Physical and Functional Antagonism between Tumor Suppressor Protein p53 and Fortilin, an Anti-apoptotic Protein*

Tumor suppressor protein p53, our most critical defense against tumorigenesis, can be made powerless by mechanisms such as mutations and inhibitors. Fortilin, a 172-amino acid polypeptide with potent anti-apoptotic activity, is up-regulated in many human malignancies. However, the exact mechanism by which fortilin exerts its anti-apoptotic activity remains unknown. Here we present significant insight. Fortilin binds specifically to the sequence-specific DNA binding domain of p53. The interaction of fortilin with p53 blocks p53-induced transcriptional activation of Bax. In addition, fortilin, but not a double point mutant of fortilin lacking p53 binding, inhibits p53-dependent apoptosis. Furthermore, cells with wild-type p53 and fortilin, but not cells with wild-type p53 and the double point mutant of fortilin lacking p53 binding, fail to induce Bax gene and apoptosis, leading to the formation of large tumor in athymic mice. Our results suggest that fortilin is a novel p53-interacting molecule and p53 inhibitor and that it is a logical molecular target in cancer therapy.

Tumor suppressor protein p53 keeps us free of cancer when it is functional. Mice lacking p53 (p53 Ϫ/Ϫ ) spontaneously develop numerous neoplasms within 6 months (1). Mutated p53 genes are seen in more than 50% of all human cancers, making them the most frequently observed genetic derangement in human cancer (2). At a molecular level, the ability of p53 to eliminate cancerous cells relies on its ability to induce apoptosis, through either the transcriptional activation of proapoptotic genes such as Noxa (3), PUMA 4 (4), and Bax (5) or the direct transcription-independent activation of Bax on mitochondria (6). Growing cancers manage to keep p53 in check either by mutating the p53 gene itself (7)(8)(9) or by expressing p53 inhibitors such as Mdm2 (9,10).
The mechanism by which fortilin functions as an anti-apoptotic molecule has been under robust investigation. First, based on the fact that fortilin physically interacts with myeloid cell leukemia protein-1 (MCL1), an anti-apoptotic Bcl-2 family member, it was suggested that fortilin stabilizes and exerts its anti-apoptotic activity through MCL1 (22). However, fortilin is capable of protecting cells from apoptosis in the absence of MCL1 (23), suggesting that fortilin is anti-apoptotic independently of MCL1. Secondly, fortilin functionally antagonizes Bax, a pro-apoptotic Bcl-2 family member (21), presumably by inserting itself into the mitochondrial membrane and preventing Bax from dimerizing within the membrane. However, there is no physical interaction demonstrable between fortilin and Bax (21), and it is still unclear how fortilin blocks Bax dimerization in the mitochondrial membrane. Thirdly, fortilin binds calcium (17, 24 -28) and blocks calcium-dependent apoptosis (29). However, the strength of the binding of fortilin to calcium was moderate at most with a dissociation constant of 7. 58 -17.5 M (29). Fourthly, fortilin binds and destabilizes transforming growth factor-␤-stimulated clone-22 (TSC-22), a pro-apoptotic molecule, and protects cells against apoptosis (30). However, it remains unclear whether the direct interaction between fortilin and TSC-22 is required for the destabilization of TSC- 22. Fortilin is localized in not only the cytosol but also in the nucleus (13). The putative mechanisms described above by which fortilin functions anti-apoptotically do not necessarily explain the nuclear presence of fortilin. We speculated that fortilin might interact with one of the nuclear pro-apoptotic molecules and block its pathway, leading to apoptosis. Because p53 rapidly accumulates in the nucleus in response to DNA damage (31) and induces apoptosis through the transcriptional activation of pro-apoptotic genes including Bax (32), Noxa (3), and PUMA (4), we asked whether fortilin physically interacted with p53 and blocked the pro-apoptotic activity of p53. The data presented here support that fortilin specifically binds p53 and blocks the p53-induced transcriptional activation of Bax and resultant apoptosis. We propose that fortilin is a novel negative regulator of p53-mediated apoptosis.

EXPERIMENTAL PROCEDURES
The detailed experimental procedures are found in the supplemental Experimental Procedures.
Cell Culture and Cell Lines-All cell lines were maintained in high glucose Dulbecco's modified Eagle's medium (DMEM) and supplemented with 10% fetal bovine serum (FBS) at 37°C in an atmosphere containing 5% CO 2 .
