Non-genotoxic Activation Research Articles

Abstract 6372: Nanodrug delivery system for non-genotoxic p53 activator INZ-C

Abstract p53 is a known tumor suppressor protein whose gene is altered in 50% of all human cancers, while its function is inactivated in the many cancers harboring wild type p53, making it an attractive target for targeted cancer therapies. In this regard, our lab had previously discovered a small molecule, Inauhzin (INZ C), as a potent non-genotoxic p53 activator. In an effort to improve the bioavailability and efficacy, we have now encapsulated the INZ C small molecule in a chitosan/cyclodextrin nanoparticle delivery system. Evaluation of in vitro/in vivo activity and protien expression suggests nanoparticle encapsulation of INZ-C (n-INZ-C) improved p53 induction and inhibition of cell growth in lung and colorectal cancers, while maintaining a minimal effect for MEF and Wi38 normal cells. These results are consistent with the significantly reduced cellular uptake observed for normal cells as compared to the cancer cells. H/E staining of tissues for mice treated with n-INZ-C reveals no toxicity to other organs as well. Additionally, n-INZ-C demonstrates the ability to block migration of cells in vitro as demonstrated by both wound healing and trans-well migration assays. Our in vivo pharmacokinetic evaluation suggests nanoparticle encapsulation doubles the half-life of INZ-C from 2.5 to 5 h. These results suggest that nanoparticle encapsulation improved activity and bioavailability of INZ-C while maintaining the non-toxic nature of INZ-C in normal cells. In addition, we unveiled GRP78 as a potential new target of INZ-C and found out that knockdown of GRP78 can alleviate the inhibitory effect of INZ-C on lung and colorectal cancer proliferation. Further studies now focus on revealing the mechanism of enhanced cellular uptake for INZ-C in cancer cells as comapred to normal cells. Thus, these results demonstrate that we have established a nanoparticle system that could enhance the bioavailability and efficacy of INZ-C as a potential anti-cancer therapeutic and also identified a novel target of INZ-C. Citation Format: Nimisha Bhattarai, Jieqiong Wang, Daniel Nguyen, Xiaoxiao Yang, Linh Helmers, Jennifer Paruch, Li Li, Yiwei Zhang, Kun Meng, Alun Wang, Janarthanan Jayawickramarajah, Binghe Wang, Shelya Zeng, Hua Lu. Nanodrug delivery system for non-genotoxic p53 activator INZ-C [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6372.

New in silico models to predict in vitro micronucleus induction as marker of genotoxicity

The evaluation of genotoxicity is a fundamental part of the safety assessment of chemicals due to the relevance of the potential health effects of genotoxicants. Among the testing methods available, the in vitro micronucleus assay with mammalian cells is one of the most used and required by regulations targeting several industrial sectors such as the cosmetic industry and food-related sectors. As an alternative to the testing methods, in recent years, lots in silico methods were developed to predict the genotoxicity of chemicals, including models for the Ames mutagenicity test, the in vitro chromosomal aberrations and the in vivo micronucleus assay. We developed several in silico models for the prediction of genotoxicity as reflected by the in vitro micronucleus assay. The resulting models include both statistical and knowledge-based models. The most promising model is the one based on fragments extracted with the SARpy platform. More than 100 structural alerts were extracted, including also fragments associated with the non-genotoxic activity. The model is characterized by high accuracy and the lowest false negative rate, making this tool suitable for chemical screening according to the regulators' needs. The SARpy model will be implemented on the VEGA platform (https://www.vegahub.eu) and will be freely available.

Comparison of genotoxic versus nongenotoxic stabilization of p53 provides insight into parallel stress-responsive transcriptional networks

ABSTRACTThe tumor suppressor protein p53 is activated in response to diverse intrinsic and extrinsic cellular stresses and controls a broad cell-protective gene network. Whether p53:DNA binding and subsequent transcriptional activation differs downstream of these diverse intrinsic and extrinsic activators is controversial. Using primary human fibroblasts, we assessed the genome-wide profile of p53 binding, chromatin structure, and transcriptional dynamics after either genotoxic or nongenotoxic activation of p53. Activation of p53 by treatment with either etoposide or the small-molecule MDM2 inhibitor nutlin 3A yields strikingly similar genome-wide binding of p53 and concomitant changes to local chromatin modifications and structure. DNA damage, but not p53 activation per se, leads to increased expression of genes in an inflammatory cytokine pathway. The NF-κB pathway inhibitor Bay 11-7082 abrogates etoposide-mediated activation of the inflammation gene signature but does not affect expression of canonical p53 target genes. Our data demonstrate that differential activation of p53 within the same cell type leads to highly similar genome-wide binding, chromatin dynamics, and gene expression dynamics and that DNA damage-mediated signaling through NF-κB likely controls the observed pro-inflammatory cytokine gene expression pattern.

