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Cancer-associated fibroblasts (CAFs) promote tumor growth and progression, and increase drug resistance through several mechanisms. We have investigated the effect of CAFs on the p53 response to doxorubicin in prostate cancer cells. We show that CAFs produce interleukin-6 (IL-6), and that IL-6 attenuates p53 induction and upregulation of the pro-apoptotic p53 target Bax upon treatment with doxorubicin. This is associated with increased levels of MDM2 mRNA, Mdm2 protein bound to p53, and ubiquitinated p53. IL-6 also inhibited doxorubicin-induced cell death. Inhibition of JAK or STAT3 alleviated this effect, indicating that IL-6 attenuates p53 via the JAK/STAT signaling pathway. These results suggest that CAF-derived IL-6 plays an important role in protecting cancer cells from chemotherapy and that inhibition of IL-6 could have significant therapeutic value.

TP53 is the most frequently mutated gene in human cancer and thus an attractive target for novel cancer therapy. Several compounds that can reactive mutant p53 protein have been identified. APR-246 is currently being tested in a phase II clinical trial in high-grade serous ovarian cancer. We have used RNA-seq analysis to study the effects of APR-246 on gene expression in human breast cancer cell lines. Although the effect of APR-246 on gene expression was largely cell line dependent, six genes were upregulated across all three cell lines studied, i.e., TRIM16, SLC7A11, TXNRD1, SRXN1, LOC344887, and SLC7A11-AS1. We did not detect upregulation of canonical p53 target genes such as CDKN1A (p21), 14-3-3σ, BBC3 (PUMA), and PMAIP1 (NOXA) by RNA-seq, but these genes were induced according to analysis by qPCR. Gene ontology analysis showed that APR-246 induced changes in pathways such as response to oxidative stress, gene expression, cell proliferation, response to nitrosative stress, and the glutathione biosynthesis process. Our results are consistent with the dual action of APR-246, i.e., reactivation of mutant p53 and modulation of redox activity. SLC7A11, TRIM16, TXNRD1, and SRXN1 are potential new pharmacodynamic biomarkers for assessing the response to APR-246 in both preclinical and clinical studies.

The tumor suppressor p53 is commonly inactivated in human tumors, allowing evasion of p53-dependent apoptosis and tumor progression. The small molecule APR-246 (PRIMA-1Met) can reactive mutant p53 in tumor cells and trigger cell death by apoptosis. The thioredoxin (Trx) and glutaredoxin (Grx) systems are important as antioxidants for maintaining cellular redox balance and providing electrons for thiol-dependent reactions like those catalyzed by ribonucleotide reductase and peroxiredoxins (Prxs). We show here that the Michael acceptor methylene quinuclidinone (MQ), the active form of APR-246, is a potent direct inhibitor of Trx1 and Grx1 by reacting with sulfhydryl groups in the enzymes. The inhibition of Trx1 and Grx1 by APR-246/MQ is reversible and the inhibitory efficiency is dependent on the presence of glutathione. APR-246/MQ also inhibits Trxs in mutant p53-expressing Saos-2 His-273 cells, showing modification of Trx1 and mitochondrial Trx2. Inhibition of the Trx and Grx systems leads to insufficient reducing power to deoxyribonucleotide production for DNA replication and repair and peroxiredoxin for removal of ROS. We also demonstrate that APR-246 and MQ inhibit ribonucleotide reductase (RNR) in vitro and in living cells. Our results suggest that APR-246 induces tumor cell death through both reactivations of mutant p53 and inhibition of cellular thiol-dependent redox systems, providing a novel strategy for cancer therapy.

