively impaired MDM2 activity (Fig. 3C). Of note, absent (for MDMX) stabilization after ALLN treatment, in mutant p53 is fully competent for binding to MDM2. We contrast to wild-type p53 MCF7 cells (Fig. 4B). Thus, in did not observe dramatic differences in the physical inter- contrast to markedly supraphysiologic MDM2 expression action between endogenous mutant p53 and MDM2, as (Fig. 3E), physiologic levels of overexpressed MDM2 in reported earlier (13, 14). The amounts of coprecipitated mutant p53 cancer cells again are functionally inhibited. p53 simply reflected the respective steady state levels (Sup- This suggests that a saturable cellular mechanism leads to plementary Fig. S2B). In sum, this strongly suggests that the MDM2 inactivation in mutant p53 cancer cells. functional impairment of endogenous MDM2 is a major factor responsible for the aberrant stabilization of mutant Tumor-specific stabilization of mutant p53 is caused in p53 in cancer cells. part by the HSP90 molecular chaperone machinery On the other hand, normal H/H mouse embryonic Guarding the proteome against misfolding, aggregation fibroblasts (MEF) harboring the R172H mutation of and illicit interactions induced by proteotoxic stress such as p53 (H/H MEFs) properly stabilize mutant p53 in response reactive oxygen species (ROS), hypoxia, and acidosis, the to genotoxic stress and proteasome inhibition, similar to heat shock family of molecular chaperones guide proper wild-type p53–harboring MEFs (Fig. 3D). Likewise, conformational folding of nascent polypeptide "clients" into mutant p53 also properly stabilizes on irradiation in normal mature proteins, assist in the productive assembly of multi- spleen and thymus of H/H mice (4). Together, this con- meric protein complexes, and regulate the cellular levels of firms that in normal cells, MDM2 retains its ability to their clients by promoting degradation. Normal chaperone control mutant p53 stability, whereas this regulation is lost function is subverted during oncogenesis to allow initiation once cells become transformed. and maintenance of malignant transformation and enable Of note, supraphysiologic levels of ectopic MDM2 read- cancer cell survival because cancer cells are in a constant ily degraded endogenous mutant p53 in cancer cells state of proteotoxic stress, both from an adverse microen- (Fig. 3E, lanes 7 and 8). Conversely, proteasome inhibition vironment (hypoxia and acidosis) and from within (con- by ALLN blocked ectopic MDM2-mediated p53 degrada- formationally aberrant oncoproteins, high levels of ROS, tion similarly in mutant and wild-type cells (Fig. 3E, spontaneous DNA damage, and aneuploidy). Thus, their compare lanes 1, 2 with 5, 6), confirming earlier reports proteins and, in particular, their oncoproteins require mas- (13, 14). This also reaffirms that the defect that causes sive chaperone support to prevent aggregation and promote mutant p53 hyperstability lies with blocked endogenous survival (22). Hence, in addition to their oncogene addic- MDM2 activity and not with its substrate. tion, cancer cells also show addiction to HSPs. Among To further test the idea that mutant p53–harboring cancer chaperones, Hsp90 is unique because many of its clients are cells suffer from a selective inhibition of their MDM2 conformationally labile signal transducers with crucial roles activity, we forcibly equilibrated MDM2 levels in mutant in growth control, cell survival, and development. Most

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A HCT 116 MDA 468 CHX 0 1 2 4 6 0 1 2 4 6 h p53 MDMX short exp MDMX long exp

*MDM2 Actin B wt p53 mut p53 120 100 -ALLN 80 +ALLN 60 RKO SW480 HCT 116 HCT 231 MDA MCF7 40 MDM2 levels ALLN - + - + - + - + - + 20 MDM2 0 Tubulin SW480 RKO wt p53 mut p53 116 HCT MCF7 231 MDA C wt p53 mut p53 DU145 MCF7 MDA 468 MDA 231 MDA HCT 116 HCT ALLN -+ -+ -+ -+ -+ MDA 231 MDA RKO MDA 468 MDA MDMX long exp CAM - + - + - +

MDMX short exp MDM2 long exp

Actin MDM2 short exp Actin

D E HCT 116 MDA 231 MDM2 - + - + - + - + mut p53 wt p53 ALLN + + - - + + - - H/H MEF MEF p53 CAM - - + - - + MDM2 ALLN - + - - + - Actin p53 1 2 3 4 5 6 7 8 Actin

