Mutant P53 Gain Of Function Research Articles

Abstract A04: Alpha-1-Antitrypsin is a secreted protein driven by mutant p53 and associated with EMT, migration and invasion in-vivo

Abstract Half of all human tumors harbor mutations in TP53 gene. Such mutations not only lead to the expression of a protein unable to impart its normal tumor suppressor ability, but also endow the mutant p53 protein with newly acquired gain-of-function (GOF) properties that drive invasion and metastasis. Although it is widely established that mutant p53-expressing tumors have a high capacity to metastasize and are associated with poor outcomes, the key molecular mechanism utilized by mutant p53 are largely unknown. Since, p53 mutations are predominantly found in pre-invasive stages of lung adenocarcinomas, the identification of critical pathways that mediate mutant p53 gain of function (GOF) properties may uncover new targets for cancer therapy. In this study, we investigated the global impact of mutant p53 induced secretome on cancer cell proteome. A1AT is a secreted protein encoded by SERPINA1, a secreted serine protease inhibitor that neutralizes the effect of proteases, and A1AT involvement in mutant p53 driven tumorigenesis is unclear. Methodology: We have developed a panel of H1299 lung adenocarcinoma derivatives that can inducibly re-express various mutant p53 proteins into a p53 null background. Expression levels were assessed by western blot analyses and real time PCR. Using iTRAQ (isobaric tag for relative and absolute quantification) and Liquid chromatography tandem mass spectrometry (LC-MS/MS) analyses, the proteome of conditioned media from H1299 cells expressing either induced p53 mutants (R175H and R248) or their isogenic un-induced counterparts were identified and quantified. Mutant p53 and p63 bindings sites were analyzed by chromatin immune-precipitation (ChIP) assays. In vitro migration/invasion and in vivo Chicken Chorio-allantoic membrane (CAM) invasion were performed in A1AT knockdown lung adenocarcinoma cells harboring p53 mutation. Public microarray, Kaplan-Meir datasets of human non-small cell lung carcinoma (NSCLCs) and A1AT immunohistochemistry (IHC) of stage I-III lung adenocarcinoma patients tissue microarray (TMA) samples (n=107) with matching normal were analyzed to correlate A1AT with prognosis and clinical significance. Results: Induction of mutant p53 in H1299 cells drove an invasive phenotype through the release of a pro-invasive secretome, providing a novel avenue through which mutant p53 may be driving invasion and metastasis. Through proteomic characterization of induced conditioned medium from H1299 versus un-induced counterparts, we identified A1AT as a novel secreted mediator and a putative metastasis marker of mutant p53 tumors. To determine the role for A1AT as a downstream mediator of mutant p53 oncogenic pathways, we developed a double inducible H1299 system whereby we can simultaneously (i) induce expression of EI-H1299 R248Q mutant p53 and (ii) induce knockdown of the expression of its target A1AT. Remarkably, silencing the expression of A1AT significantly attenuated mutant p53 dependent migration and invasion both in vitro and in vivo. Importantly, knockdown of A1AT did not alter the basal level of motility in the absence of induced mutant p53, suggesting that the role of A1AT is specific to the mutant p53 pathway. Knockdown of A1AT significantly altered epithelial-mesenchymal transition (EMT) markers expression and reduced the ability of p53 mutant cells to grow in an anchorage independent environment. Treatment of cells with conditioned medium containing secreted A1AT enhanced the cell invasion while A1AT knockdown elicited the opposite effect. A1AT-blocking antibody attenuated the mutant p53 driven migration and invasion, strongly suggesting that mutant p53 is using secretory gene targets to potentiate its gain-of-function. In addition, A1AT intracellular levels were directly regulated by mutant p53 through the involvement with p63, indicating that A1AT is a direct target of mutant p53 transcriptomic regulation. In public microarray dataset, A1AT mRNA expression was higher in lung adenocarcinoma (AC) and associated with shorter overall survival. Consistent with these findings, immunohistochemistry of 107 lung adenocarcinoma TMA showed upregulation of A1AT expression compared with matching normal tissues. The A1AT signal was cytoplasmic in tumor but not in the core of the tumor. A1AT overexpression in tumor cells correlated with increased tumor size (P<0.046), and strongly with increased tumor progression (advanced T stage; P<0.0006) and shorter survival (P<0.001). In addition, we detected a clear correlation between elevated p53 staining and high levels of A1AT (P<0.018). This indicates A1AT expression is predominantly driven by mutant p53 gain-of-function. Conclusion: We conclude A1AT is an essential mediator of mutant p53 driven gain-of-function properties and our results highlight crucial roles of A1AT in driving oncogenic transformations. We suggest that A1AT is a potential therapeutic target in lung adenocarcinoma patient tumors expressing mutant p53. Citation Format: Reshma Shakya, Andrew G. Turner, Wendy Cooper, Noor A. Lokman, Carmela Ricciardelli, Gerard Tarulli, Paul M. Neilsen, David F. Callen. Alpha-1-Antitrypsin is a secreted protein driven by mutant p53 and associated with EMT, migration and invasion in-vivo. [abstract]. In: Proceedings of the AACR Special Conference: Developmental Biology and Cancer; Nov 30-Dec 3, 2015; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(4_Suppl):Abstract nr A04.

