Abstract 881: Identification of autoantibody biomarkers to wild-type and mutant p53 in pancreatic and ovarian cancer

Exposure of mammalian cells to genotoxic stress results in activation of the c-jun amino-terminal kinase (JNK)-stress-activated protein kinase (SAPK) pathway and induction of DNA repair enzymes and cell cycle-regulatory proteins such as p53 and p21waf1. The p53 tumor suppressor protein transmits signals that activate p21waf1 gene expression. The p21waf1 protein then restricts cell-cycle progression, thereby allowing time for DNA repair to occur. In this study, we investigated the effects of modulation of the level of wild-type and mutant p53 protein on basal JNK1 activity in the A1-5 rat fibroblast cell line. This cell line contains a p53 gene coding for a temperature-sensitive p53 protein, which allows us to regulate the relative level of wild-type and mutant p53 protein produced in cells. Using the immune complex kinase assay to measure JNK1 activity, we demonstrated that cells expressing the wild-type-conformation p53 protein (when grown at 32.5 degrees C) exhibited a very low level of JNK1 activity. When cells were grown at 37 degrees C or 39 degrees C to express predominantly mutant p53 protein, basal level of JNK1 activity was significantly higher than at 32.5 degrees C. We also demonstrated protein-protein interactions between the p53, p21waf1, and JNK1 proteins in this cell line. Both wild-type p53 protein (expressed at 32.5 degrees C) and mutant p53(val135) protein (expressed at 37 degrees C and 39 degrees C) were present in immunocomplexes of JNK1 protein. Under conditions where wild-type p53 protein was present to induce p21waf1 expression (at 32.5 degrees C), a higher level of p21waf1 protein was also detected in the JNK1 immunocomplexes than in those at 37 degrees C and 39 degrees C. We next investigated the effect that co-association of p53 protein and p21waf1 protein would have on JNK1 activity. We measured basal levels of JNK1 activity in cells expressing wild-type p53 and p21waf1, or in p21waf1-null cells, and demonstrated that cells expressing both p53 and p21waf1 proteins exhibited an approximately threefold lower basal level of JNK1 activity when compared with p21waf1-null cells. To confirm that p21waf1 protein expression in cells resulted in reduced JNK1 activity, we transfected p21waf1-/- cells with a p21waf1 expression vector. We observed that JNK1 activity was inhibited after exogenous p21waf1 protein was expressed in these cells. Our results provide evidence for modulation of the JNK1 pathway by p53 and p21waf1 proteins and support the hypothesis that modulation of JNK1 activity occurred through protein-protein interactions between JNK1, p53, and p21waf1 proteins.

The p53 protein is an important tumor suppressor that can function to inhibit cell growth through the activation of a number of different responses, including cell cycle arrest, senescence and apoptosis. Recent evidence has suggested that under conditions of low or transient stress, p53 can also contribute to the survival of stressed cells - allowing for the prevention or repair of damage. Each of these activities can restrain malignant progression, and most cancers show loss of the normal functions of p53. In many cancers this is due to a mutation in the p53 gene that leads to the expression of a mutant p53 protein. Interestingly, these tumor associated mutant p53s not only lose wild type p53 activity but can also acquire the ability to promote cell motility and migration, and so contribute to the development of metastases. We have found that tumor associated mutant p53s can promote invasion and loss of directionality when cells migrate in vitro. These activities are independent of the loss of wild type p53 function, and reflect activation of integrin and EGFR trafficking that depends on Rab-coupling protein and which results in constitutive activation of EGFR/integrin signalling. These findings open the possibility that blocking alpha5/beta1 integrin and/or the EGF receptor will have therapeutic benefit in mutant p53 expressing cancers. We are now proposing to extend these observations by testing whether this activity of mutant p53 is restricted to the EGFR, or may also promote the activity of other cell surface receptors too. Simultaneous loss of p53 and p63 recapitulates the phenotype of mutant p53, suggesting that this function of mutant p53 reflects, at least in part, the inhibition of p63. However, mutant p53 is likely to have additional functions that contribute to the ability to induce an invasive phenotype, and we are presently investigating the activity of other mutant p53 binding proteins. While mutant p53s are often expressed at very high levels in tumors, normal cells express mutant p53s at low level, like the wild type p53 protein. The turnover of mutant p53 in these cells is dependent on the function of the MDM2 ubiquitin ligase, which also regulates the stability of wild type p53, and deletion or inhibition of MDM2 leads to the stabilization of both mutant and wild type p53 proteins. A number of small molecule drugs that can inhibit MDM2's ability to target p53 for degradation have been described, with the hope that these will be useful in treating those tumors that retain wild type p53. However, studies have suggested that many apparently normal tissues harbour p53 mutations, leading to the possibility that systemic treatment with MDM2 inhibitors would result in the stabilization of these mutant p53s, with unanticipated, deleterious consequences. We are therefore investigating how mutant p53 turnover is regulated, with the view to testing whether the stabilization of mutant p53 would affect the behaviour of otherwise normal cells. Citation Format: Patricia Muller, Patricia Roxburgh, Patrick Caswell, Jim Norman, Karen H. Vousden. Functions of wild type and mutant p53 [abstract]. In: Proceedings of the AACR 101st Annual Meeting 2010; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr SY29-01