Nude Mouse Tumor Xenograft Assays-All mouse experiments were performed under the approved Institutional Animal Care and Use Committee (IACUC) as described previously (37).
Statistical Analyses-The degree of the spread of data was expressed by Ϯ S.D. p Ͻ 0.05 was considered to be statistically significant.
U2OS cells harbor wild-type p53 (38). To validate the interaction between fortilin and p53 in vivo, we generated U2OS cells overexpressing hemagglutinin (HA)-tagged fortilin (U2OS fortilin-HA ) or HA tag alone (U2OS Empty-HA ) and subjected the cleared total cell lysates from these cells to immunoprecipitation using anti-HA antibody (Fig. 1B, top panel, lanes 1  and 3). HA-tagged fortilin was successfully immunoprecipitated from the lysate from U2OS fortilin-HA , but not from U2OS Empty-HA (Fig. 1B, top panel, lanes 2 and 4). In this system, the p53 signal was detectable only in the presence of successfully immunoprecipitated fortilin-HA (Fig. 1B, bottom panel,  lanes 2 and 4). We then repeated the same experiment using a different anti-HA antibody preparation (rat 3F10 primary antibody and anti-rat IgG magnetic beads for Fig. 1B; 3F10 matrix beads for supplemental Fig. S1). In this system, the anti-HA antibody successfully immunoprecipitated fortilin-HA (supplemental Fig. S1, top panel, lane 4). The p53 signal was detectable in the presence of fortilin-HA (supplemental Fig. S1, middle and bottom panels, lane 4), but not in its absence (supplemental Fig. S1, middle and bottom panels, lane 3).
We then performed a reverse in vivo co-immunoprecipitation assay by equally dividing the cleared total cell lysates from U2OS fortilin-HA cells into three microcentrifuge tubes and incubating them with the bare agarose beads, beads coated with normal mouse IgG, or beads coated with anti-p53 antibody (FL-393AC). Beads coated with anti-p53 antibody, but not other types of beads, successfully immunoprecipitated native p53 ( Finally, we performed the same in vivo reverse co-immunoprecipitation assay on cells expressing only native fortilin and p53. The equally divided aliquots of the cleared total cell lysates from wild-type U2OS cells were treated with either a mixture of DO1 and Pab421 antibodies or control mouse normal IgG. Native p53 was successfully immunoprecipitated by anti-p53 antibodies, but not by control IgG (Fig. 1D, top panel, lanes 1 and 2). In this system, the signal of native fortilin was detectable only in the presence of immunoprecipitated p53 (Fig. 1D, bottom panel, lane 1), but not in its absence (lane 2).
These data (Fig. 1, A-D) consisting of in vitro pulldown assays and in vivo forward and reverse immunoprecipitation Western blot assays, clearly suggest that fortilin specifically interacts with p53. To evaluate whether ultraviolet (UV) irradiation and resultant DNA damage affect the intensity of the fortilin-p53 interaction, we UV-irradiated U2OS fortilin-HA cells, immunoprecipitated HA-tagged fortilin, and evaluated the amount of p53 co-immunoprecipitated by fortilin-HA. UV irradiation increased p53 expression in a dose-dependent fashion (supplemental Fig. S2A, Input). More p53 was co-immunoprecipitated by fortilin-HA in the presence of mild to moderate UV irradiation (5-40 mJ/cm 2 ), suggesting that UV irradiation leads to more fortilin-p53 interaction (supplemental Fig. S2, A and B). Further investigation is necessary to determine whether this is due to increased affinity between fortilin and p53 (such as through the post-translational modification of fortilin and/or p53) or increased availability of fortilin and/or p53 for each other.
To evaluate the spatial localization of fortilin in relation to that of p53, UV-irradiated HeLa cells were immunostained using anti-p53 (DO1) and anti-fortilin (MBL International) antibodies. Bound antibodies were detected by anti-mouse Alexa Fluor 488 and anti-rabbit rhodamine red-X secondary antibodies, respectively. The p53 signal was mostly localized in the nucleus, whereas the fortilin signal was present in both the nucleus and the cytosol, although a higher amount was found in the nucleus (Fig. 1E, DRAQ5, ␣-p53, and ␣-fortilin).
The merged image suggests that the two molecules co-localize in the nucleus, but not in the nucleoli or cytosol (Fig. 1E, merge), suggesting that fortilin interacts with p53 in the nucleus.