Gain-of-function miRNA signature by mutant p53 associates with poor cancer outcome.

Missense mutation of p53 not only impairs its tumor suppression function, but also causes oncogenic gain of function (GOF). The molecular underpinning of mutant p53 (mutp53) GOF is not fully understood, especially for the potential roles of non-coding genes. Here we identify the microRNA expression profile (microRNAome) of mutp53 on Arg282 by controlled microarray experiments, and clarify the prognostic significance of mutp53-regulated miRNAs in cancers. A predominant repression effect on miRNA expression was found for mutant p53, with 183 significantly downregulated and only 12 upregulated miRNAs. Mutp53 and wild-type (wtp53) commonly upregulate let-7i, and other two miRNAs were upregulated by wtp53 but repressed by mutp53 (miR-610 and miR-3065–3p). Based the mutp53-regulated miRNA signature, a non-negative matrix factorization (NMF) model classified gastric cancer (GC) cases into subgroups with significantly different Disease-free survival (Kaplan-Meier test, P = 0.013). In contrast, the NMF model based on all miRNAs did not associate with cancer outcome. The mutp53 miRNA signature associated with the outcomes of breast cancer (P = 0.024) and hepatocellular cancer (P = 0.012). The miRPath analysis revealed that mutp53-suppressed miRNAs associate with Hippo, TGF-β and stem cell signaling pathways. Taken together, our results highlight a miRNA-mediated GOF mechanism of mutant p53 on Arg282, and suggest the prognostic potential of mutp53-associated miRNA signature.

Mutant p53: Multiple Mechanisms Define Biologic Activity in Cancer.

The functional importance of p53 as a tumor suppressor gene is evident through its pervasiveness in cancer biology. The p53 gene is the most commonly altered gene in human cancer; however, not all genetic alterations are biologically equivalent. The majority of alterations involve p53 missense mutations that result in the production of mutant p53 proteins. Such mutant p53 proteins lack normal p53 function and may concomitantly gain novel functions, often with deleterious effects. Here, we review characterized mechanisms of mutant p53 gain of function in various model systems. In addition, we review mutant p53 addiction as emerging evidence suggests that tumors may depend on sustained mutant p53 activity for continued growth. We also discuss the role of p53 in stromal elements and their contribution to tumor initiation and progression. Lastly, current genetic mouse models of mutant p53 in various organ systems are reviewed and their limitations discussed.

DNA damage-induced ephrin-B2 reverse signaling promotes chemoresistance and drives EMT in colorectal carcinoma harboring mutant p53.