The heat shock proteins (HSPs) are encoded by genes whose expression is substantially increased during stress conditions, such as heat shock, alcohol, inhibitors of energy metabolism, heavy metals, oxidative stress, fever or inflammation. During these conditions, HSPs increase cell survival by protecting and disaggregating stress‐labile proteins (Skowyra et al ., 1990), as well as the proteolysis of the damaged proteins (Wickner et al ., 1999). Under non‐stress conditions, HSPs have multiple housekeeping functions, such as folding and translocating newly synthesized proteins, activation of specific regulatory proteins, including transcription factors, replication proteins and kinases, protein degradation, protein signalling, including steroid hormone activation and tumour immunogenicity, and antigen presentation (for reviews see Helmbrecht et al ., 2000; Jolly and Morimoto, 2000). This broad spectrum of functions gave rise to the term ‘molecular chaperone’, an entity that acts to assist other proteins folding and maturating in the cell. It should also be emphasized that not all HSPs are molecular chaperones and not all chaperones are HSPs (Ellis and Hartl, 1999). HSPs are designated nomenclature according to their approximate molecular weight, e.g. the 70 kDa HSP is known as the molecular chaperone Hsp70. The 70 kDa heat shock‐related proteins comprise a family of highly conserved molecular chaperones that regulate a wide variety of cellular processes during normal and stress conditions (Boorstein et al ., 1994). Hsp70 is one of the most abundant of these proteins, accounting for as much as 1–2% of total cellular protein (Herendeen et al ., 1979). In humans, there are at least 11 distinct genes that code for Hsp70 isoforms, which are located on several different chromosomes (Tavaria et al ., 1996). The major, constitutively expressed hsp70 isoform is called hsc70 (gene product known as the clathrin‐uncoating ATPase or Hsp73) (Welch, 1992). The transcription of inducible forms of hsp70 or hsp72 are under …

The Molecular Epidemiology of Oncoproteins: Serum p53 Protein in Patients With Asbestosis