MDA 468

p53 MDM2 merge DAPI

Figure 3. Selective impairment of MDM2 E3 ligase activity in mutant p53 cancer cells. A, bona fide physiologic substrates of MDM2 (p53, MDMX, and MDM2 itself) exhibit degradation deficiencies in mutant p53 (MDA 468) compared with wild-type p53 (HCT 116) cells. CHX chase for the indicated times. Actin, loading control; *, a nonspecific band. B, nonresponsiveness of MDM2 and MDMX to proteasome inhibition and DNA damage in mutant p53 cancer cells indicates selective impairment of the MDM2 E3 ligase activity in mutant p53 cancer cells. Left, MDM2 activity is selectively impaired in mutant p53 cancer cells. Although wild-type p53 cells stabilized MDM2 more than 3-fold on ALLN, no MDM2 stabilization occurred in mutant p53 cells. Right, corresponding densitometry quantitation of relative MDM2 levels is shown. Bottom, in contrast to wild-type p53 cells, MDMX levels failed to stabilize in mutant p53 cells in response to ALLN. Cells treated with 25 mmol/L ALLN for 12 hours followed by immunoblotting. C, DNA damage destabilizes MDM2 because of autoubiquitination in wild-type p53 but not in mutant p53 cancer cells. Camptothecin (CAM; 5 mmol/L) for 2 hours. Immunoblot. D, normal mutant p53–harboring p53H/H MEFs (which express somewhat higher constitutive levels in culture than wild-type p53 MEFs), properly stabilize mutant p53 in response to genotoxic stress and proteasome inhibition. Immunoblots of cells treated with 5 mmol/L camptothecin or 25 mmol/L ALLN for 6 hours. E, endogenous mutant p53 is readily degraded by overexpressed levels of ectopic MDM2. Top, immunoblots after transient transfection of MDM2 with and without 25 mmol/L ALLN for 6 hours. Bottom, immunofluorescence of mutant p53 MDA 468 cells transfected with MDM2 (red). The transfected cell does not exhibit p53 staining (green). A to E, immunoblots, actin, and tubulin as loading controls. Abbreviations: wt p53, wild-type p53; mut p53, mutant p53, DAPI, 40,6-diamidino-2-phenylindole.

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HSP90-Mediated MDM2 and CHIP Inactivation Causes Mutant p53 Stabilization in Cancer

A B MDA 231 wt p53 MDM2

MCF7 wt p53 mut p53 Clone 9 Clone 10 MCF7 Vector only Vector Clone 21

ALLN - + - + - + - + - + MDM2 long exp MDA231 MDM2 siRNA MDA468 Scr siRNA MDM2 short exp MDM2 MDMX

p53 p53-Ub Tubulin

p53

Tubulin C - ALLN + ALLN 125 125 100 100 75 75 50 50 25 25 MDM2 levels MDM2 levels 0 1 2 3 4 5 0 1234 IgG control clone 21 clone 21 MCF7 MCF7 MDM2 MCF7 MCF7 Clone 9 Clone 9 Clone 21 Clone 10 Clone 21 p53 Clone 10 Vector only Vector