Mutation in the TP53 gene positively correlates with increased incidence of chemoresistance in different cancers. In this study, we investigated the mechanism of chemoresistance and epithelial-to-mesenchymal transition (EMT) in colorectal cancer involving the gain-of-function (GOF) mutant p53/ephrin-B2 signaling axis. Bioinformatic analysis of the NCI-60 data set and subsequent hub prediction identified EFNB2 as a possible GOF mutant p53 target gene, responsible for chemoresistance. We show that the mutant p53-NF-Y complex transcriptionally upregulates EFNB2 expression in response to DNA damage. Moreover, the acetylated form of mutant p53 protein is recruited on the EFNB2 promoter and positively regulates its expression in conjunction with coactivator p300. In vitro cell line and in vivo nude mice data show that EFNB2 silencing restores chemosensitivity in mutant p53-harboring tumors. In addition, we observed high expression of EFNB2 in patients having neoadjuvant non-responder colorectal carcinoma compared with those having responder version of the disease. In the course of deciphering the drug resistance mechanism, we also show that ephrin-B2 reverse signaling induces ABCG2 expression after drug treatment that involves JNK-c-Jun signaling in mutant p53 cells. Moreover, 5-fluorouracil-induced ephrin-B2 reverse signaling promotes tumorigenesis through the Src-ERK pathway, and drives EMT via the Src-FAK pathway. We thus conclude that targeting ephrin-B2 might enhance the therapeutic potential of DNA-damaging chemotherapeutic agents in mutant p53-bearing human tumors.

Small-Molecule NSC59984 Restores p53 Pathway Signaling and Antitumor Effects against Colorectal Cancer via p73 Activation and Degradation of Mutant p53.

The tumor-suppressor p53 prevents cancer development via initiating cell-cycle arrest, cell death, repair, or antiangiogenesis processes. Over 50% of human cancers harbor cancer-causing mutant p53. p53 mutations not only abrogate its tumor-suppressor function, but also endow mutant p53 with a gain of function (GOF), creating a proto-oncogene that contributes to tumorigenesis, tumor progression, and chemo- or radiotherapy resistance. Thus, targeting mutant p53 to restore a wild-type p53 signaling pathway provides an attractive strategy for cancer therapy. We demonstrate that small-molecule NSC59984 not only restores wild-type p53 signaling, but also depletes mutant p53 GOF. NSC59984 induces mutant p53 protein degradation via MDM2 and the ubiquitin-proteasome pathway. NSC59984 restores wild-type p53 signaling via p73 activation, specifically in mutant p53-expressing colorectal cancer cells. At therapeutic doses, NSC59984 induces p73-dependent cell death in cancer cells with minimal genotoxicity and without evident toxicity toward normal cells. NSC59984 synergizes with CPT11 to induce cell death in mutant p53-expressing colorectal cancer cells and inhibits mutant p53-associated colon tumor xenograft growth in a p73-dependent manner in vivo. We hypothesize that specific targeting of mutant p53 may be essential for anticancer strategies that involve the stimulation of p73 in order to efficiently restore tumor suppression. Taken together, our data identify NSC59984 as a promising lead compound for anticancer therapy that acts by targeting GOF-mutant p53 and stimulates p73 to restore the p53 pathway signaling.

BAG2 promotes tumorigenesis through enhancing mutant p53 protein levels and function.

Tumor suppressor p53 is the most frequently mutated gene in tumors. Many mutant p53 (mutp53) proteins promote tumorigenesis through the gain-of-function (GOF) mechanism. Mutp53 proteins often accumulate to high levels in tumors, which is critical for mutp53 GOF. Its underlying mechanism is poorly understood. Here, we found that BAG2, a protein of Bcl-2 associated athanogene (BAG) family, promotes mutp53 accumulation and GOF in tumors. Mechanistically, BAG2 binds to mutp53 and translocates to the nucleus to inhibit the MDM2-mutp53 interaction, and MDM2-mediated ubiquitination and degradation of mutp53. Thus, BAG2 promotes mutp53 accumulation and GOF in tumor growth, metastasis and chemoresistance. BAG2 is frequently overexpressed in tumors. BAG2 overexpression is associated with poor prognosis in patients and mutp53 accumulation in tumors. These findings revealed a novel and important mechanism for mutp53 accumulation and GOF in tumors, and also uncovered an important role of BAG2 in tumorigenesis through promoting mutp53 accumulation and GOF.