Two of the most common events in triple-negative breast cancers (TNBC) are mutation of the tumor suppressor gene TP53 and development of aneuploidy. This is often the case in aggressive and molecularly heterogeneous cancer subtypes such as TNBC, where ~80% of cases harbor a mutation in TP53. In addition to losing wild-type (WT) tumor-suppressive function, mutant p53 proteins are proposed to acquire gain-of-function (GOF) activity, leading to novel oncogenic phenotypes. Mutant p53 proteins can accumulate to high levels in tumors, and this stabilization is thought to be a pre-requisite for mutant p53 to exert its GOF effects. Mechanisms underlying the accumulation of mutant p53 are not well understood. To study mutant p53 GOF mechanisms and phenotypes, we used CRISPR/Cas9-mediated genome editing and developed two isogenic TNBC cell line models (one non-transformed and one tumor-derived) that express the most frequently occurring p53 missense mutations (R175H and R273H), are deficient for functional p53 protein (null), or retain the wild-type (WT) protein. In these engineered models, endogenous p53 expression is regulated by the native p53 promoter, thus providing a controlled system for rigorous functional experimentation across different p53 states. Through functional genomics analyses comparing isogenic TNBC cell lines, which initially differed only by the TP53 genotype, we have evaluated the relationship between mutant p53 and development of aneuploidy and determined that development of aneuploidy and not TP53 genotype contributed to several previously reported mutant p53 GOF phenotypes in vitro and in vivo1. Immunoblotting, immunofluorescence, and immunohistochemistry experiments revealed that mutant p53-expressing cell lines display a range of p53 protein levels, with several maintaining low levels of p53, similar to the cell lines which contain WT p53. In addition, mutant p53 levels correlate with aneuploidy, but not mean nuclear area or whole-genome doubling status, and protein levels increase over time in cell lines that become aneuploid. Using our various clonal cell lines that exhibit differing p53 protein stability, we are currently determining if p53 stability is dysregulated in aneuploid cell lines after copy number gain or loss of genes encoding critical p53 regulators, leading to the accumulation of the mutant protein. Analysis of cancer cell line genetic dependencies and human tumor data from The Cancer Genome Atlas (TCGA) has identified genetic and clinical correlates with increased mutant p53 protein levels. Utilizing biochemical and bioinformatic approaches, we aim to uncover mechanisms for increased mutant p53 stability and identify novel regulators of both WT and mutant p53 proteins. The dissection of mechanisms that contribute to stabilization of p53 protein will not only give insight into the regulation and tumor suppressive function of WT p53, but it has the potential for clinical translation in human cancers that have high-frequency p53 mutation and high levels of mutant p53 protein, as targeting mutant p53 through pharmacologic inhibition or antibody-based therapies is an active area of investigation. 1Article in press in Nature Communications Citation Format: Lindsay Redman-Rivera, Hailing Jin, Brian D Lehmann, Jennifer A Pietenpol. Using isogenic model systems to determine mechanisms regulating mutant p53 protein stability in breast cancer cells [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-09-01.

The p53 tumour suppressor protein is a labile transcription factor that is activated and stabilized in response to a wide range of cellular stresses, through a mechanism involving disruption of its interaction with MDM2, a negative regulatory partner. Induction of p53 by DNA damage additionally involves a series of phosphorylation and acetylation modifications, some of which are thought to regulate MDM2 binding. Here we report the effects of introducing mutations at several known or putative N-terminal phosphorylation sites on the transactivation function of p53. These studies highlight phosphorylation of Ser15, a key phosphorylation target during the p53 activation process, as being critical for p53-dependent transactivation. Biochemical data indicate that the mechanism by which phosphorylation of Ser15 stimulates p53-dependent transactivation occurs through increased binding to the p300 coactivator protein. The data also indicate that Ser15-dependent regulation of transactivation is independent of any involvement in modulating MDM2 binding, and that Ser15 phosphorylation alone is not sufficient to block the p53-MDM2 interaction.