PCNA MDA 231 MDM2 clones MDA 231 MDM2 clones IP: MDM2 input

Figure 4. MDM2 activity is functionally impaired in mutant p53 cancer cells A and B, decreased levels of MDM2 are not the main determinant of mutant p53 stability in cancer cells. A, MDM2 levels in wild-type p53 breast cancer cells (MCF7) were downregulated by siRNA to match those of mutant p53 cells (MDA 231, MDA 468). However, knockdown of MDM2 in MCF7 cells elevates wild-type p53 levels only by less than 2-fold and does not reach the 20-fold stabilization present in mutant p53 cells. B, conversely, the lower MDM2 levels in mutant p53 MDA 231 cells were corrected back to those of wild-type p53 MCF7 cells. Stable MDM2 clones express 2-fold higher MDM2 levels. However, in all established MDA 231 clones, mutant p53 levels remained unaffected and ubiquitination nondetectable, even after challenge with ALLN (3 independent clones of 7 shown). Cells treated with 25 mmol/L ALLN for 12 hours. MDM2 and MDMX display the same (self)-degradation defect as seen in Figure 3, in contrast to wild-type p53 MCF7 cells. Bottom, densitometry quantitation of MDM2 levels of above blot in the absence or presence of ALLN by densitometry of long and short exposures, respectively. Tubulin, loading control. C, ectopic MDM2 undergoes effective complex formation with endogenous mutant p53. MDM2 immunoprecipitation (IP) from MDA 231–MDM2 clone 21 or wild-type p53 MCF7 cells, followed by immunoblots. PCNA, loading control. Abbreviations: wt p53, wild-type p53; mut p53, mutant p53. importantly, HSP90 plays a key role in the conformational (7). Importantly, upregulation of HSPs and, in particular, stabilization and maturation of mutant oncogenic signaling Hsp90 is an almost ubiquitous feature of human cancers proteins. These encompass steroid hormone receptors, (22). Moreover, structural and affinity differences exist, as receptor tyrosine kinases (i.e., HER-2), signaling kinases revealed by the fact that Hsp90 purified from tumor cells (Bcr-Abl, Akt, and Raf-1), and mutant p53 (8, 22). Hsp90 has a 100-fold stronger binding affinity to small molecule is the core protein of the multicomponent chaperone ligands of its ATP-binding pocket than does Hsp90 protein machinery HSP90 (that includes Hsp70 and others), a purified from normal cells. Of note, tumor Hsp90 is powerful antiapoptotic system that is highly upregulated entirely engaged in multichaperone complexes due to an and activated in cancer. Hsp90 is a dynamic ATPase. ATP increased load of mutant clients, whereas normal cell Hsp90 binding to the N-terminal domain of Hsp90 and its sub- is largely uncomplexed and free (22, 23). sequent hydrolysis drives a conformational cycle that is Importantly, many mutant p53 proteins are damaged in essential for the HSP90 chaperone activity. Cochaperone their conformation-sensitive core domain and form abundant Hsp40 stimulates the associated Hsp70 ATPase activity; stable complexes with Hsp90 in tumor cells (8, 10). In con- chaperone Hsp70 helps fold nascent polypeptides, and trast, wild-type p53 is unable to form stable Hsp90 complexes adaptor Hop mediates interaction of Hsp90 with Hsp70 and does so only transiently and with a few components. For