Mode of action and human relevance of rodent liver tumours produced by nongenotoxic CAR and PPARα activators

Mode of action and human relevance of rodent liver tumours produced by nongenotoxic CAR and PPARα activators

P53 Nongenotoxic Activation and mTORC1 Inhibition Lead to Effective Combination for Neuroblastoma Therapy.

Purpose: mTORC1 inhibitors are promising agents for neuroblastoma therapy; however, they have shown limited clinical activity as monotherapy, thus rational drug combinations need to be explored to improve efficacy. Importantly, neuroblastoma maintains both an active p53 and an aberrant mTOR signaling.Experimental Design: Using an orthotopic xenograft model and modulating p53 levels, we investigated the antitumor effects of the mTORC1 inhibitor temsirolimus in neuroblastoma expressing normal, decreased, or mutant p53, both as single agent and in combination with first- and second-generation MDM2 inhibitors to reactivate p53.Results: Nongenotoxic p53 activation suppresses mTOR activity. Moreover, p53 reactivation via RG7388, a second-generation MDM2 inhibitor, strongly enhances the in vivo antitumor activity of temsirolimus. Single-agent temsirolimus does not elicit apoptosis, and tumors rapidly regrow after treatment suspension. In contrast, our combination therapy triggers a potent apoptotic response in wild-type p53 xenografts and efficiently blocks tumor regrowth after treatment completion. We also found that this combination uniquely led to p53-dependent suppression of survivin whose ectopic expression is sufficient to rescue the apoptosis induced by our combination.Conclusions: Our study supports a novel highly effective strategy that combines RG7388 and temsirolimus in wild-type p53 neuroblastoma, which warrants testing in early-phase clinical trials. Clin Cancer Res; 23(21); 6629-39. ©2017 AACR.

Safety evaluation of an oat grain alkaloid gramine by genotoxicity assays

Gramine is a natural indole alkaloid that has been isolated from different raw plants occurring mainly in Avena sativa, etc. The study was aimed to investigate the possible in vitro antioxidant, in vitro mutagenic, in vitro antimutagenic, and in vivo genotoxic activity of gramine using ferric reducing ability of plasma (FRAP) assay, Metal chelating, Ames bacterial reverse mutation test, and the mouse bone marrow micronucleus assay as well as chromosomal aberration. Four concentrations of gramine viz. 250, 500, 1000, and 2000 μg/mL were evaluated for its antioxidant activity in FRAP Assay and Metal Chelating Test. Four concentrations of gramine (1250 μg/plate, 2500 μg/plate, 5000 μg/plate, and 10 000 μg/plate) were employed in Salmonella typhimurium strains to study the mutagenicity in the presence and absence of standard mutagens, 2-aminofluorene (2-AF), sodium azide (SA), and 2-nitrofluorene (2-NF). Three doses, i.e. 0.1, 0.2, and 0.3 × the LD50 of gramine (i.e. 50 mg/kg, 100 mg/kg, and 150 mg/kg) were administered orally to either sex of Swiss albino mice for 48 h to study the genotoxic activity in micronucleus assay as well as chromosomal aberration. Gramine showed potent antioxidant activity in both the assay. Gramine at the given dose lacks mutagenicity as well as found to possess antimutagenic efficacy. Interestingly, S9 enzymes increase the antimutagenic activity in a dose-dependent manner. There was no significant increase in the frequency of micronucleated polychromatic erythrocytes (MNPCEs), as well as no significant difference in the percentage of chromosomal aberrations was observed between the gramine groups and the negative groups but percentage of polychromatic erythrocytes (PCEs) is found to be higher in all the gramine groups. These results indicate significant antioxidant, non-mutagenic as well as non-genotoxic activity of gramine in vitro and in vivo in the given doses.

P53 induces formation of NEAT1 lncRNA-containing paraspeckles that modulate replication stress response and chemosensitivity.