Abstract 1221: Pontin, a new mutant p53 binding protein, promotes gain-of-function of mutant p53

Abstract Tumor suppressor p53 is frequently mutated in human cancers. Many tumor-associated mutant p53 (mutp53) proteins gain new functions in promoting tumorigenesis, defined as gain of function (GOF). The mechanisms for mutp53 GOF are not well-understood. Mutp53 has been reported to interact with some proteins and it has been suggested that some interactions of mutp53 with its binding partners are important for mutp53 GOF through regulating the levels or functions of mutp53 and/or its binding proteins. To understand the molecular mechanisms for mutp53 GOF, we searched for mutp53 binding proteins in thymic lymphomas of R172H mutp53 knock-in (p53R172H/R172H) mice with significant mutp53 protein accumulation by employing an unbiased immunoprecipitation (IP) combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS) screening approach. Normal tissues of p53R172H/R172H mice with low mutp53 levels and tumors of p53-/- mice were used as controls. Using this approach, we identified Pontin, a highly conserved AAA+ ATPase important for various cellular functions, as a new mutp53 binding protein. This Pontin-mutp53 is conserved in both humans and mice. Importantly, this Pontin-mutp53 interaction promotes mutp53 GOF in invasion, migration, and anchorage-independent growth of tumor cells. Furthermore, we found that the C-terminus of Pontin is required for Pontin-mutp53 interaction and mutp53 GOF. The ATPase domain in the C-terminus of Pontin is crucial for its promoting effect on mutp53 GOF; blocking the ATPase activity of Pontin by a Pontin specific ATPase inhibitor or an ATPase deficient dominant-negative Pontin expression vector greatly diminished mutp53 GOF. We found that Pontin was largely co-localized with mutp53 in the nucleus and promotes mutp53 GOF through regulation of mutp53 transcriptional activity; knock-down of Pontin abolished the transcriptional regulation of mutp53 towards a group of genes involved in many important signaling pathways, including gap junction signaling, IGF-1 signaling, EGF signaling etc. Furthermore, overexpression of Pontin is associated with poor survival in cancer patients, especially those containing mutp53 in tumors. This work demonstrates that Pontin promotes mutp53 GOF in tumorigenesis in an ATPase-dependent manner, and raises interesting and intriguing possibilities to block mutp53 GOF in tumors through targeting Pontin or its ATPase activity. In summary, our results highlight an important role and mechanism for Pontin, a new p53 partner, in promoting mutp53 GOF in tumorigenesis. Citation Format: Yuhan Zhao, Cen Zhang, Xuetian Yue, Xiaoyan Li, Juan Liu, Haiyang Yu, Qifeng Yang, Zhaohui Feng, Wenwei Hu. Pontin, a new mutant p53 binding protein, promotes gain-of-function of mutant p53. [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 1221. doi:10.1158/1538-7445.AM2015-1221

Pontin, a novel interactor of mutant p53 that promotes mutant p53 gain of function

ABSTRACTMany tumor-associated mutant p53 proteins gain new activities to promote tumorigenesis, which is termed as gain of function of mutant p53 (GOF). Currently, the mechanisms of mutant p53 GOF remain elusive. We recently identified the AAA+ ATPase pontin as a critical regulator of mutant p53 GOF, thus providing a novel mechanism for mutant p53 GOF.

The predictive value of ERCC1 and p53 for the effect of panobinostat and cisplatin combination treatment in NSCLC.