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example, the A1–5 fibroblasts expressing the temperature- type p53. In fact, 17AAG has opposite effects, downregulat- sensitive p53 A135V mutant showed that the HSP90 com- ing mutant p53 whereas upregulating wild-type p53 ponents Hsp90, Hsp70, cochaperone p23, and cyclophilin 40 (Fig. 5D). This 17AAG effect on wild-type p53 is consistent only coimmunoprecipitate with mutant p53 (at 37C) but with previous data (28). In primary tumor cells derived from not with wild-type p53 (at 30C; refs. 8–10). mouse models of medulloblastoma that either retain or are This stable mutant p53–specific interaction with HSP90 null for wild-type p53 function, inhibition of Hsp90 by a chaperones in cancer cells has been speculated to be linked derivative molecule, 17DMAG, induced wild-type p53– to aberrant stabilization of mutant p53. In a preliminary dependent apoptosis (29). immunoprecipitation study, Peng and colleagues presented circumstantial evidence, although no direct proof, that Inhibition of MDM2 and CHIP by HSP90 is largely MDM2 might be inactivated by being trapped within a responsible for stabilization of mutant p53 trimeric complex of mutant p53–MDM2–Hsp90 and pro- In normal cells, HSP90 chaperones regulate the protein posed that Hsp90 binding conceals the Arf-binding site on levels of their clients in part by directly recruiting ubiquitin MDM2, thereby somehow inhibiting its ligase function (24). ligases and presenting them for proteasome-mediated degra- Interpretation of this study, however, was made difficult by dation. The chaperone-dependent E3 ligase CHIP binds to the fact that MDM2 was found stabilized in tumor cells with Hsp70 and is a resident part of the HSP90 complex, mutant p53 for reasons that are unclear. Although we and normally promoting degradation of clients such as gluco- others find MDM2 downregulated in mutant p53 tumor corticoid and androgen receptors, c-ErbB2 (30), and phos- cells, our findings nevertheless fully endorse that mutant p53 phorylated tau (31). Importantly, degradative function of hyperstability in cancer cells is strongly dependent on heat CHIP can become defective in tumors. shock support because it inhibits mutant p53 ligases, as As shown above, interference with the HSP90 chaperone described below. HSF1, the master transcriptional regulator function by 17AAG triggers mutant p53 degradation by of the inducible heat shock response, controls all stress- freeing mutant p53 from the Hsp90 complex, which appar- inducible chaperones including HSP90 (25). HSF1 is fre- ently enables the reactivation of E3 ligases (Figs. 5B–F). quently upregulated in human tumors, and the HSF1- Which endogenous ligase(s) are responsible? Our evidence mediated stress response plays a causal, broadly supportive implicates both MDM2 and CHIP reactivation in 17AAG- role in mammalian oncogenesis (22, 25). We find that mediated degradation. First, 17AAG-reactivated MDM2 shRNA-mediated knockdown of HSF1 in mutant p53 cancer leads to self-degradation of MDM2 and its physiologic cells, which in turn downregulates Hsp90 and Hsp70 pro- substrate MDMX (Fig. 5F). Moreover, 17AAG-mediated tein, induces rapid destabilization of mutant p53 (Fig. 5A and mutant p53 destabilization is partially reversed (rescued) by Supplementary Fig. S3) and reduces its half-life (Supplemen- nutlin (Fig. 5G) or by siRNA-mediated MDM2 or CHIP tary Fig. S3A). Moreover, this relationship follows a direct knockdown (Fig. 5H) and almost completely rescued by dose response. The stronger the HSF1 knockdown and synergistic interference with both ligases (combined siRNAs therefore the Hsp90 and Hsp70 knockdown (obtained by and nutlin, Fig. 5H, lane 5). In further support, HSF1 repeat rounds of retroviral HSF1-shRNA infection), the knockdown–mediated mutant p53 degradation is again stronger the mutant p53 destabilization (Fig. 5A). partially reversed by nutlin (Supplementary Fig. S3B) and ATP binding to the N-terminal domain of Hsp90 with simultaneous knockdown of either MDM2 or CHIP ligases subsequent ATP hydrolysis drives a conformational cycle (Supplementary Fig. S3C). Together, our findings indicate that is essential for client binding and chaperone activity of that both MDM2 and CHIP are the major endogenous HSP90. Specific Hsp90 inhibitors such as the ansamycin E3 ligases for mutant p53, although CHIP seems to be the antibiotics geldanamycin and 17AAG competitively bind to more effective one. Both are presumably active in normal the N-terminal ATP-binding pocket and stop the chaperone cells of p53H/H KI mice harboring mutant p53. In cancer cycle, leading to client protein degradation. 17AAG is a cells, mutant p53 is trapped in stable interactions with potent and highly specific Hsp90 inhibitor currently in upregulated and activated HSP90 that effectively inhibits phase I to III clinical trials for refractory multiple myeloma MDM2 and CHIP activity, leading to its aberrant stabiliza- and several solid cancers including breast cancer (26). We tion. Ectopic expression studies previously implicated CHIP find that in all mutant p53 human cancer cells tested, as an alternative E3 ligase for ubiquitination and degradation 17AAG specifically induces endogenous mutant p53 to be of mutant p53 (14, 32, 33). Likewise, pharmacologic inter- released from HSP90 (e.g., Fig. 5B), followed by its efficient ference with Hsp90 by 17AAG reactivates both ligases to ubiquitination (Fig. 5C) and degradation (Fig. 5D; refs. 10, degrade mutant p53. This is similar to the functional 27). To further support the specificity of the 17AAG data, we redundancy of chaperone-associated E3 ligases that promote examined the effect of Hsp90 levels on mutant p53 stability. degradation of glucocorticoid and androgen receptors, as Indeed, downregulation of Hsp90 protein by siRNA desta- revealed after deletion of CHIP (34). bilizes mutant p53 in MDA 231 and 5637 cancer cells (Fig. 5E). Of note, 17AAG does not downregulate Hsp90 17AAG reduces cell viability more profoundly in levels in mutant p53 cells (Fig. 5D). Accordingly, 17AAG mutant p53 compared with wild-type p53 cancer cells markedly shortens the half-life of mutant p53 (Fig. 5F). To further test the notion that mutant p53 levels are the Interestingly, this is not the case for cancer cells with wild- major determinant of its oncogenic GOF and to test the