Fortilin Blocks p53-dependent Apoptosis by Preventing the Transcriptional Activation of Bax Gene by p53-To evaluate the role of fortilin in the transcriptional activation of p53 target genes, we developed a unique luciferase assay system in which each CHO cell stably harbors a single copy of luciferase reporter plasmid containing the p53-responsive element (RE) of Bax gene (CHO Bax-luciferase ) (32). We transiently transfected the CHO Bax-luciferase cells with p53 (or control) and with fortilin (or control) mammalian expression plasmids. Western blot analyses confirmed adequate expression of both p53 and fortilin proteins (Fig. 3A, WB). Background luciferase activity measured in CHO Bax-luciferase cells was low in the absence of p53 either with or without fortilin overexpression (Fig. 3A, lanes 1 and 2,  respectively). When p53 was overexpressed, luciferase activity increased more than 2-fold (lane 3), suggesting that p53 appropriately activated the p53-RE of the Bax promoter. In this system, co-overexpression of fortilin significantly reduced the activation by p53 of the p53-RE of the Bax promoter. (Fig. 3A, lane 4 versus lane 3), suggesting that fortilin blocked p53-induced transcriptional activation of Bax gene.
We next compared the susceptibilities of U2OS cells treated with siRNA against fortilin (siRNA Fortilin ) or siRNA Luciferase to UV irradiation (43). In the presence of fortilin, UV irradiation did not significantly increase the DNA fragmentation of U2OS cells (Fig. 3C, lanes 1 and 2). In the absence of fortilin, however, UV irradiation drastically increased the DNA fragmentation (Fig. 3C, lanes 3 and 4), suggesting that fortilin protected the cells against UV-induced apoptosis.
Next, we quantified the Bax message levels within the UVirradiated U2OS cells that were treated with either siRNA Fortilin or siRNA Luciferase , using a quantitative real-time RT-PCR assay. As expected, siRNA Fortilin , but not siRNA Luciferase , drastically decreased fortilin message levels in both UV-irradiated and control U2OS cells (Fig. 3D, lanes 2 and 4 versus lanes 1 and  3, respectively). In that system, Bax message levels were significantly higher in siRNA Fortilin -treated cells than in siRNA Luciferase -treated cells (Fig. 3E, lanes 3 versus 4), suggesting that the lack of fortilin led to the greater transcriptional activation of Bax gene in response to UV irradiation.
Next, we quantified p53 and Bax protein levels in response to UV irradiation using ELISA, in the presence and absence of fortilin. The intracellular p53 concentrations drastically increased as UV doses increased (Fig. 3F). In that system, Bax protein concentrations were significantly higher in siRNA Fortilin -treated cells than in siRNA Luciferase -treated cells, FIGURE 2. Fortilin binds the sequence-specific DNA binding domain through its N and C terminus ends. A, in vitro co-precipitation of p53 deletion mutants by fortilin in GST pulldown assay. Input, autoradiography of the 1/20 portion of binding mixture; Pulled down proteins, Coomassie Blue staining of GST-fusion proteins pulled-down by GST-Sepharose beads; Co-precipitants, 35 S-labeled proteins co-precipitated by pulled down GST-fortilin or GST only. B, summary of interaction between fortilin and the wild-type and deletion mutants of p53. aa, amino acids. C and D, GST pulldown assay of fortilin deletion mutants. p53 (1-393), full-length p53; p53 , the SSDBD of p53. E, GST pulldown assay of fortilin point mutants. Binding to p53, a densitometric analysis of signal intensity of co-precipitants. F, identification of a fortilin double point mutant that lacks binding to p53. *, point mutation; Ϫ, no p53 binding; ϩ/Ϫ, minimum p53 binding; ϩ, decreased p53 binding; ϩϩ, p53 binding similar to wild-type fortilin; ϩϩϩ, greater p53 binding. The same GST pulldown assay was performed at least three times, and the data were found to be consistent among these experiments. G, the proximity of N and C terminus ends of fortilin to each other. H, the spatial representation of Tyr 4 and Glu 168 on the surface of fortilin molecule. Positively and negatively charged amino acid residues are colored blue and red, respectively. I, the SSDBD of p53 bound to DNA. J, docking of fortilin to the DNA binding surface of p53.