Cisplatin is one of the most common chemotherapeutic drugs for non-small cell lung cancer (NSCLC). However, the response rate is limited because of drug resistance. Histone deacetylase inhibitors (HDACis), which can alter DNA accessibility by regulating chromatin structure and inducing apoptosis, exhibit a synergistic action with cisplatin. However, no biomarkers that can predict the efficacy of the combination of HDACis and cisplatin have been reported. Our study found that panobinostat, an HDAC inhibitor, increased the cisplatin sensitivity of several NSCLC cell lines with low ERCC1 expression but not those with high ERCC1 expression or gain-of-function (GOF) p53 mutation despite of ERCC1 expression level. ERCC1 knockdown increased the cisplatin sensitivity of NSCLC cell lines with high ERCC1 expression without GOF p53 mutations. In addition, in low ERCC1 expression NSCLC cell lines, knockdown of GOF mutant p53 enhanced cisplatin sensitivity. Further double knockdown of ERCC1 and GOF mutant p53 but not ERCC1 knockdown alone increased the cisplatin sensitivity of cells with both high ERCC1 expression and GOF p53 mutations. Therefore, this study demonstrated that ERCC1 expression combined with p53 mutation status may determine the efficacy of cisplatin and HDACi combined therapy and guide the development of future NSCLC therapies.

Mutant p53 - Heat Shock Response Oncogenic Cooperation: A New Mechanism of Cancer Cell Survival.

The main tumor suppressor function of p53 as a “guardian of the genome” is to respond to cellular stress by transcriptional activation of apoptosis, growth arrest, or senescence in damaged cells. Not surprisingly, mutations in the p53 gene are the most frequent genetic alteration in human cancers. Importantly, mutant p53 (mutp53) proteins not only lose their wild-type tumor suppressor activity but also can actively promote tumor development. Two main mechanisms accounting for mutp53 proto-oncogenic activity are inhibition of the wild-type p53 in a dominant-negative fashion and gain of additional oncogenic activities known as gain-of-function (GOF). Here, we discuss a novel mechanism of mutp53 GOF, which relies on its oncogenic cooperation with the heat shock machinery. This coordinated adaptive mechanism renders cancer cells more resistant to proteotoxic stress and provides both, a strong survival advantage to cancer cells and a promising means for therapeutic intervention.

Pontin, a new mutant p53-binding protein, promotes gain-of-function of mutant p53.

Tumor-suppressor p53 is frequently mutated in human cancers. Many tumor-associated mutant p53 (mutp53) proteins gain new functions in promoting tumorigenesis, defined as gain-of-function (GOF). The mechanisms for mutp53 GOF are not well understood. Here, we report Pontin, a highly conserved AAA+ ATPase important for various cellular functions, as a new mutp53-binding protein. This Pontin-mutp53 interaction promotes mutp53 GOF in invasion, migration and anchorage-independent growth of tumor cells. The ATPase domain of Pontin is crucial for its promoting effect on mutp53 GOF; blocking the ATPase activity of Pontin by a Pontin-specific ATPase inhibitor or an ATPase-deficient dominant-negative Pontin expression vector greatly diminished mutp53 GOF. Pontin promotes mutp53 GOF through regulation of mutp53 transcriptional activity; knockdown of Pontin abolished the transcriptional regulation of mutp53 toward a group of genes. Furthermore, overexpression of Pontin in tumors is associated with the poor survival in cancer patients, especially those containing mutp53. Our results highlight an important role and mechanism for Pontin, a new mutp53 partner, in promoting mutp53 GOF in tumorigenesis.

