Depletion of Mutant p53 and Cytotoxicity of Histone Deacetylase Inhibitors Mikhail V. Blagosklonny, 1,2 Shana Trostel, 3 Ganesh Kayastha, 3 Zoya N. Demidenko, 1 Lyubomir T. Vassilev, 4 Larisa Y. Romanova, 5 Susan Bates, 3 and Tito Fojo 3 1 New York Medical College, Valhalla, New York; 2 Cancer Center, Ordway Research Institute, Albany, New York; 3 The Center for Cancer Research, National Cancer Institute, NIH, Bethesda, Maryland; 4 Discovery Oncology, Roche Research Center, Hoffmann-La Roche, Inc., Nutley, New Jersey; and 5 Department of Radiation Oncology, Harvard Medical School, Charlestown, Massachusetts Abstract Mutant p53 is a cancer-specific target for pharmacologic intervention. We show that histone deacetylase inhibitors such as FR901228 and trichostatin A completely depleted mutant p53 in cancer cell lines. This depletion was preceded by induction of p53-regulated transcription. In cells with mutant p53 pretreated with histone deacetylase inhibitors, DNA damage further enhanced the p53 trans-function. Further- more, histone deacetylase inhibitors were preferentially cytotoxic to cells with mutant p53 rather than to cells lacking wild-type p53. We suggest that, by either restoring or mimicking p53 trans-functions, histone deacetylase inhibitors initiate degradation of mutant p53. Because mutant p53 is highly expressed, a sudden restoration of p53-like functions is highly cytotoxic to cells with mutant p53. In a broader perspective, this shows how selectivity may be achieved by targeting a non-cancer-specific target, such as histone deacetylases, in the presence of a cancer-specific alteration, such as mutant p53. (Cancer Res 2005; 65(16): 7386-92) Introduction The p53 tumor suppressor is mutated in 50% of human cancers (1). Although mutant p53 renders cancer cells resistant to certain anticancer drugs, it is also a potential cancer-specific target for pharmacologic intervention (2–4). Given that mutant p53 is highly overexpressed, its sudden reactivation may be very toxic for a cell. Reintroduction of wild-type (wt) p53 by adenoviruses (Ad-p53) is predominantly cytotoxic to cancer cells that lack wt p53 (mutant p53 and null; ref. 5). However, it is currently technically impossible to introduce wt p53 all tumor cells. Another strategy is to reactivate mutant p53 using small molecular therapeutic agents that change conformation of mutant p53 (2–4). Although several compounds were described, their selectivity, specificity, and mechanism of action are still unclear (2, 6). Another strategy is to deplete mutant p53. For example, Hsp90-active agents (e.g., geldanamycin) moderately decrease mutant p53 (7). Although depletion of mutant p53 per se cannot and does not restore p53 functions, it may abolish dominant-positive effects (8, 9). Conversely, restoration of p53 function, in theory, will deplete mutant p53. In addition, functional reactivation of mutant p53, which is initially overexpressed, will be especially toxic to such a cell. Wt p53 transactivates Mdm-2, which targets p53 for degradation. In addition, p21 may also be required for p53 degradation (10, 11). By inducing Mdm-2 and p21, wt p53 stimulates its own degradation via the proteasome. Because mutant p53 cannot trans-activate Mdm-2 or p21, mutant p53 is not degraded and accumulates at high levels. Therefore, restoration of p53 function should result in degradation of mutant p53. Support of this idea can be found in observations that introduction of wt p53 leads to degradation of mutant p53 (12), and in experiments in which normal p53 function is imitated (13). For example, compounds that block degradation of Mdm-2 and p21 cause depletion of mutant p53 (13). The histone deacetylase inhibitor FR901228 (FK228, depsipeptide) is currently undergoing clinical trials (14). Histone deacetylases inhibit the trans-activating functions of wt p53 (15, 16). It was expected that FR901228 would be predominantly cytotoxic to cells with wt p53. However, as shown by correlative studies, including data from the National Cancer Institute drug screen, FR901228 was less active in cells with wt p53 (17, 18). Without mechanistic explanation, the significance of the correlation between wt p53 status and relative resistance to FR901228 remained elusive. The effects of drugs on p53 were usually studied in cells with wt p53; histone deacetylase inhibitors exert neither consistent nor signifi- cant effects on wt p53 levels (19–21). Here we report that trichostatin A and FR901228 induce p53-regulated transcription in cells with mutant p53 resulting in complete depletion of the p53 protein. Furthermore, mutant p53, rather than loss of wt p53, was associated with increased cytotoxicity of histone deacetylase inhibitors. Materials and Methods Cell lines. Cancer cell lines with mutant p53, MDA-MB-231, DU145, and SKBr3 (mutations at residues 280, 274/223, and 175H, respectively), were obtained from American Type Culture Collection (Manassas, VA). Clones of A2780-1A9 cells with different p53 statuses, A2780-1A9 (wt p53), PTX22 (mutant p53), and PTX5 (pseudo-null p53), were previously described (22, 23). Reagents. Paclitaxel (Taxol) was a Bristol-Myers product (Bristol-Myers, Princeton, NJ). Doxorubicin was obtained from Sigma (St. Louis, MO). Trichostatin A was obtained from Wako Pure Chemical Industries, Ltd. (Japan) and prepared as a 1 mg/mL stock in DMSO. FR901228 (FK228, depsipeptide) was obtained from the Chemistry and Synthesis Branch of National Cancer Institute (Bethesda, MD) and prepared as a 1 mg/mL stock solution in water. The active enantiomer of nutlin-3 (nutlin-3a) or the inactive enantiomer nutlin-3b was provided by Hoffmann-La Roche, Inc. (Nutley, NJ). These drugs were dissolved in DMSO and kept as 10 mmol/L stock solutions at À20jC. Immunoblot analysis. Proteins were resolved on SDS-PAGE or NuPAGE 4% to 12% Bis-Tris gel with MOPS running buffer (NOVEX, San Diego, CA) according to the instructions of the manufacturer. Immunoblotting was done using rabbit polyclonal anti-human poly(ADP-ribose) polymerase (Upstate Biotechnology, Lake Placid, NY), mouse monoclonal anti-human p21 [Calbiochem (Cambridge, MA), or Transduction Laboratories (Lex- ington, KY)], rabbit polyclonal anti–acetylated histone H3 (Upstate Requests for reprints: Mikhail V. Blagosklonny, Cancer Center, Ordway Research Institute, 150 New Scotland Avenue, Albany, NY 12208. Phone: 914-347-2801; Fax: 914- 347-2804; E-mail: blagosklonny@hotmail.com. I2005 American Association for Cancer Research. doi:10.1158/0008-5472.CAN-04-3433 Cancer Res 2005; 65: (16). August 15, 2005 7386 www.aacrjournals.org Research Article Research. on March 14, 2016. © 2005 American Association for Cancer cancerres.aacrjournals.org Downloaded from