To test whether the above observation is true in a different cell line, we stably knocked down p53 and/or fortilin using a lentiviral shRNA system in PMJ2-PC cells, a peritoneal macrophage cell line, and generated the four polyclonal cell lines, PMJ2-PC p53ϩfortilinϩ , PMJ2-PC p53ϩfortilinϪ , PMJ2-PC p53-fortilinϩ , and PMJ2-PC p53-fortilinϪ (supplemental Fig. S5A). These cell lines were subjected to UV irradiation, and the degree of DNA frag-mentation and caspase 3 activity was quantified. Upon UV irradiation, all four cell lines exhibited a significant increase in DNA fragmentation (supplemental Fig. S5B, lanes 2, 4, 6, and 8 in comparison with supplemental Fig. S5B, lanes 1, 3, 5, and 7) and caspase 3 activities (supplemental Fig. S5C, lanes 2, 4, 6, and 8 in comparison with supplemental Fig. S5C, lanes 1, 3, 5, and  7). Strikingly, the lack of fortilin was associated with an ϳ6-fold increase in DNA fragmentation (supplemental Fig. S5B, lane 2 versus lane 4) and 2.5-fold increase in caspase activities (supplemental Fig. S5C, lane 2 versus lane 4), only in the presence of p53 (supplemental Fig. S5, B and C, lanes 2 and 4) but not in the absence of p53 (supplemental Fig. S5, B and C, lanes 6 and 8). In other words, fortilin failed to protect cells against either DNA fragmentation or caspase 3 activation in the absence of p53 (supplemental Fig. S5, B and C, lanes 6 and 8). These data suggest that fortilin specifically blocks UV-induced, p53-dependent apoptosis through Bax.
Next, we infected U2OS cells with retroviral vector containing wild-type fortilin (Ret-fortilin), fortilin(Y4A,E168A) (Ret-fortilin⌬), or empty vector (Ret-empty, control). We then subjected them to UV radiation and performed both MTT and caspase 3 activity assays. Neither Ret-fortilin nor Ret-fortilin⌬ contained epitope tags. Western blot analysis confirmed that both U2OS Ret-fortilin and U2OS Ret-fortilin⌬ expressed more fortilins than did U2OS Ret-Empty and that UV irradiation increased p53 expression in all three cell lines (Fig. 4B). In this system, the cell death rate was significantly lower for U2OS Ret-fortilin cells than for U2OS Ret-empty cells (Fig. 4C, p Ͻ 0.05 by analysis of variance). Remarkably, U2OS Ret-fortilin⌬ cells were more susceptible to UV irradiation than were U2OS Ret-fortilin or U2OS Ret-empty cells (Fig.  4C, p Ͻ 0.05 by analysis of variance). Consistently, U2OS Ret-fortilin⌬ cells had more caspase 3 activation than did U2OS Ret-empty cells, whose caspase activity surpassed that of U2OS Ret-fortilin (Fig. 4D, p Ͻ 0.05 by analysis of variance). These results, when taken together, suggest that fortilin is required to bind to p53 to block p53-mediated cell death (Fig. 4, C and D). U2OS cells harbor wild-type p53 (38), whereas SAOS cells do not express p53 (44), offering a unique opportunity to evaluate the role of p53 and fortilin in UV-induced apoptosis. We first generated and characterized SAOS Ret-Empty , SAOS Ret-Fortilin , and SAOS Ret-Fortilin⌬ (Fig. 4E) and subjected them and the three U2OS counterparts to UV irradiation and the DNA fragmentation assay. As expected, fortilin, but not fortilin⌬, protected U2OS cells against UV-induced DNA fragmentation (Fig. 4F,  lanes 2, 4, and 6). Remarkably, however, fortilin did not protect SAOS cells any better than did fortilin⌬ against UV-induced DNA fragmentation (Fig. 4F, lanes 8, 10, and 12). These results suggest that fortilin protected cells more than fortilin⌬ only in the presence of wild-type p53 (Fig. 4F, lane 4 versus lane 6 and  lane 10 versus lane 12). Also shown was that UV irradiation induced apoptosis predominantly through p53-dependent pathways in this system (Fig. 4F, lane 2 versus lane 8). Together, these data suggest that fortilin is required to bind p53 to block p53-dependent apoptosis.