In a search for mediators of the p53 tumor suppressor pathway, which induces pleiotropic and often antagonistic cellular responses, we identified the long noncoding RNA (lncRNA) NEAT1. NEAT1 is an essential architectural component of paraspeckle nuclear bodies, whose pathophysiological relevance remains unclear. Activation of p53, pharmacologically or by oncogene-induced replication stress, stimulated the formation of paraspeckles in mouse and human cells. Silencing Neat1 expression in mice, which prevents paraspeckle formation, sensitized preneoplastic cells to DNA-damage-induced cell death and impaired skin tumorigenesis. We provide mechanistic evidence that NEAT1 promotes ATR signaling in response to replication stress and is thereby engaged in a negative feedback loop that attenuates oncogene-dependent activation of p53. NEAT1 targeting in established human cancer cell lines induced synthetic lethality with genotoxic chemotherapeutics, including PARP inhibitors, and nongenotoxic activation of p53. This study establishes a key genetic link between NEAT1 paraspeckles, p53 biology and tumorigenesis and identifies NEAT1 as a promising target to enhance sensitivity of cancer cells to both chemotherapy and p53 reactivation therapy.

Pre-clinical efficacy and synergistic potential of the MDM2-p53 antagonists, Nutlin-3 and RG7388, as single agents and in combined treatment with cisplatin in ovarian cancer

Ovarian cancer is the fifth leading cause of cancer-related female deaths. Due to serious side effects, relapse and resistance to standard chemotherapy, better and more targeted approaches are required. Mutation of the TP53 gene accounts for 50% of all human cancers. In the remaining malignancies, non-genotoxic activation of wild-type p53 by small molecule inhibition of the MDM2-p53 binding interaction is a promising therapeutic strategy. Proof of concept was established with the cis-imidazoline Nutlin-3, leading to the development of RG7388 and other compounds currently in early phase clinical trials. This preclinical study evaluated the effect of Nutlin-3 and RG7388 as single agents and in combination with cisplatin in a panel of ovarian cancer cell lines. Median-drug-effect analysis showed Nutlin-3 or RG7388 combination with cisplatin was additive to, or synergistic in a p53-dependent manner, resulting in increased p53 activation, cell cycle arrest and apoptosis, associated with increased p21WAF1 protein and/or caspase-3/7 activity compared to cisplatin alone. Although MDM2 inhibition activated the expression of p53-dependent DNA repair genes, the growth inhibitory and pro-apoptotic effects of p53 dominated the response. These data indicate that combination treatment with MDM2 inhibitors and cisplatin has synergistic potential for the treatment of ovarian cancer, dependent on cell genotype.

Therapeutic Response to Non-genotoxic Activation of p53 by Nutlin3a Is Driven by PUMA-Mediated Apoptosis in Lymphoma Cells.

Nutlin3a is a small-molecule antagonist of MDM2 that promotes non-genotoxic activation of p53 through p53 protein stabilization and transactivation of p53 target genes. Nutlin3a is the forerunner of a class of cancer therapeutics that have reached clinical trials. Using transgenic and gene-targeted mouse models lacking the critical p53 target genes, p21, Puma, and Noxa, we found that only loss of PUMA conferred profound protection against Nutlin3a-induced killing in both non-transformed lymphoid cells and Eμ-Myc lymphomas in vitro and in vivo. CRISPR/Cas9-mediated targeting of the PUMA gene rendered human hematopoietic cancer cell lines markedly resistant to Nutlin3a-induced cell death. These results demonstrate that PUMA-mediated apoptosis, but not p21-mediated cell-cycle arrest or senescence, is a critical determinant of the therapeutic response to non-genotoxic p53 activation by Nutlin3a. Importantly, in human cancer, PUMA expression may predict patient responses to treatment with MDM2 antagonists.

Abstract 1785: Chemical inhibition or transient knockdown of wild-type p53 induced phosphatase 1 (WIP1/PPM1D) potentiates the response to MDM2 inhibitors in a p53-dependent manner