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HSP90-Mediated MDM2 and CHIP Inactivation Causes Mutant p53 Stabilization in Cancer

Figure 5. Stabilization of mutant p53 in cancer cells is caused by the HSP90 chaperone machinery that inhibits the MDM2 and CHIP E3 ligase activity. A, HSF1 knockdown, which leads to downregulation of Hsp90 and Hsp70 proteins, induces destabilization of mutant p53. The eukaryotic genome contains separate genes encoding constitutively expressed and inducible Hsp90. Only inducible Hsp90 transcription is controlled by HSF1 (23). Note that the Hsp90 antibody recognizes both constitutive and inducible Hsp90, explaining the only partial obliteration of Hsp90 after HSF1 knockdown. B, Hsp90 interference by 17AAG causes release of mutant p53 from the Hsp90 complex. Hsp90 (or IgG control) immunoprecipitation (IP; 1 mg protein each) from MDA 231 cells before and after 17AAG treatment (5 mmol/L for 24 hours). HAUSP, loading control. C, 17AAG induces ubiquitination of mutant p53 in a dose-dependent manner. Cells were treated with the indicated concentrations of 17AAG for 24 hours. D, Left, 17AAG induces degradation of mutant p53 (and MDMX) in a dose-dependent manner. 17AAG induces degradation of mutant but not wild-type p53 in cancer cells. Right, cells treated as indicated for 24 hours; 2 mg of mutant p53 and 20 mg of wild-type p53 cell extracts probed. 17AAG induces degradation of mutant p53 but does not decrease Hsp90 levels. Under normal conditions, HSF1 is also a client of HSP90 but held in an inactive HSF1–HSP90 complex. 17AAG inhibits HSP90 chaperone activity and promotes the release of HSF1 for transcriptional activation of HSPs, including Hsp90. This explains the slight increasein Hsp90. HAUSP, loading control. E, downregulation of Hsp90 protein by siRNA destabilizes mutant p53 in MDA 231 and 5637 cancer cells. Scr si, scrambled siRNA. Three days posttransfection, cells were harvested and analyzed by immunoblots. Actin, loading control. F, 17AAG decreases the half-life of mutant p53, MDM2, and MDMX. CHX chase of MDA 231 and MDA 468. Cells treated with 5 mmol/L 17AAG for 16 hours, then 17AAG plus 50 mg/mL CHX for the indicated times. G, nutlin partially prevents 17AAG-induced destabilization of mutant p53, indicating MDM2 reactivation on 17AAG. PANC1 cells harboring mutant p53 were treated with 5 mmol/L 17AAG with or without 10 mmol/L nutlin for 16 hours. Immunoblot. Actin, loading control. H, siRNA-mediated knockdown of MDM2 and CHIP rescue 17AAG-induced destabilization of mutant p53. Abbreviations: wt p53, wild-type p53; mut p53, mutant p53.

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ACB 500 1.2 MDA231 400 1 p53 shRNA - + 300 0.8 p53 0.6 actin Invasion 200 0.4 200,000 MDA 231 100 Tumor weight gr , 150,000 MDA 231 p53sh 0.2

100,000 0 0

Cell count MDA231 MDA231p53sh 50,000 control p53 shRNA p53 shRNA - + 0 p53 024487296 120 144 168 p53 Time (h) tubulin actin 1 2 3 4 1 2 3 4 Tumour #

D SW480 E 110 1 100 0.8 90 0.6 80 0.4 70 0.2 60

Tumor Weight, grams 0 50 Parental + Tet shp53 + Tet (% control) Cell Viability 40 Untreated Tet 17AAG Tet + 17AAG

TET - + - + 17AAG --+ + p53 Parental + Tet shp53 + Tet actin

G 120 100 F 110 80 100 60 T47D 40 90 Cell Viability T47D+R280K Mock 20 80 0 17AAG 2 µmol/L µ 70 Untreated 0.625 mol/L 17AAG 17AAG 5 µmol/L 60

Cell Viability (% of Cell Viability control) T47D T47D + R280K 50 17AAG - + - + p53 HAUSP