Abstract 3234: Covalent capture of protein binding partners using an azide-tagged, photo-reactive stapled alpha helical p53 peptide

Abstract Mutations in the DNA binding region of tumor suppressor p53 render it transcriptionally inactive (dominant negative) but can also manifest in transcriptionally-independent gain-of-function (GOF) effects, possibly due to mutp53 interacting with other, as yet, unknown targets. Identification of these targets would help to elucidate the pathways and vulnerabilities involved in mutp53 GOF. Covalently linking a known handle to these unknown proteins is one method to aid in their isolation and identification. A hydrocarbon-stapled alpha helical peptide of the p53 transactivation domain (residues 14-29), SAH-p53-8, has been shown to strongly interact with known p53 targets that utilize this domain for binding (HDM2, Kd = 55 nM, HDMX, Kd = 2.3 nM). Substitution of a key interacting residue for the unnatural amino acid benzoylphenylalanine (Bpa) results in a stapled peptide that retains the biochemical properties of SAH-p53-8 (affinities of 38.1 nM and 58.3 nM, respectively) and can covalently capture known targets via photochemical reaction. The introduction of an azido group - a versatile, biochemically-inert capping group - to the N-terminus of this photo-reactive peptide allows for the attachment of various tags to the covalently bound protein(s) via a copper-catazlyzed azide alkyne cycloaddition. Tags include both fluorophores for visualization and affinity tags for protein isolation. This poster will cover the biochemical proof-of-principle of this protein isolation method and exploration into more complex cell lysate environments. Citation Format: Amanda L. Whiting, Joe J. Mitala, Kathryn M. Headley, Joe Reilly, Bethanie L. Morrison, Kevin A. Murray, Federico Bernal. Covalent capture of protein binding partners using an azide-tagged, photo-reactive stapled alpha helical p53 peptide. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3234. doi:10.1158/1538-7445.AM2014-3234

The Activating Transcription Factor 3 Protein Suppresses the Oncogenic Function of Mutant p53 Proteins

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.

Mutant p53 gain of function can be at the root of dedifferentiation of human osteosarcoma MG63 cells into 3AB-OS cancer stem cells

Osteosarcoma is a highly metastatic tumor affecting adolescents, for which there is no second-line chemotherapy. As suggested for most tumors, its capability to overgrow is probably driven by cancer stem cells (CSCs), and finding new targets to kill CSCs may be critical for improving patient survival. TP53 is the most frequently mutated tumor suppressor gene in cancers and mutant p53 protein (mutp53) can acquire gain of function (GOF) strongly contributing to malignancy. Studies thus far have not shown p53-GOF in osteosarcoma. Here, we investigated TP53 gene status/role in 3AB-OS cells—a highly aggressive CSC line previously selected from human osteosarcoma MG63 cells—to evaluate its involvement in promoting proliferation, invasiveness, resistance to apoptosis and stemness. By RT-PCR, methylation-specific PCR, fluorescent in situ hybridization, DNA sequence, western blot and immunofluorescence analyses, we have shown that—in comparison with parental MG63 cells where TP53 gene is hypermethylated, rearranged and in single copy—in 3AB-OS cells, TP53 is unmethylated, rearranged and in multiple copies, and mutp53 (p53-R248W/P72R) is post-translationally modified and with nuclear localization. p53-R248W/P72R-knockdown by short-interfering RNA reduced the growth and replication rate of 3AB-OS cells, markedly increasing cell cycle inhibitor levels and sensitized 3AB-OS cells to TRAIL-induced apoptosis by DR5 up-regulation; moreover, it strongly decreased the levels of stemness and invasiveness genes. We have also found that the ectopic expression of p53-R248W/P72R in MG63 cells promoted cancer stem-like features, as high proliferation rate, sphere formation, clonogenic growth, high migration and invasive ability; furthermore, it strongly increased the levels of stemness proteins. Overall, the findings suggest the involvement of p53-R248W/P72R at the origin of the aberrant characters of the 3AB-OS cells with the hypothesis that its GOF can be at the root of the dedifferentiation of MG63 cells into CSCs.