Abstract The mechanisms that determine p53 cell fate decision after its non-genotoxic activation by MDM2 inhibitors are poorly understood. Wild-type p53-inducible phosphatase-1 (WIP1/PPM1D) forms an autoregulatory loop with p53 and is involved in the homeostasis of the p53 stress response network. WIP1 is activated, gained/amplified and overexpressed in a range of p53 wild-type malignancies. We investigated cellular growth/proliferation of p53 wild-type (wt) and matched p53 mutant (mut)/null cell line pairs, differing in WIP1 genetic status, in response to Nutlin-3 + GSK2830371 (WIP1i)/WIP1 siRNA-mediated knockdown. We also assessed the effects of WIP1i or knockdown in combination with Nutlin-3 on p53 post-translational modifications, p53 downstream transcriptional targets and apoptosis. Phosphorylation of p53S15 (pp53S15) was used as a marker of WIP1 catalytic activity. WIP1-amplified MCF-7p53wt cells were very sensitive to the WIP1i, with a 50% growth inhibitory concentration (GI50) of 2.65μM + 0.54 (SEM). The three p53 wild-type and mutant/null cell line pairs showed relatively little sensitivity to single agent WIP1i (GI50>10μM). However the WIP1i, at a non-growth inhibitory dose (2.5μM) in vitro, potentiated their response to Nutlin-3 in a p53-dependent manner. HCT116p53+/+; WIP1L450fs*1/+ (L450fs*1 is a WIP1 activating mutation), NGPp53wt and SJSA-1p53wt cell lines showed a 2.4-fold (p = 0.007), 2.1-fold (p = 0.04) and 1.3-fold (p = 0.02) decrease in their Nutlin-3 GI50 values respectively in the presence of WIP1i at 2.5μM. In contrast Nutlin-3 GI50 did not change in HCT116p53-/−, N20R1p53Mut and SN40R2p53Mut, NGP and SJSA-1 Nutlin-3 resistant daughter cell lines respectively. WIP1 transient siRNA knockdown also resulted in potentiation of cells to Nutlin-3. In WIP1-non-amplified (WNA) cells treated with either Nutlin-3 at the GI50 dose or 2.5μM WIP1i, pp53S15 was not detected, however in combination, these drugs resulted in dramatic levels of pp53S15. Interestingly protein expression of p53 targets MDM2 and p21 were not affected in WNA cells by the increased p53S15 phosphorylation; whereas BAX expression was elevated. The Nutlin-3 + WIP1i/siRNA knockdown combination also resulted in a significant increase in cell death markers between 24-48Hrs post-treatment in a p53-dependent manner. Although the strongest WIP1i potentiation of Nutlin-3 was observed in HCT116p53+/+; WIP1L450fs*1/+ cells which possess an activating WIP1 mutation, the NGPp53wt and SJSA-1p53wt cell lines with wild-type WIP1 also showed potentiation. These findings highlight the mechanistic importance of the p53-WIP1 autoregulatory loop in cell fate determination after non-genotoxic p53 activation by Nutlin-3. Potent inhibitors of WIP1 may potentiate the response to other therapeutics aiming to activate p53 mediated tumour suppression. Citation Format: Arman Esfandiari, Nicola J. Curtin, John Lunec. Chemical inhibition or transient knockdown of wild-type p53 induced phosphatase 1 (WIP1/PPM1D) potentiates the response to MDM2 inhibitors in a p53-dependent manner. [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 1785. doi:10.1158/1538-7445.AM2015-1785

Abstract 4474: Dual targeting of the stress-p53 pathway as a potential anti-cancer therapy

Abstract The “ribosomal stress (RS)-p53 pathway” is triggered by any stressor or genetic alteration that disrupts ribosomal biogenesis, and mediated by several ribosomal proteins (RPs), such as RPL11 and RPL5, which inhibit MDM2 and activate p53. IMPDH2 is a rate-limiting enzyme in de novo guanine nucleotide biosynthesis and crucial for maintaining cellular guanine deoxy- and ribonucleotide pools needed for DNA and RNA synthesis. It is highly expressed in many malignancies. We previously showed that inhibition of IMPDH2 leads to p53 activation by causing RS. Surprisingly, our current study reveals that Inauzhin (INZ), a novel non-genotoxic p53 activator by inhibiting SIRT1, can also inhibit cellular IMPDH2 activity, and reduce the levels of cellular GTP and GTP-binding nucleostemin that is essential for rRNA processing. Consequently, INZ induces RS and the RPL11/RPL5-MDM2 interaction, activating p53. These results support the new notion that INZ suppresses cancer cell growth by dually targeting SIRT1 and IMPDH2. Note: This abstract was not presented at the meeting. Citation Format: Hua Lu, Qi Zhang, Shelya X. Zeng, Rui-Zhi Wu, Yiwei Zhang, Daniel Nguyen, Xiang Zhou, Jun-ming Liao, Bo Cao. Dual targeting of the stress-p53 pathway as a potential anti-cancer therapy. [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 4474. doi:10.1158/1538-7445.AM2015-4474

Non-genotoxic activation of p53 through the RPL11-dependent ribosomal stress pathway.