Figure 6. 17AAG reduces cell viability more profoundly in mutant p53 compared with wild-type p53 cancer cells. A to D, knockdown of mutant p53 by shRNA inhibits proliferation and invasion of human cancer cells in vitro and in vivo. shp53-mediated stable knockdown of mutant p53 in MDA 231 compared with parentals, assayed for proliferation in vitro (A) and in tumor xenografts in nude mice (C and D). C, average tumors weight (n ¼ 12 each) at day 20. Bars represent mean standard error. All tumors show significant reduction of mutant p53 levels (examples shown). B, knockdown of mutant p53 in MDA 231 cells by tetracycline-inducible shp53 inhibits their invasion. Matrigel Boyden chambers and immunoblot (bottom). D, knockdown of mutant p53 in SW480 colon cancer cells by tetracycline-inducible shp53 inhibits their growth in vivo. Tumor xenografts were harvested at day 12. Bottom, p53 immunostaining (DO1) in parental and knockdown xenografts after oral tetracycline fed to nude mice. The shp53 tumor shown corresponds to the single larger tumor in graph above. E, knockdown of mutant p53 by 17AAG (2 mmol/L) and/or tetracycline-inducible shp53 RNAi decreases cell viability of mutant p53 SW480 cells proportional to the extent of mutant p53 destabilization (bottom). CTB assay. Cells treated with tetracycline for 24 hours or left untreated, then concurrently with empty vehicle and/or 2 mmol/L 17AAG for 24 hours. F, 17AAG inhibits cell viability more profoundly in mutant p53 than in wild-type p53 cancer cells. CTB assay. Cells treated with 2 or 5 mmol/L 17AAG for 24 hours or left untreated. G, 17AAG loses killing efficacy when its ability to degrade mutant p53 is overwhelmed by excess ectopic mutant p53. The indicated T47D cells were treated with 0.625 mmol/L 17AAG or mock-treated for 48 hours. Top, cell viability assay (CTB). Bottom, corresponding immunoblot of empty vector or mutant p53 R280K overexpressing T47D cells. HAUSP, loading control.

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HSP90-Mediated MDM2 and CHIP Inactivation Causes Mutant p53 Stabilization in Cancer