Tumour-associated mutant p53 drives the Warburg effect

Tumor cells primarily utilize aerobic glycolysis for energy production, a phenomenon known as the Warburg effect. Its mechanism is not well-understood. The tumor suppressor gene p53 is frequently mutated in tumors. Many tumor-associated mutant p53 (mutp53) proteins not only lose tumor suppressive function, but also gain new oncogenic functions that are independent of wild type p53, defined as mutp53 gain-of-function (GOF). Here we show that tumor-associated mutp53 stimulates the Warburg effect in cultured cells and mutp53 knock-in mice as a new mutp53 GOF. Mutp53 stimulates the Warburg effect through promoting GLUT1 translocation to plasma membrane, which is mediated by the activated RhoA and its downstream effector ROCK. Inhibition of the RhoA/ROCK/GLUT1 signaling largely abolishes mutp53 GOF in stimulating the Warburg effect. Furthermore, inhibition of glycolysis in tumor cells greatly compromises mutp53 GOF in promoting tumorigenesis. Thus, our results reveal a new mutp53 GOF and a mechanism for controlling the Warburg effect.

Spliced MDM2 isoforms promote mutant p53 accumulation and gain-of-function in tumorigenesis

Tumor suppressor p53 is frequently mutated in tumors. Mutant p53 (Mutp53) proteins often gain new activities in promoting tumorigenesis, defined as gain-of-function (GOF). Mutp53 often accumulates at high levels in tumors, which promotes mutp53 GOF in tumorigenesis. The mechanism of mutp53 accumulation is poorly understood. Here we find that MDM2 isoforms promote mutp53 accumulation in tumors. MDM2 isoform B (MDM2-B), the MDM2 isoform most frequently over-expressed in human tumors, interacts with full-length MDM2 to inhibit MDM2-mediated mutp53 degradation, promoting mutp53 accumulation and GOF in tumorigenesis. Furthermore, MDM2-B over-expression correlates with mutp53 accumulation in human tumors. In mutp53 knock-in mice, a MDM2 isoform similar to human MDM2-B is over-expressed in the majority of tumors, which promotes mutp53 accumulation and tumorigenesis. Thus, over-expression of MDM2 isoforms promotes mutp53 accumulation in tumors, contributing to mutp53 GOF in tumorigenesis. Furthermore, promoting mutp53 accumulation and GOF is an important mechanism by which MDM2 isoforms promote tumorigenesis.

Differential regulation of the REGγ–proteasome pathway by p53/TGF-β signalling and mutant p53 in cancer cells

Proteasome activity is frequently enhanced in cancer to accelerate metastasis and tumorigenesis. REGγ, a proteasome activator known to promote p53/p21/p16 degradation, is often overexpressed in cancer cells. Here we show that p53/TGF-β signalling inhibits the REGγ–20S proteasome pathway by repressing REGγ expression. Smad3 and p53 interact on the REGγ promoter via the p53RE/SBE region. Conversely, mutant p53 binds to the REGγ promoter and recruits p300. Importantly, mutant p53 prevents Smad3/N-CoR complex formation on the REGγ promoter, which enhances the activity of the REGγ–20S proteasome pathway and contributes to mutant p53 gain of function. Depletion of REGγ alters the cellular response to p53/TGF-β signalling in drug resistance, proliferation, cell cycle progression and proteasome activity. Moreover, p53 mutations show a positive correlation with REGγ expression in cancer samples. These findings suggest that targeting REGγ–20S proteasome for cancer therapy may be applicable to human tumours with abnormal p53/Smad protein status. Furthermore, this study demonstrates a link between p53/TGF-β signalling and the REGγ–20S proteasome pathway, and provides insight into the REGγ/p53 feedback loop.