Fortilin Negates the Tumor-suppressing Effects of p53 through Its Binding to p53 in Growing Tumors in Nude Mice-To determine whether fortilin facilitates tumor growth through its binding and inhibition of p53, we injected USOS and SAOS cells containing Ret-Empty, Ret-fortilin, or Ret-fortilin⌬ into nude mice and followed the tumor growth (n ϭ 10 each). All 10 nude mice injected with U2OS Ret-fortilin and six injected with U2OS Ret-Empty formed tumors at the 6th week where U2OS Ret-fortilin formed larger tumors than did U2OS Ret-Empty (Fig. 5, A-C). Strikingly, U2OS Ret-fortilin⌬ did not form any tumors at the 6th week (Fig. 5, A and B) with only four animals showing detectable tumors at the 12th week (Fig. 5C). None of the nude mice injected with SAOS Ret-Empty , SAOS Ret-Fortilin , or SAOS Ret-Fortilin⌬ cells in Matrigel developed significant tumors at the 6th week, thus preventing us from assessing the effect of fortilin in tumor growth in the p53-null environment of SAOS at this time point (data not shown). At the 15th week, however, five, three, and four nude mice injected with SAOS Ret-Empty , SAOS Ret-Fortilin , and SAOS Ret-Fortilin⌬ cells, respectively, exhibited significant tumors. The average tumor volumes from SAOS Ret-Empty , SAOS Ret-Fortilin , or SAOS Ret-Fortilin⌬ cells were 235 Ϯ 17.1, 23.3 Ϯ 40.7, and 95.8 Ϯ 154.9 mm 3 , respectively. Although the small sample size (n ϭ 12 total) precluded an adequate statistical comparison on the tumor volumes among these three groups, these tumor materials were sufficient for and thus subjected to immunohistochemical evaluation as described below in Fig. 6. In summary, the U2OS cell data above (Fig. 5) strongly suggest that fortilin facilitates tumor growth through its interaction with and inhibition of p53.
Fortilin Decreases Bax Expression and Apoptosis in p53-positive Tumors-To test whether the presence of fortilin interfered with the ability of p53 to induce Bax and apoptosis within the growing tumors in the nude mouse model above, we immunostained the tissue with anti-fortilin (␣-fortilin) and anti-Bax (␣-Bax). We also performed TUNEL staining on these samples. Twelve (12) tumor samples from the nude mice injected with SAOS cells were also evaluated. Both U2OS and SAOS cells infected with Ret-fortilin and Ret-fortilin⌬ showed the robust expression of these constructs in comparison with cells infected with the control vector (Ret-Empty) (Fig. 6, A-C). In this sys-tem, the immunoreactivity of Bax was significantly less in tumors from U2OS Ret-fortilin than those from U2OS Ret-fortilin⌬ (Fig. 6, D, top panel, and E), whereas the immunoreactivity of Bax was identical among all the tumors from SAOS Ret-Empty , SAOS Ret-fortilin , and SAOS Ret-fortilin⌬ (Fig. 6D, bottom panel, and F). In addition, U2OS Ret-fortilin , but not U2OS Ret-fortilin⌬ , had a significantly lower TUNEL index than did U2OS Ret-Empty (Fig. 6, G, top panel, and H). Intriguingly, there was no difference in TUNEL indices of SAOS Ret-Empty , SAOS Ret-fortilin , and SAOS Ret-fortilin⌬ (Fig. 6, G, bottom panel, and I), which were uniformly lower than those of the U2OS cells. These data, when taken together, suggest that fortilin facilitates tumor growth by binding p53 and blocking its ability to induce Bax and apoptosis.