Thus, these data suggest that 17AAG might have more H potent anticancer effects in mutant p53 tumors compared Stable chaperone Liberated mutp53 with wild-type p53 tumors. In support of a causal link complex with mutp53 between 17AAG-targeting mutant p53 and 17AAG cyto- mutp53 ub toxicity, 17AAG largely loses its killing efficacy (Fig. 6G, 17AAG ub ub top) when its ability to degrade mutant p53 is overwhelmed MDM2 ub MDM2 by excess amounts of ectopically expressed mutant p53 ("overstuffed"; Fig. 6G, bottom). At the concentration used, HSP90 HSP90 mutp53 CHIP the excessively high level of mutant p53 has exhausted the ability of 17AAG to degrade it and concomitantly squelches HIP? HOP? CHIP CHIP the ability of 17AAG to affect cell viability. As expected, HSP70HSP70 mutp53 ub 17AAG retains some remnant efficacy, suggesting a partial CHIP? ub ub p53-independent component of 17AAG action. ub In sum, given that the tumor-specific aberrant accumulation of mutant p53 is the basis for its GOF in malignancy and chemoresistance (6, 7, 35), understanding its underlying Figure 6. (Continued) H, proposed model of regulation of mutant – p53 stability by the HSP90 multichaperone machinery in cancer cells mechanism is critical for therapy for mutant p53 harboring (see text for explanation). Abbreviations: mut p53, mutant p53, cancers. On the basis of our results, we propose the following Tet, Tetracycline. model as a likely scenario (Fig. 6H). Normal tissues in p53H/ H knock-in mice that harbor missense mutant p53 are able to efficiently control their mutant p53 levels, despite the fact that dependence of established tumors on maintaining these their MDM2 levels are diminished because MDM2 is only high levels, we evaluated the consequences of downregu- supported by constitutive P1 promoter–driven transcription lating mutant p53 by (i) shp53 and (ii) pharmacologic (4). Of note, mutant p53 tumor cells are facing the same destabilization via 17AAG. In strong support that GOF MDM2 situation, that is, lower MDM2 levels that are only P1 indeed depends on highly stabilized mutant p53, we and promoter driven because of impaired p53 transcriptional others consistently find that downregulation of mutant activity. Therefore, tumor-specific stabilization of mutant p53 by shRNA strongly inhibits the malignant phenotype p53 proteins—which contributes to driving the tumor phe- of human cancer cells in vitro and in vivo (Figs. 6A–D). notype-–largely or exclusively depends on a second alteration For example, stable and tetracycline-inducible knockdown that these cells undergo on their transformation. This altera- of endogenous mutant p53 in breast (MDA 231) and tion is the addiction of malignant cells to support from the colon (SW480) cancer cells by shp53 RNAi dramatically activated heat shock machinery for their survival. In contrast inhibits cell proliferation (Fig. 6A) and invasion (Fig. 6B) to wild-type p53, the aberrant conformation of many mutant in culture and strongly inhibits tumor growth in nude p53 proteins makes them dependent on heat shock support so mouse xenografts in vivo (Figs. 6C and D). In agreement, that they stably engage in complexes with the highly activated mutant p53 knockdown in SK-BR3, HT29, SW480, HSP90 chaperone to prevent their aggregation. Intimately MiaPaCa-2, and MDA 231 cells also caused strong inhi- linked to this conformational stabilization, however, is the fact bition in proliferation, clonogenicity, and soft agar assays that this interaction also acts as a large protective "cage" against in vitro (6, 7). It also induced strong chemosensitization degradation, thereby enabling the GOF of mutant p53. The toward conventional genotoxicdrugs(6,7)andinhibited E3 ligases MDM2 and CHIP, which in principle are capable metastatic spread in mouse xenografts (35). Collectively, of degrading mutant p53, are also trapped in this complex in these data imply that tumors are addicted to their high an inactive state. Because mutant p53 is fully competent to levels of mutant p53 and support the rationale that bind to MDM2, HSP90 likely binds to preexisting mutant suppression of mutant p53 levels in vivo might achieve p53–MDM2 complexes. Alternatively, chaperone-bound clinically significant effects, particularly when combined mutant p53 could recruit MDM2. Depleting HSP90 com- with other anticancer therapies. ponents or binding of 17AAG to HSP90 destroys the com- Importantly, we find that destabilization of mutant p53 plex, releases mutant p53, and enables MDM2/CHIP- via Hsp90 interference by 17AAG markedly inhibits the mediated degradation. However, although unlikely, formally viability of SW480 colon cancer cells (Fig. 6E, compare it cannot be completely excluded that despite the same column 1 with column 3). This is again dose dependent MDM2 situation as in normal tissues, the lower MDM2 because 17AAG at 2 mmol/L cooperates with further reduc- levels in tumor cells might also play a minor role in mutant tion of mutant p53 levels by tetracycline-inducible shp53 in p53 hyperstability. In aggregate, these data provide encoura- reducing cell viability (Fig. 6E, compare column 2 with ging evidence for the possibility of mutant p53–directed column 4). Importantly, in a side by side comparison of anticancer therapy that targets an essential cofactor of its mutant and wild-type p53–harboring cancer lines, 17AAG stabilization rather than mutant p53 itself. We present a reduces cell viability more profoundly in mutant p53 cancer rationale for further pharmacologic improvement in small cells. Moreover, 17AAG at the same effective concentration molecule inhibitors of HSP90 chaperones. Such drugs, gen- is nontoxic toward normal cells such as MRC5 (Fig. 6F). erally well tolerated and some already in clinical trials, might

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represent an attractive mutant p53–targeting strategy for those Grant Support 50% of cancer patients, particularly when combined with other anticancer agents. The work was funded by grants from the National Cancer Institute (CA93853 to U.M. Moll) and the Carol Baldwin Breast Cancer Research Fund (to N.D. Marchenko). Disclosure of Potential Conflicts of Interest Received November 23, 2010; revised February 7, 2011; accepted March 9, 2011; No potential conflicts of interest were disclosed. published OnlineFirst April 8, 2011.

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588 Mol Cancer Res; 9(5) May 2011 Molecular Cancer Research

Functional Inactivation of Endogenous MDM2 and CHIP by HSP90 Causes Aberrant Stabilization of Mutant p53 in Human Cancer Cells

Dun Li, Natalia D. Marchenko, Ramona Schulz, et al.

Mol Cancer Res 2011;9:577-588. Published OnlineFirst April 8, 2011.

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