Not all p53 gain-of-function mutants are created equal

Not all p53 gain-of-function mutants are created equal

Mutant p53 Prolongs NF-κB Activation and Promotes Chronic Inflammation and Inflammation-Associated Colorectal Cancer

The tumor suppressor p53 is frequently mutated in human cancer. Common mutant p53 (mutp53) isoforms can actively promote cancer through gain-of-function (GOF) mechanisms. We report that mutp53 prolongs TNF-α-induced NF-κB activation in cultured cells and intestinal organoid cultures. Remarkably, when exposed to dextran sulfate sodium, mice harboring a germline p53 mutation develop severe chronic inflammation and persistent tissue damage, and are highly prone to inflammation-associated colon cancer. This mutp53 GOF is manifested by rapid onset of flat dysplastic lesions that progress to invasive carcinoma with mutp53 accumulation and augmented NF-κB activation, faithfully recapitulating features frequently observed in human colitis-associated colorectal cancer (CAC). These findings might explain the early appearance of p53 mutations in human CAC.

Abstract 2171: A new small molecule compound restoring p53 pathway induces cell death via active p73 and degradation of mutant p53 in colorectal cancer cells.

Abstract Tumor suppressor p53 is critical for protection against cancer. Over 50% of human cancers harbor mutant p53 which is hyper-stabilized and blocks p53 family member p73/p63 activity. Mutations not only abrogate the p53 tumor suppressor function, but also endow mutant p53 with gain-of-function (GOF) as pro-oncogene which contributes to tumor progression and chemotherapy resistance. Targeting mutant p53 to restore the p53 pathway is a promising strategy for cancer therapy. Small molecule restoring p53 pathway is mostly based on conformational change in mutant p53 or upregulation of wild-type p53. It is barely reported about small molecule compound with ability to both restore p53 pathway and deplete mutant p53 GOF. In this study, we describe a new small molecular compound, NCI-8 that specifically depletes mutant p53 protein and activates p73 to induce p53 responsive transcriptional activity and cell death in mutant p53 colorectal cancer cells. NCI-8 treatment increased p53 responsive bioluminescence and p53 target gene expression such as P21, Puma and DR5 specifically in mutant p53 colorectal cancer cells. Accompanied with p53 pathway restoration, NCI-8 down-regulated mutant p53 in cancer cells with no evident wild-type conformational change in mutant p53. NCI-8- mediated down-regulation of mutant p53 was rescued by MG132, a proteasome inhibitor and nutlin-3, an MDM2 inhibitor. NCI-8 induced ubiquitination of mutant p53. Taken together these results indicate that NCI-8 induces mutant p53 protein degradation via an MDM2-mediated ubiquitin-proteasome pathway. Further, we examined the role of p73 in NCI-8 action to p53 pathway restoration. NCI-8 enhanced p53-responsive bioluminescence with10-fold increase in cells overexpressing p73, but only 2-fold increase in p53-overexpressing cells. NCI-8- induced p53-responsive bioluminescence was significantly reduced by knock-down of p73, but not by knock-down of mutant p53. Knock-down of p73 significantly reduced NCI-8 induction of p53 target genes in mutant p53 cancer cells. These results indicate that p73 is required for NCI-8 to restore the p53 pathway in mutant p53-expressing cancer cells. Importantly, NCI-8 induced cell death in cancer cells, but not in normal cells. No genotoxicity of NCI-8 was detected in cancer and normal cells. Knock-down of p73 reduced cell apoptosis in mutant p53-expressing cancer cells treated with NCI-8, while, overexpression of p73 sensitized these cancer cells to NCI-8 induced cell death. Combination treatments demonstrate that NCI-8 synergizes with CPT11chemotherapy to induce cell death in mutant p53 colorectal cancer cells. These results suggest that p53 pathway restoration and cell death induced by NCI-8 occur via activation of p73 and depletion of mutant p53 GOF. NCI-8 is a promising lead compound specifically targeting mutant p53 for the development of new anti-cancer drugs. Citation Format: Shengliang Zhang, Lanlan Zhou, Bo Hong, Noel Warfel, Antonius Van den heuvel, David Dicker, Levy Kopelovich, Wafik El-Deiry. A new small molecule compound restoring p53 pathway induces cell death via active p73 and degradation of mutant p53 in colorectal cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2171. doi:10.1158/1538-7445.AM2013-2171