Nucleolar disruption has recently emerged as a relevant means to activate p53 through inhibition of HDM2 by ribosome-free RPL11. Most drugs that induce nucleolar disruption also possess important genotoxic activity, which can have lasting mutagenic effects. Therefore, it is of interest to identify compounds that selectively produce nucleolar disruption in the absence of DNA damage. Here, we have performed a high-throughput screening to search for nucleolar disruptors. We have identified an acridine derivative (PubChem CID-765471) previously known for its capacity to activate p53 independently of DNA damage, although the molecular mechanism underlying p53 activation had remained uncharacterized. We report that CID-765471 produces nucleolar disruption by inhibiting ribosomal DNA transcription in a process that includes the selective degradation of the RPA194 subunit of RNA polymerase I. Following nucleolar disruption, CID-765471 activates p53 through the RPL11/HDM2 pathway in the absence of detectable DNA damage. In a secondary screening of compounds approved for medical use, we identify two additional acridine derivatives, aminacrine and ethacridine, that operate in a similar manner as CID-765471. These findings provide the basis for non-genotoxic chemotherapeutic approaches that selectively target the nucleolus.

Non-genotoxic hepatocarcinogenic rodent CAR and PPARα activators. What is the human relevance?

Non-genotoxic hepatocarcinogenic rodent CAR and PPARα activators. What is the human relevance?

Acetylation of p53 stimulates miRNA processing and determines cell survival following genotoxic stress

It is widely accepted that different forms of stress activate a common target, p53, yet different outcomes are triggered in a stress-specific manner. For example, activation of p53 by genotoxic agents, such as camptothecin (CPT), triggers apoptosis, while non-genotoxic activation of p53 by Nutlin-3 (Nut3) leads to cell-cycle arrest without significant apoptosis. Such stimulus-specific responses are attributed to differential transcriptional activation of various promoters by p53. In this study, we demonstrate that CPT, but not Nut3, induces miR-203, which downregulates anti-apoptotic bcl-w and promotes cell death in a p53-dependent manner. We find that acetylation of K120 in the DNA-binding domain of p53 augments its association with the Drosha microprocessor and promotes nuclear primary miRNA processing. Knockdown of human orthologue of Males absent On the First (hMOF), the acetyltransferase that targets K120 in p53, abolishes induction of miR-203 and cell death mediated by CPT. Thus, this study reveals that p53 acetylation at K120 plays a critical role in the regulation of the Drosha microprocessor and that post-transcriptional regulation of gene expression by p53 via miRNAs plays a role in determining stress-specific cellular outcomes.

Cytotoxic, pro-apoptotic, pro-oxidant, and non-genotoxic activities of a novel copper(II) complex against human cervical cancer

Cisplatin remains one of the most effective current chemotherapeutic agents; however, metal complexes synthesis has increased in order to produce new anti-neoplastic drugs with DNA binding and apoptotic activities in tumor cells and less toxicity for patients. In this study, we evaluated the cytotoxic activity of a novel copper(II) complex (LQM402) against cervical cancer cell lines and found that LQM402 exhibited selective cytotoxicity against HeLa and Ca Ski cells. FITC-annexin assay and DNA fragmentation indicated that apoptosis could be involved in HeLa cell death. Caspase 3/7 and cytochrome c analysis by immunoblotting suggest the intrinsic pathway. LQM402 is a lipid peroxidation inductor according to TBARS production. Additionally, the Ames and micronucleus tests demonstrated non-genotoxic activity for this compound in Salmonella typhimurium and CD1 mice, respectively. Therefore, LQM402 may be a promising and safe anti-cervical cancer compound.