The TP53 tumor suppressor gene is frequently inactivated in human tumors by missense mutations in the DNA binding domain. TP53 mutations lead to protein unfolding, decreased thermostability and loss of DNA binding and transcription factor function. Pharmacological targeting of mutant p53 to restore its tumor suppressor function is a promising strategy for cancer therapy. The mutant p53 reactivating compound APR-246 (PRIMA-1

International audience; The standard classification used to define the various cancer genes confines tumor protein p53 (TP53) to the role of a tumor suppressor gene. However, it is now an indisputable fact that many p53 mutants act as oncogenic proteins. This statement is based on multiple arguments including the mutation signature of the TP53 gene in human cancer, the various gains-of-function (GOFs) of the different p53 mutants and the heterogeneous phenotypes developed by knock-in mouse strains modeling several human TP53 mutations. In this review, we will shatter the classical and traditional image of tumor protein p53 (TP53) as a tumor suppressor gene by emphasizing its multiple oncogenic properties that make it a potential therapeutic target that should not be underestimated. Analysis of the data generated by the various cancer genome projects highlights the high frequency of TP53 mutations and reveals that several p53 hotspot mutants are the most common oncoprotein variants expressed in several types of tumors. The use of Muller's classical definition of mutations based on quantitative and qualitative consequences on the protein product, such as 'amorph', 'hypomorph', 'hypermorph' 'neomorph' or 'antimorph', allows a more meaningful assessment of the consequences of cancer gene modifications, their potential clinical significance, and clearly demonstrates that the TP53 gene is an atypical cancer gene.

The tumor suppressor gene TP53 is inactivated by mutation in a large fraction of human tumors. Around 10% of TP53 mutations are nonsense mutations that lead to premature termination of translation and expression of truncated unstable and non-functional p53 protein. Aminoglycosides G418 (geneticin) and gentamicin have been shown to induce translational readthrough and expression of full-length p53. However, aminoglycosides have severe side effects that limit their clinical use. Here, we show that combination treatment with a proteasome inhibitor or compounds that disrupt p53-Mdm2 binding can synergistically enhance levels of full-length p53 upon aminoglycoside-induced readthrough of R213X nonsense mutant p53. Full-length p53 expressed upon combination treatment is functionally active as assessed by upregulation of p53 target genes, suppression of cell growth, and induction of cell death. Thus, our results demonstrate that combination treatment with aminoglycosides and compounds that inhibit p53 degradation is synergistic and can provide significantly improved efficacy of readthrough when compared with aminoglycosides alone. This may have implications for future cancer therapy based on reactivation of nonsense mutant TP53.

The TP53 tumor suppressor gene is frequently inactivated in human tumors by missense mutations in the DNA binding domain. TP53 mutations lead to protein unfolding, decreased thermostability and loss of DNA binding and transcription factor function. Pharmacological targeting of mutant p53 to restore its tumor suppressor function is a promising strategy for cancer therapy. The mutant p53 reactivating compound APR-246 (PRIMA-1Met) has been successfully tested in a phase I/IIa clinical trial. APR-246 is converted to the reactive electrophile methylene quinuclidinone (MQ), which binds covalently to p53 core domain. We identified cysteine 277 as a prime binding target for MQ in p53. Cys277 is also essential for MQ-mediated thermostabilization of wild-type, R175H and R273H mutant p53, while both Cys124 and Cys277 are required for APR-246-mediated functional restoration of R175H mutant p53 in living tumor cells. These findings may open opportunities for rational design of novel mutant p53-targeting compounds.

Background Mutation in the tumor suppressor gene TP53 is an early event in the development of high-grade serous (HGS) ovarian cancer and is identified in more than 96 % of HGS cancer patients. APR-246 (PRIMA-1MET) is the first clinical-stage compound that reactivates mutant p53 protein by refolding it to wild type conformation, thus inducing apoptosis. APR-246 has been tested as monotherapy in a Phase I/IIa clinical study in hematological malignancies and prostate cancer with promising results, and a Phase Ib/II study in combination with platinum-based therapy in ovarian cancer is ongoing. In the present study, we investigated the anticancer effects of APR-246 in combination with conventional chemotherapy in primary cancer cells isolated from ascitic fluid from 10 ovarian, fallopian tube, or peritoneal cancer patients, 8 of which had HGS cancer. Methods Cell viability was assessed with fluorometric microculture cytotoxicity assay (FMCA) and Combination Index was calculated using the Additive model. p53 status was determined by Sanger sequencing and single strand conformation analysis, and p53 protein expression by western blotting. Results We observed strong synergy with APR-246 and cisplatin in all tumor samples carrying a TP53 missense mutation, while synergistic or additive effects were found in cells with wild type or TP53 nonsense mutations. Strong synergy was also observed with carboplatin or doxorubicin. Moreover, APR-246 sensitized TP53 mutant primary ovarian cancer cells, isolated from a clinically platinum-resistant patient, to cisplatin; the IC50 value of cisplatin decreased 3.6 fold from 6.5 to 1.8 μM in the presence of clinically relevant concentration of APR-246. Conclusion These results suggest that combination treatment with APR-246 and DNA-damaging drugs could significantly improve the treatment of patients with TP53 mutant HGS cancer, and thus provide strong support for the ongoing clinical study with APR-246 in combination with carboplatin and pegylated liposomal doxorubicin in patients with recurrent HGS cancer.