Wild-type p53 protein was shown to bind specifically to DNA sequences within SV40 (Bargonetti et al. 1991), the human ribosomal gene cluster (RGC) (Kern et al. 1991a), and the murine muscle creatine kinase gene (MCK) (Zambetti et al. 1992). However, a direct comparison of these three sites was not performed. Here we demonstrate, by filter binding and gel mobility-shift assays, that wild-type p53 binds with similar affinities to MCK and RGC sites but less tightly to the SV40 site. We examined the effects of two candidate regulators of p53 function, SV40 large T antigen and oncogenic mutant p53, on the binding of wild-type p53 to RGC DNA. We show that wild-type T antigen prevents p53 from binding to the RGC site under all conditions tested. Moreover, two temperature-sensitive mutant SV40 T antigens, which fail to transform cells at the nonpermissive temperature, prevent p53 from binding to the RGC site at the permissive, but not at the restrictive, temperature. The ability of complexes containing wild-type p53 and tumor-derived mutant p53 proteins to bind to RGC DNA varies according to the position of the mutation. Complexes containing wild-type and either his175 or his273 mutant p53 proteins are completely unable to bind to the RGC DNA sequence. Interestingly, a complex containing wild-type p53 and the trp248 mutant p53 characteristic of Li-Fraumeni syndrome patients displays nearly wild-type levels of binding. Perhaps this mutant allele can be tolerated in these individuals because the wild-type mutant p53 complex maintains the ability to bind to DNA. Our data indicate that the oncogenic potential of both T antigen and some mutant p53 proteins is the result of their ability to block binding of wild-type p53 to DNA.

While the wild type form of p53 possesses strong tumor-suppressive activities, the p53 proteins that are commonly mutated in cancer often endow more malignant properties to the cancers they inhabit.1,2 There are several lines of evidence supporting such oncogenic gain of function of mutant p53. Compared with p53-null mice, knock-in mice harboring mutant p53 proteins display different and more metastatic tumor spectra. Such mutant proteins are frequently present at far higher levels than the wild-type protein in tumors; in fact, the p53 protein present in the knock-in mice accumulates in tumors despite being inherently unstable in normal tissues,3 suggesting that stabilization of mutant p53 protein is required for its oncogenic activity. Consistently, knockdown of mutant p53 protein in human cancer cell lines leads to reduced cell proliferation, invasion, motility, tumorigenicity and resistance to anticancer drugs.1,2 Since epidemiological studies indicate that high levels of mutant p53 proteins correlate with tumor aggressiveness and poorer outcomes, it is important to understand how mutant p53 is stabilized in tumors and how it can be eliminated. The Avantaggiati group in a recent issue of Cell Cycle have recently provided important new insight into this question.4 They demonstrated that glucose restriction (GR) results in deacetylation and destabilization of endogenous mutant p53, but not of wild-type p53 protein. As protein degradation is mediated primarily by two pathways; the 26S proteasome and autophagy, the authors sought to identify which pathway is responsible for the degradation. They found that while the proteasome inhibitor MG132 treatment does not abolish GR-induced mutant p53 degradation, knockdown of autophagy genes such as Beclin-1, ATG5, ATG7 or pharmacological inhibition of autophagy prevents the degradation. Further, mutant p53 physically interacts with components of the autophagy machinery in a GR-dependent manner, suggesting that mutant p53 is a substrate for autophagic degradation. Interestingly, a C-terminal acetylation-mimicking mutant version of p53 (G245A-6KQ) is resistant to GR-dependent degradation. Taken together, these findings suggest that GR induces posttranslational modifications of lysines within mutant p53 proteins, which subsequently target them for autophagy-dependent degradation. The authors next examined the effects of GR-induced degradation of mutant p53 on autophagy and cancer cell death. As indicated by two markers of autophagy (LC3 conversion and p62 degradation), GR activates this process, and the subsequent mutant p53 protein degradation leads to a maximal induction of autophagy and cell death. Consistent with their previous observations, expression of G245A-6KQ mutant p53 confers at least partial resistance to GR-induced cell death. Next, the authors used two mutant p53 mouse models to investigate the effects of a low carbohydrate (LC) diet on p53 stability and tumorigenicity in vivo. In line with their ex vivo data, in knock-in mice harboring the tumor-derived p53 mutation (A135V) placed on an LC diet, p53 protein is destabilized in mammary glands, ovaries and adipose tissues, while p53 in wild-type mice is stabilized. In xenografted mice, mutant p53 expressing cancer cells show enhanced tumorigenicity compared with those that are either p53-null or bearing wild-type p53.2 Strikingly, Rodriguez et al. found that an LC diet leads to a marked decrease in size of xenografted tumors with mutant p53, while this diet does not decrease tumors arising from the GR-resistant mutant p53G245A-6KQ expressing cells—it actually increases their growth. The findings of Rodriguez et al.4 raise several interesting questions. First, virtually every residue with the ~200 amino acid DNA-binding domain of p53 has been found to be mutated in different tumors, albeit with differing frequencies. Autophagy is activated when the proteasome fails to eliminate misfolded and aggregated proteins,5 and different mutant p53 proteins vary in their propensity for aggregation.6 Is there a correlation between mutant p53 proteins’ tendency to aggregate and their ability to be degraded after GR? Second, MDM2, the prime E3 ligase that ubiquitinates wild-type p53, cannot bind to p53 in Nutlin-3a-treated cells.7 Since the authors found that while mutant p53 is ubiquitinated after GR, Nutlin-3a actually blocks GR-induced mutant p53 degradation, what is the role of MDM2 in the setting of their study? Last, but not least, AMP-activated protein kinase (AMPK) has been known to regulate autophagy8 and may also be involved in GR-induced mutant p53 degradation. Is AMPK critical for the upstream signaling pathway to GR-induced mutant p53 degradation? Answers to these and other questions will provide the next chapters in this exciting story. (Fig. 1) Figure 1. Glucose restriction induces post-translational modifications of mutant p53 (ubiquitination, Ub-mutant p53; acetylation, Ac-mutant p53), which, in turn, leads to its degradation by activated autophagy and ensuing autophagic cell death ...