Nutlin-3a, an MDM2 antagonist and p53 activator, helps to preserve the replicative potential of cancer cells treated with a genotoxic dose of resveratrol

Resveratrol is a natural compound that has been intensely studied due to its role in cancer prevention and potential as an anti-cancer therapy. Its effects include induction of apoptosis and senescence-like growth inhibition. Here, we report that two cancer cell lines (U-2 OS and A549) differ significantly in their molecular responses to resveratrol. Specifically, in U-2 OS cells, the activation of the p53 pathway is attenuated when compared to the activation in A549 cells. This attenuation is accompanied by a point mutation (458: CGA→TGA) in the PPM1D gene and overexpression of the encoded protein, which is a negative regulator of p53. Experimentally induced knockdown of PPM1D in U-2 OS cells resulted in slightly increased activation of the p53 pathway, most clearly visible as stronger phosphorylation of p53 Ser37. When treated with nutlin-3a, a non-genotoxic activator of p53, U-2 OS and A549 cells both responded with substantial activation of the p53 pathway. Nutlin-3a improved the clonogenic survival of both cell lines treated with resveratrol. This improvement was associated with lower activation of DNA-damage signaling (phosphorylation of ATM, CHK2, and histone H2AX) and higher accumulation of cells in the G1 phase of the cell cycle. Thus, the hyperactivation of p53 by nutlin-3a helps to preserve the replicative potential of cells exposed to resveratrol.

Nanoparticles Loaded with Nutlin-3 Display Cytotoxicity Towards p53<sup>wildtype</sup> JVM-2 But Not Towards p53<sup>mutated</sup> BJAB Leukemic Cells

The small molecule Nutlin-3 is a potent antagonist of the murine double minute 2 (MDM2)/p53 interaction exhibiting promising therapeutic anti-cancer activity. Nutlin-3 has been proposed as an anti-neoplastic agent for the treatment of onco-hematological diseases characterized by a lower incidence of p53 mutation with respect to solid tumors. Indeed, based on its selective non-genotoxic p53 activation, Nutlin-3 might represent an alternative to the current cytotoxic chemotherapy. To overcome the poor bioavailability of Nutlin-3, we have assessed the potential efficacy of Nutlin-3 loaded poly(lactide-co-glycolide) (PLGA) nanoparticles (NP) against hematological malignancies. To test the specificity of the anti-leukemic activity, we have used leukemic cell lines characterized by different p53 status (JVM-2 and BJAB). NP loaded with Nutlin-3 (NP-Nutlin) were rapidly taken up by the leukemic cells and were as effective as native Nutlin-3 in promoting both induction of apoptosis and cell cycle arrest in p53(wild-type) JVM-2 cells, but not in p53(mutated) BJAB cells. Moreover, injection of NP-Nutlin, but not of free Nutlin-3, in a JVM-2-derived xenograft mouse model, reduced the subcutaneous tumor volume and promoted induction of apoptosis in the tumor mass. Overall, the chemical and structural characteristics of the NP-Nutlin-3, as well as their biological activity in vitro and in vivo, made them promising for further preclinical evaluations as potentially useful anti-leukemic carriers.

The MDM2 inhibitor Nutlin-3 attenuates streptozotocin-induced diabetes mellitus and increases serum level of IL-12p40

Besides its well-established oncosuppressor activity, a key function of p53 in regulating metabolic pathways has been recently identified. Nevertheless, the role of p53 with respect to diabetes mellitus (DM) appears highly controversial. To address this issue, we have used the cis-imidazoline compound Nutlin-3, an inhibitor of MDM2/p53 interaction, which represents a potent and selective non-genotoxic activator of the p53 pathway both in in vivo and in vitro experimental settings. Experimental DM was induced by intraperitoneal injections of low concentrations of streptozotocin (STZ) in C57BL/6N mice (n = 20). A group of control vehicle-injected mice (n = 10) and of STZ-treated mice (n = 10) was co-injected with Nutlin-3. Mice co-injected with STZ + Nutlin-3 exhibited attenuated features of DM with respect to animals treated with STZ alone. Indeed, STZ + Nutlin-3-treated mice were characterized by significantly (p < 0.05) lower levels of hyperglycemia, reduced weight loss, and increased spleen weight. In addition, STZ alone promoted a marked decrease in the levels of several circulating cytokines, including interleukin-12 (IL-12)p40. On the other hand, co-injection of STZ + Nutlin-3 significantly (p < 0.01) counteracted IL-12p40 down-modulation. In vitro experiments performed on the RAW264.7 macrophagic cell line model, used as cellular source of IL-12p40, demonstrated that Nutlin-3 treatment increased IL-12p40 release, strongly suggesting a direct effect of Nutlin-3 on the immune system. Overall, these data demonstrate that systemic administration of Nutlin-3 ameliorates the severity of STZ-induced DM and increases the levels of circulating IL-12p40.

Abstract 1721: Testicular germ cell tumors are hypersensitive to p53 activation based on their Oct-4/Noxa-mediated cellular context rather than on differential p53 activity.