TP53 is the most frequently mutated gene in cancer. The p53 protein activates transcription of genes that promote cell cycle arrest or apoptosis, or regulate cell metabolism and other processes . Missense mutations in TP53 abolish specific DNA binding of p53 and allow evasion of apoptosis and accelerated tumor progression. Mutant p53 often accumulates at high levels in tumor cells. Pharmacological reactivation of mutant p53 has emerged as a promising strategy for improved cancer therapy. Small molecules that restore wild type activity of mutant p53 have been identified using various approaches. One of these, APR-246, is a prodrug that is converted to the Michael acceptor methylene quinuclidinone (MQ) that binds covalently to cysteines in p53, leading to refolding and restoration of wild type p53 function. MQ also targets the cellular redox balance by inhibiting thioredoxin reductase (TrxR1) and depleting glutathione (GSH). This dual mechanism of action may account for the striking synergy between APR-246 and platinum compounds. APR-246 is the only mutant p53-targeting compound in clinical development. A phase I/IIa clinical trial in hematological malignancies and prostate cancer showed good safety profile and clinical effects in some patients. APR-246 is currently tested in a phase Ib/II trial in patients with high-grade serous (HGS) ovarian cancer.

Drug resistance is a major problem in cancer therapy. A growing body of evidence demonstrates that the tumor microenvironment, including cancer-associated fibroblasts (CAFs), can modulate drug sensitivity in tumor cells. We examined the effect of primary human CAFs on p53 induction and cell viability in prostate cancer cells on treatment with chemotherapeutic drugs. Co-culture with prostate CAFs or CAF-conditioned medium attenuated DNA damage and the p53 response to chemotherapeutic drugs and enhanced prostate cancer cell survival. CAF-conditioned medium inhibited the accumulation of doxorubicin, but not taxol, in prostate cancer cells in a manner that was associated with increased cancer cell glutathione levels. A low molecular weight fraction (

p53 and p63 share extensive sequence and structure homology. p53 is frequently mutated in cancer, whereas mutations in p63 cause developmental disorders manifested in ectodermal dysplasia, limb defects, and orofacial clefting. We have established primary adult skin keratinocytes from ectrodactyly, ectodermal dysplasia, and cleft lip/palate (EEC) syndrome patients with p63 mutations as an in vitro human model to study the disease mechanism in the skin of EEC patients. We show that these patient keratinocytes cultured either in submerged 2D cultures or in 3D skin equivalents have impaired epidermal differentiation and stratification. Treatment of these patient keratinocytes with the mutant p53-targeting compound APR-246/PRIMA-1 MET (p53 reactivation and induction of massive apoptosis) that has been successfully tested in a phase I/II clinical trial in cancer patients partially but consistently rescued morphological features and gene expression during epidermal stratification in both 2D and 3D models. This rescue coincides with restoration of p63 target-gene expression. Our data show that EEC patient keratinocytes with p63 mutations can be used for characterization of the abnormal molecular circuitry in patient skin and may open possibilities for the design of novel pharmacological treatment strategies for patients with mutant p63 -associated developmental abnormalities.