To facilitate the purification of wild type p53 protein, we established a recombinant p53 vaccinia viral expression system. Using this efficient eukaryotic expression vector, we found that the expressed p53 proteins retained their specific structural characteristics. A comparison between wild type and mutant p53 proteins showed the conservation of the typical subcellular localization and the expression of specific antigenic determinants. Furthermore, wild type p53 exhibited a typical binding with large T antigen, whereas no binding was detected with mutant p53. Both wild type and mutant p53 proteins were highly stable and constituted 5-7% of total protein expressed in the infected cells. These expression recombinant viruses offer a simple, valuable system for the purification of wild type and mutant p53 proteins that are expressed abundantly in eukaryotic cells.

Mutant p53 proteins accumulate to high levels in human tumors and in preneoplastic lesions in the skin and fallopian tube. However examination of tissues from mice and fish that are homozygous for mutant p53 surprisingly showed that the protein was present only at low levels except in the tumors that arose in these animals. The mutant protein did accumulate, however, following treatment with ionizing radiation in the same tissues in which the wild-type protein is induced. Here we study in detail the accumulation of mutant and wild-type p53 proteins following ionizing radiation in zebrafish embryos. We found that the mutant protein was induced by lower levels of radiation and reached higher levels than the wild-type protein. Morpholino knockdown of the zebrafish homologs of Mdm2 and Mdm4 caused dramatic accumulation of mutant p53 protein. The most remarkable results were observed by examining p53 protein levels over an extended time course. Mutant p53 protein increased and persisted for days after irradiation and this was accompanied by persistent elevation of phosphorylated H2AX (γH2AX), implying that the resolution of DNA damage signaling in these embryos is severely compromised by mutations in p53. Thus mutation in p53 results in an exaggerated and persistent damage response, which could in turn drive the process of cancer development as high levels of mutant p53 can act as an oncoprotein to drive invasion and metastasis.