Abstract TGCTs are highly apoptosis-prone cancers which can be cured by Cisplatin-based chemotherapy. We have previously shown that p53 plays a major role in this phenotype. Furthermore, we demonstrated that sensitivity of these tumors is dictated by p53 accumulation in general and is not restricted to Cisplatin. Hypersensitivity seems to be an intrinsic feature of pluripotent TGCTs, since both differentiation and loss of the pluripotency factor Oct-4 induce resistance. We sought to investigate the link between the Oct-4-mediated pluripotent context and the proapoptotic p53 response in these tumors. For this, we tested if loss of Oct-4 also reduces p53-mediated apoptosis upon non-genotoxic p53 activation. Indeed, RNAi-mediated silencing of Oct-4 significantly reduced sensitivity to the p53 activator Nutlin-3. Hence, we investigated a possible differential activation of p53 in the context of Oct-4-positive TGCT cells. For this, a qPCR system that allows simultaneous acquisition of 46 bona fide p53 target genes was used to detect p53’s transactivation capacity in NTERA-2D1 and 2102EP cells upon Cisplatin treatment in the presence and absence of Oct-4. In spite of p53’s central role in Cisplatin-induced apoptosis, no significant alterations in transactivation of p53 target genes were observed upon Cisplatin treatment in both cell lines. We also analyzed the cellular distribution of p53 upon Cisplatin treatment and found that p53 was not only accumulated in the nucleus but was increased to a similar extent in the cytoplasm. Importantly, RNAi-mediated silencing of Oct-4 did neither influence accumulation of p53 in the nucleus nor in the cytoplasm. These data indicate that Oct-4 does not directly modulate p53 activity but provides a cellular context that augments the proapoptotic activity of p53. We have previously proposed that Oct-4-dependent high constitutive Noxa protein levels account for the low apoptotic threshold in TGCTs. In contrast to Cisplatin or Nutlin-3, treatment with MG132 resulted in apoptosis in Oct-4-depleted NTERA-2D1 and 2102EP cells. This was due to simultaneous accumulation of p53 and Noxa, as RNAi-mediated silencing of either protein rescued the cells from death. It is of importance that p53 knockdown did not compromise Noxa accumulation upon MG132 treatment, indicating that both p53 activation and the Noxa-mediated apoptosis-prone context are required for high sensitivity. In conclusion, our data demonstrate that in spite of its impact on hypersensitivity, p53 activity is not altered in Oct-4-positive TGCT cells when compared to relatively resistant Oct-4-depleted cells. Although a robust p53 response is essential for hypersensitivity of TGCTs, its predominantly proapoptotic characteristic requires the apoptosis-prone cellular context of pluripotent Oct-4-positive TGCT cells, which includes high Noxa protein levels. Citation Format: Matthias Gutekunst, Thomas Mueller, Andrea Weilbacher, Michael A. Dengler, Moshe Oren, Walter E. Aulitzky, Heiko van der Kuip. Testicular germ cell tumors are hypersensitive to p53 activation based on their Oct-4/Noxa-mediated cellular context rather than on differential p53 activity. [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 1721. doi:10.1158/1538-7445.AM2013-1721

Activation of p53 pathway by Nutlin-3a inhibits the expression of the therapeutic target α5 integrin in colon cancer cells

Integrins emerge nowadays as crucial actors of tumor aggressiveness and resistance to therapies. Integrin α5β1, the fibronectin receptor, determines malignant properties of colon carcinoma which is one of the most important causes of cancer-related deaths in the world. Here we show that inhibition of α5 integrin subunit expression by siRNA or α5β1 integrin function by specific antagonist affects the survival of HCT116 colon cancer cells. We also evidence that pharmacological reactivation of the tumor suppressor p53 by Nutlin-3a inhibits specifically the expression of the α5 integrin subunit both at the transcriptional and protein level. Inversely repression of α5 integrin modulates p53 activity. A clear relationship between p53 activation by Nutlin-3a, α5 repression and cell survival is shown. No such effects are obtained in cells lacking p53 or when another non-genotoxic activator of p53, RITA, is used. Our results emphasize the crucial role of α5β1 integrin in colon tumors. Data also suggest that interfering with the integrin α5β1 through the reactivation of p53 by Nutlin-3a may be of valuable interest as a new therapeutic option for colon tumors expressing high level of the integrin and a wild type p53.