Reactivation of mutant p53 in human tumor cells should induce cell death by apoptosis and thus eliminate the tumor. Several small molecules that reactivate mutant p53 have been identified. Here we show that STIMA-1, a low molecular weight compound with some structural similarities to the previously identified molecule CP-31398, can stimulate mutant p53 DNA binding in vitro and induce expression of p53 target proteins and trigger apoptosis in mutant p53-expressing human tumor cells. Human diploid fibroblasts are significantly more resistant to STIMA-1 than mutant or wild type p53-carrying tumor cells. STIMA-1 may provide new insights into possible mechanisms of mutant p53 reactivation and thus facilitate the development of novel anticancer drugs that target mutant p53-carrying tumors.

Reactivation of the tumor suppressor activity to mutant p53 should trigger massive apoptosis and eliminate tumors. The low molecular weight compounds PRIMA-1 and the structural analog PRIMA-1MET reactivate human mutant p53 in vitro and suppress growth of human tumor xenografts in SCID mice. However, little is known about their effect on mouse mutant p53 in mouse tumor cells. We have examined the effect of PRIMA-1MET on mouse sarcomas, mammary carcinomas and chemically induced fibrosarcomas. PRIMA-1MET showed potent growth suppression in mutant p53-carrying mouse tumors in vitro and a significant anti-tumor effect in syngeneic mice in vivo. These results demonstrate that PRIMA-1MET targets mouse tumors carrying mutant p53 and provide strong support for the anti-tumor efficiency of PRIMA-1METin vivo.

The low-molecular-weight compound APR-246 (PRIMA-1(MET)) restores wild-type conformation and function to mutant p53, and triggers apoptosis in tumor cells. We show here that APR-246 also targets the selenoprotein thioredoxin reductase 1 (TrxR1), a key regulator of cellular redox balance. APR-246 inhibited both recombinant TrxR1 in vitro and TrxR1 in cells. A Sec-to-Cys mutant of TrxR1 was not inhibited by APR-246, suggesting targeting of the selenocysteine residue in wild-type TrxR1. Preheated APR-246 and its conversion product methylene quinuclidinone (MQ) were much more efficient TrxR1 inhibitors than APR-246 itself, indicating that MQ is the active compound responsible for TrxR1 enzyme inhibition. TrxR1 inhibited by MQ was still functional as a pro-oxidant NADPH oxidase. Knockdown of TrxR1 caused a partial and reproducible attenuation of APR-246-induced tumor cell death independently of p53 status. Cellular TrxR1 activity was also inhibited by APR-246 irrespective of p53 status. We show that APR-246 can directly affect cellular redox status via targeting of TrxR1. Our findings provide an explanation for the previously observed effects of APR-246 on tumor cells lacking mutant p53.

Wig-1 is a transcriptional target of the p53 tumor suppressor and encodes an mRNA stability-regulating protein. We show here that Wig-1 knockdown causes a dramatic inhibition of N-Myc expression and triggers differentiation in neuroblastoma cells carrying amplified N-Myc. Transient Wig-1 knockdown significantly delays development of N-Myc-driven tumors in mice. We also show that N-Myc expression is induced upon moderate p53-activating stress, suggesting a role of the p53-Wig-1-N-Myc axis in promoting cell cycle re-entry upon p53-induced cell cycle arrest and DNA repair. Moreover, our findings raise possibilities for the improved treatment of poor prognosis neuroblastomas that carry amplified N-Myc.

Epstein-Barr virus (EBV) immortalized human lymphoblastoid cell lines express six virally encoded nuclear proteins, designated EBV nuclear antigens 1-6 (EBNA-1-6). We show that the EBNA-5 protein (alternatively designated EBNA-LP) that is required for B-cell transformation can form a molecular complex with the retinoblastoma (RB) and p53 tumor suppressor proteins. Using EBNA-5 deletion mutants, we have found that a 66-amino acid-long peptide, encoded by the W repeat of the EBV genome, is sufficient for binding. Point mutations of RB and p53 that inhibit their complexing with other DNA viral oncoproteins do not affect their binding to EBNA-5. p53 competes with RB for EBNA-5 binding. Our data suggest that the mechanisms involved in EBV transformation may include impairment of RB and p53 function.

p53 mutations are found in 50% of human cancers. Molecular epidemiology has shown strong correlations between the spectrum of p53 mutations and exposure to exogenous carcinogens. This spectrum is influenced quantitatively and qualitatively by various upstream genetic filters that modulate carcinogen activation, detoxification, and/or DNA repair. In this review, we will discuss how other factors such as tissue specificity, SNP of genes associated with the p53 pathway, other genetic alterations, or p53 mutant heterogeneity can act as a second set of downstream filters that also have a profound impact on the spectrum of p53 mutations.