Mutant p53 proteins (mutp53) often acquire oncogenic activities, conferring drug resistance and/or promoting cancer cell migration and invasion. Although it has been well established that such a gain of function is mainly achieved through interaction with transcriptional regulators, thereby modulating cancer-associated gene expression, how the mutp53 function is regulated remains elusive. Here we report that activating transcription factor 3 (ATF3) bound common mutp53 (e.g. R175H and R273H) and, subsequently, suppressed their oncogenic activities. ATF3 repressed mutp53-induced NFKB2 expression and sensitized R175H-expressing cancer cells to cisplatin and etoposide treatments. Moreover, ATF3 appeared to suppress R175H- and R273H-mediated cancer cell migration and invasion as a consequence of preventing the transcription factor p63 from inactivation by mutp53. Accordingly, ATF3 promoted the expression of the metastasis suppressor SHARP1 in mutp53-expressing cells. An ATF3 mutant devoid of the mutp53-binding domain failed to disrupt the mutp53-p63 binding and, thus, lost the activity to suppress mutp53-mediated migration, suggesting that ATF3 binds to mutp53 to suppress its oncogenic function. In line with these results, we found that down-regulation of ATF3 expression correlated with lymph node metastasis in TP53-mutated human lung cancer. We conclude that ATF3 can suppress mutp53 oncogenic function, thereby contributing to tumor suppression in TP53-mutated cancer.

p53 protein conformation is an important determinant of its localization and activity. Changes in p53 conformation can be monitored by reactivity with wild-type conformation-specific (pAb-1620) or mutant conformation-specific (pAb-240) p53 antibodies. Wild-type p53 accumulated in a mutant (pAb-240 reactive) form when its proteasome-dependent degradation was blocked during recovery from stress treatment and in cells co-expressing p53 and MDM2. This suggests that conformational change precedes wild-type p53 degradation by the proteasome. MDM2 binding to the p53 N terminus could induce a conformational change in wild-type p53. Interestingly, this conformational change was opposed by heat-shock protein 90 and did not require the MDM2 RING-finger domain and p53 ubiquitination. Finally, ubiquitinated p53 accumulated in a pAb-240 reactive form when p53 degradation was blocked by proteasome inhibition, and a p53-ubiquitin fusion protein displayed a mutant-only conformation in MDM2-null cells. These results support a model in which MDM2 binding induces a conformational change that is opposed by heat-shock protein 90 and precedes p53 ubiquitination. The covalent attachment of ubiquitin may "lock" p53 in a mutant conformation in the absence of MDM2-binding and prior to its degradation by the proteasome.

Regulation of Transcription Functions of the p53 Tumor Suppressor by the mdm-2 Oncogene

Background:Transforming growth factor-beta (TGF-β) induces the epithelial-to-mesenchymal transition (EMT) leading to increased cell plasticity at the onset of cancer cell invasion and metastasis. Mechanisms involved in TGF-β-mediated EMT and cell motility are unclear. Recent studies showed that p53 affects TGF-β/SMAD3-mediated signalling, cell migration, and tumorigenesis. We previously demonstrated that Nox4, a Nox family NADPH oxidase, is a TGF-β/SMAD3-inducible source of reactive oxygen species (ROS) affecting cell migration and fibronectin expression, an EMT marker, in normal and metastatic breast epithelial cells. Our present study investigates the involvement of p53 in TGF-β-regulated Nox4 expression and cell migration.Methods:We investigated the effect of wild-type p53 (WT-p53) and mutant p53 proteins on TGF-β-regulated Nox4 expression and cell migration. Nox4 mRNA and protein, ROS production, cell migration, and focal adhesion kinase (FAK) activation were examined in three different cell models based on their p53 mutational status. H1299, a p53-null lung epithelial cell line, was used for heterologous expression of WT-p53 or mutant p53. In contrast, functional studies using siRNA-mediated knockdown of endogenous p53 were conducted in MDA-MB-231 metastatic breast epithelial cells that express p53-R280K and MCF-10A normal breast cells that have WT-p53.Results:We found that WT-p53 is a potent suppressor of TGF-β-induced Nox4, ROS production, and cell migration in p53-null lung epithelial (H1299) cells. In contrast, tumour-associated mutant p53 proteins (R175H or R280K) caused enhanced Nox4 expression and cell migration in both TGF-β-dependent and TGF-β-independent pathways. Moreover, knockdown of endogenous mutant p53 (R280K) in TGF-β-treated MDA-MB-231 metastatic breast epithelial cells resulted in decreased Nox4 protein and reduced phosphorylation of FAK, a key regulator of cell motility. Expression of WT-p53 or dominant-negative Nox4 decreased TGF-β-mediated FAK phosphorylation, whereas mutant p53 (R280K) increased phospho-FAK. Furthermore, knockdown of WT-p53 in MCF-10A normal breast epithelial cells increased basal Nox4 expression, whereas p53-R280K could override endogenous WT-p53 repression of Nox4. Remarkably, immunofluorescence analysis revealed MCF-10A cells expressing p53-R280K mutant showed an upregulation of Nox4 in both confluent and migrating cells.Conclusions:Collectively, our findings define novel opposing functions for WT-p53 and mutant p53 proteins in regulating Nox4-dependent signalling in TGF-β-mediated cell motility.