DISCUSSION
The current study for the first time provides a clear mechanistic insight as to exactly how fortilin protects cells against apoptosis. This is because we show here that fortilin, a unique anti-apoptotic molecule that does not resemble either Bcl-2 or inhibitor of apoptosis (IAP) family member proteins (13), specifically interacts with p53 (Fig. 1, A-E), prevents p53 from transcriptionally activating Bax gene (Figs. 3, A, E, and G, and 6, D and E) and inducing apoptosis (Fig. 3, B, C, supplemental Fig.  S5, B and C), and sustains tumor growth in a whole animal (Fig.  5, A-C) by suppressing p53-dependent Bax induction and resultant apoptosis (Fig. 6, D-I). Intriguingly, in the absence of wild-type p53, fortilin⌬, which lacks p53 binding, behaved in the same fashion as wild-type fortilin, failing to protect cells from Bax induction or apoptosis (Figs. 4F and 6, D, F, G, and I), suggesting that fortilin is minimally involved in the non-p53 pathways. Immunocytochemical staining shows that the fortilin-p53 interaction likely takes place in the nucleus (Fig. 1E). The detailed analyses on deletion and point mutants of fortilin and p53 showed that the N and C terminus ends of fortilin participate in the binding to the SSDBD of p53 (Fig. 2, A-J). The ability of fortilin to inhibit the binding of p53 to its consensus sequence (Fig. 4A) and to block p53-mediated apoptosis was dependent on its binding to p53 (Fig. 4, B-F). We thus propose that fortilin is a key negative regulator of the p53-Bax apoptosis pathway. In a supplemental experiment, we evaluated the ability of fortilin to prevent p53 from inducing PUMA (4) and Noxa (3), p53-inducible genes. We found that fortilin inhibits the induction by p53 of Noxa, but not PUMA (supplemental Fig.  S6). A possible differential inhibition by fortilin of p53 target genes, Bax and Noxa versus PUMA, is currently under investigation in our laboratory. In the final stage of our manuscript preparation, we were made aware of a study published on the interaction between fortilin and p53 where the authors used fortilin as bait, employed the yeast two-hybrid screening system, and identified a portion of p53 as a fortilin interacting molecule (45). Our work confirms the interaction and provides the totally new mechanistic insight as to how fortilin protects cells against p53-mediated apoptosis at all three levels, in vitro, in vivo, and whole animal.
p53 is designed to play two important, but fundamentally opposing, roles in response to stress (59). p53, under the low, constitutive levels of DNA damage, induces cell cycle arrest through the transcriptional activation of p21 CIP1/WAF1 , allowing the cells to repair themselves. Upon the higher levels of DNA damage, on the contrary, p53 switches from promoting survival and repair to the induction of apoptosis (59,60), allowing the organism to eliminate hopelessly damaged cells from itself. The exact mechanism by which p53 differentially activates the cell cycle regulatory versus the apoptosis pathway is unknown, although the partial and total acetylation of p53 by histone acetyltransferases may be involved in the activation of the cell cycle regulatory and pro-apoptotic genes, respectively (47). In addition, the molecules that bind the SSDBD of p53, such as Hzf (51), Brn-3a (52,53), and ASPP (54), are shown to differentially regulate cell cycle progression and apoptosis. Although it is tempting to speculate that fortilin, an SSDBD binding molecule, also differentially regulates cell cycle progression and apoptosis, the role of fortilin in the regulation by p53 of cell cycle progression is currently unknown and under investigation.
Yang et al. (15) reported that fortilin interacts with Bcl-xL using both GST pulldown and co-immunoprecipitation assays. However, in our current assay (Fig. 1A) as well our previous work (22), fortilin binds MCL1, but not Bcl-xL. It is likely that fortilin interacts with p53 and MCL1 more strongly than with Bcl-xL and that our wash condition was too stringent to retain the interaction between fortilin and Bcl-xL.
The binding of Mdm2 to p53 (55) results in ubiquitination and proteasome-mediated degradation of p53, making the halflife of p53 very short (61)(62)(63) and intracellular levels of p53 very low. Cell stress such as DNA damage decreases the degree of sumoylation of Mdm2 and increases Mdm2 degradation, leading to the stabilization of p53 (64). Unlike Mdm2, neither fortilin overexpression nor depletion showed any consistent or detectable changes in the p53 levels by either Western blots or ELISA (Figs. 3, A, B, and F, and 4, B and E and supplemental Fig.  S5A). The fortilin double point mutant identified in the current study (fortilin⌬) may prove to be a viable reagent as the role of fortilin in the stability of p53 is further investigated.
Our discovery of the physical and functional interaction between fortilin and p53 has significant clinical implications. First, targeting of the fortilin-p53 interaction by small molecules may result in the reactivation of p53 and the induction of apoptosis within cancer cells that harbor wild-type p53, although such strategies may not work in cancers that harbor a mutated p53. Such anti-fortilin small molecules may also be useful in further improving the response of cancer cells to chemotherapeutic agents, ionizing radiation, and Nutlin-3A (65) and MI-219 (66), small molecules that disrupt the p53-Mdm2 interaction. In addition, such anti-fortilin small molecules may be useful in the prevention and treatment of atherosclerosis refractory to 3-hydroxy-3-methyl-glutaryl-CoA reductase inhibitors (statins) because the lack of functioning p53, more specifically, macrophage p53 (67,68), is associated with accelerated atherosclerosis (69).