Restoration of wild-type p53 expression triggers cell death and eliminates tumors in vivo. The identification of mutant p53-reactivating small molecules such as PRIMA-1 opens possibilities for the development of more efficient anticancer drugs. Although the biological effects of PRIMA-1 are well demonstrated, little is known about its molecular mechanism of action. We show here that PRIMA-1 is converted to compounds that form adducts with thiols in mutant p53. Covalent modification of mutant p53 per se is sufficient to induce apoptosis in tumor cells. These findings might facilitate the design of more potent and specific mutant p53-targeting anticancer drugs. This work was supported by the Swedish Cancer Society (Cancerfonden), Cancerfo¨ reningen, Karolinska Institutet, Magn. Bergvalls Stiftelse, and the EU 6th framework program (FP6).

The p53-induced Wig-1 gene encodes a double stranded RNA-binding zinc finger protein. We generated Saos-2 osteosarcoma cells expressing tetracycline-inducible Flag-tagged human Wig-1. Induction of Wig-1 expression by doxycycline inhibited cell growth in a long-term assay but did not cause any changes in cell cycle distribution nor increased fraction of apoptotic cells. Using co-immunoprecipitation and mass spectrometry, we identified two Wig-1-binding proteins, hnRNP A2/B1 and RNA Helicase A, both of which are involved in RNA processing. The binding was dependent on the presence of RNA. Our results establish a link between the p53 tumor suppressor and RNA processing via hnRNPA2/B1 and RNA Helicase A.Structured summaryMINT-6542926, MINT-6542899:WIG1 (uniprotkb:Q9HA38) physically interacts (MI:0218) with hnRNP A2/B1 (uniprotkb:P22626) by anti bait coimmunoprecipitation (MI:0006)MINT-6542945:RHA (uniprotkb:Q08211) physically interacts (MI:0218) with hnRNP A2/B1 (uniprotkb:P22626) by anti bait coimmunoprecipitation (MI:0006)MINT-6542918, MINT-6542891:WIG1 (uniprotkb:Q9HA38) physically interacts (MI:0218) with RHA (uniprotkb:Q08211) by anti bait coimmunoprecipitation (MI:0006)MINT-6542867:WIG1 (uniprotkb:Q9HA38) physically interacts (MI:0218) with RHA (uniprotkb:Q08211) by anti tag coimmunoprecipitation (MI:0007)MINT-6542879:WIG1 (uniprotkb:Q9HA38) physically interacts (MI:0218) with hnRNP A2/B1(uniprotkb:P22626) by anti tag coimmunoprecipitation (MI:0007)

TP53, a critical tumour suppressor gene, is mutated in over half of all cancers resulting in mutant-p53 protein accumulation and poor patient survival. Therapeutic strategies to target mutant-p53 cancers are urgently needed. We show that accumulated mutant-p53 protein suppresses the expression of SLC7A11, a component of the cystine/glutamate antiporter, system xC−, through binding to the master antioxidant transcription factor NRF2. This diminishes glutathione synthesis, rendering mutant-p53 tumours susceptible to oxidative damage. System xC− inhibitors specifically exploit this vulnerability to preferentially kill cancer cells with stabilized mutant-p53 protein. Moreover, we demonstrate that SLC7A11 expression is a novel and robust predictive biomarker for APR-246, a first-in-class mutant-p53 reactivator that also binds and depletes glutathione in tumours, triggering lipid peroxidative cell death. Importantly, system xC− antagonism strongly synergizes with APR-246 to induce apoptosis in mutant-p53 tumours. We propose a new paradigm for targeting cancers that accumulate mutant-p53 protein by inhibiting the SLC7A11–glutathione axis.