The tumor suppressor p53 is mutated in over 50% of human cancers leading not only to loss of wild-type p53 function, but also to gain-of-function (GOF) mutant p53 which acts as an oncogene. Mutant p53 is expressed at very high levels and can inhibit residual wild-type p53 activity as well as the function of p53 family member proteins such as p73 or p63. Therefore, targeting mutant p53 by either restoring the p53 pathway or depleting its GOF is an attractive strategy for cancer therapy. NCI-8 is a small molecule compound with dual abilities to induce p53 signal pathway and destabilize mutant p53 protein (deplete GOF) in mutant p53 expressing colorectal cancer cells. It remains unclear how NCI-8 regulates mutant p53 protein degradation. ERK2 is a member of the MAP kinase family which plays a critical role in regulating cell growth and differentiation by phosphorylating substrates including wild-type p53. We demonstrate that NCI-8-induces mutant p53 protein degradation via activation of ERK2 signaling. We observed a sustained phosphorylation of ERK2 upon NCI-8 treatment of cancer cells, but not in normal cells. MEK inhibitor U0126 treatment completely blocked NCI-8-mediated phosphorylation of ERK2 in cancer cells. These results, taken together, suggest that NCI-8 activates the ERK2 signaling pathway in cancer cells. There was a correlation between mutant p53 degradation and phosphorylation of ERK2 in cancer cells treated with NCI-8. We further examined the role of ERK2 phosphorylation in NCI-8- mediated mutant p53 protein degradation. Cancer cells were treated with U0126 to block ERK2 phosphorylation or transiently transfected with siRNA to knockdown ERK2. U0126 treatment or knockdown of ERK2 rescued mutant p53 from NCI-8-mediated protein degradation, suggesting that NCI-8-mediated phosphorylation of ERK2 is required for mutant p53 degradation. We further found that U0126 treatment inhibited NCI-8 induction of p21, puma and Noxa expressions in cancer cells. Consistently, transient over-expression of exogenous mutant p53 blocked NCI-8-mediated restoration of p53 pathway signaling in mutant-p53 expressing cancer cells. These results suggest that NCI-8 restores p53 signal pathway via ERK2-dependent mutant p53 protein degradation. Correlated with the U0126-rescue of mutant p53 protein, the percentage of cells with sub-G1 content induced by NCI-8 is decreased in response to U0126 treatment in cancer cells, suggesting that cell death induced by NCI-8 depends on ERK2-mediated mutant p53 degradation. Furthermore, combinational indices showed that U0126 antagonized NCI-8- induced cell death, but no antagonism was found in combinational treatment of NCI-8 and EGFR inhibitors in cancer cells. Our data indicate an important role of NCI-8-phosphorylated ERK2 in regulation of mutant p53 protein degradation and cell death in cancer cells, and provide a rationale for clinical testing of NCI-8 and ERK2 pathway-related factors in cancer therapy. Citation Format: Shengliang Zhang, Lanlan Zhou, David T. Dicker, Wafik S. EL-Deiry. Small molecule compound NCI-8 induces ERK2-dependent mutant-p53 protein degradation. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2640. doi:10.1158/1538-7445.AM2015-2640