Disruption of the MDM2�Cp53 interaction strongly potentiates p53-dependent apoptosis in cisplatin-resistant human testicular carcinoma cells via the Fas/FasL pathway

Wild-type p53 has a major role in the response and execution of apoptosis after chemotherapy in many cancers. Although high levels of wild-type p53 and hardly any TP53 mutations are found in testicular cancer (TC), chemotherapy resistance is still observed in a significant subgroup of TC patients. In the present study, we demonstrate that p53 resides in a complex with MDM2 at higher cisplatin concentrations in cisplatin-resistant human TC cells compared with cisplatin-sensitive TC cells. Inhibition of the MDM2–p53 interaction using either Nutlin-3 or MDM2 RNA interference resulted in hyperactivation of the p53 pathway and a strong induction of apoptosis in cisplatin-sensitive and -resistant TC cells. Suppression of wild-type p53 induced resistance to Nutlin-3 in TC cells, demonstrating the key role of p53 for Nutlin-3 sensitivity. More specifically, our results indicate that p53-dependent induction of Fas membrane expression (∼threefold) and enhanced Fas/FasL interactions at the cell surface are important mechanisms of Nutlin-3-induced apoptosis in TC cells. Importantly, an analogous Fas-dependent mechanism of apoptosis upon Nutlin-3 treatment is executed in wild-type p53 expressing Hodgkin lymphoma and acute myeloid leukaemia cell lines. Finally, we demonstrate that Nutlin-3 strongly augmented cisplatin-induced apoptosis and cell kill via the Fas death receptor pathway. This effect is most pronounced in cisplatin-resistant TC cells.     

Nutlin-3a: A Potential Therapeutic Opportunity for TP53 Wild-Type Ovarian Carcinomas

Epithelial ovarian cancer is a diverse molecular and clinical disease, yet standard treatment is the same for all subtypes. TP53 mutations represent a node of divergence in epithelial ovarian cancer histologic subtypes and may represent a therapeutic opportunity in subtypes expressing wild type, including most low-grade ovarian serous carcinomas, ovarian clear cell carcinomas and ovarian endometrioid carcinomas, which represent approximately 25% of all epithelial ovarian cancer. We therefore sought to investigate Nutlin-3a—a therapeutic which inhibits MDM2, activates wild-type p53, and induces apoptosis—as a therapeutic compound for TP53 wild-type ovarian carcinomas. Fifteen epithelial ovarian cancer cell lines of varying histologic subtypes were treated with Nutlin-3a with determination of IC₅₀ values. Western Blot (WB) and quantitative real-time polymerase chain reaction (qRT-PCR) analyses quantified MDM2, p53, and p21 expression after Nutlin-3a treatment. DNA from 15 cell lines was then sequenced for TP53 mutations in exons 2-11 including intron-exon boundaries. Responses to Nutlin-3a were dependent upon TP53 mutation status. By qRT-PCR and WB, levels of MDM2 and p21 were upregulated in wild-type TP53 sensitive cell lines, and p21 induction was reduced or absent in mutant cell lines. Annexin V assays demonstrated apoptosis in sensitive cell lines treated with Nutlin-3a. Thus, Nutlin-3a could be a potential therapeutic agent for ovarian carcinomas expressing wild-type TP53 and warrants further investigation.     

MDM2 inhibitor Nutlin-3a suppresses proliferation and promotes apoptosis in osteosarcoma cells

Restoring p53 activity by inhibiting the interaction between p53 and the mouse double minutes clone 2 (MDM2) offers an attractive approach to cancer therapy. Nutlin-3a is a small-molecule inhibitor that inhibits MDM2 binding to p53 and subsequent p53-dependent DNA damage signaling. In this study, we determined the efficacy of Nutlin-3a in inducing p53-mediated cell death in osteosarcoma (OS) cell lines both in vivo and in vitro. Targeted disruption of the p53-MDM2 interaction by Nutlin-3a stabilizes p53 and selectively activates the p53 pathway only in OS cells with wild-type p53, resulting in a pronounced anti-proliferative and cytotoxic effect due to G1 cell cycle arrest and apoptosis both in vitro and in vivo. p53 dependence of these alternative outcomes of Nutlin-3a treatment was shown by the abrogation of these effects when p53 was knocked-down by small interfering RNA. These data suggest that the disruption of p53-MDM2 interaction by Nutlin-3a might be beneficial for OS patients with MDM2 amplification and wt p53 status.     

Nutlin-3 induces apoptosis,disrupts viral latency and inhibits expression of angiopoietin-2 in Kaposi sarcoma tumor cells

Kaposi sarcoma (KS) tumors often contain a wild-type p53. However, the function of this tumor suppressor in KS tumor cells is inhibited by both MDM2 and latent nuclear antigen (LANA) of Kaposi sarcoma-associated herpes virus (KSHV). Here, we report that MDM2 antagonist Nutlin-3 efficiently reactivates p53 in telomerase-immortalized human umbilical vein endothelial cells (TIVE) that had been malignantly transformed by KSHV as well as in KS tumor cells. Reactivation of p53 results in a G₁ cell cycle arrest, leading to inhibition of proliferation and apoptosis. Nutlin-3 inhibits the growth of “KS-like” tumors resulting from xenografted TIVE-KSHV cells in nude mice. In addition, Nutlin-3 strongly inhibits expression of the pro-angiogenic and pro-inflammatory cytokine angiopoietin-2 (Ang-2). It also disrupts viral latency by inducing expression of KSHV lytic genes. these results suggest that Nutlin-3 might serve as a novel therapy for KS.Key words: Kaposi sarcoma (KS), nutlin-3, p53, cell cycle arrest, apoptosis, angiopoietin-2     

p53 modulation as a therapeutic strategy in gastrointestinal stromal tumors

The KIT-inhibitor imatinib mesylate (IM) has greatly improved the treatment of metastatic gastrointestinal stromal tumors (GIST). IM exhibits strong antiproliferative effects but fails to induce sufficient levels of apoptosis resulting in low pathologic complete remission rates and a high rate of secondary progression in the metastatic setting. Upregulation of p53 by MDM2 inhibitors has been shown to induce apoptosis in p53 wildtype tumors. Analyzing a series of 62 mostly untreated, localized and metastatic GIST we detected a low rate (3%) of inactivating p53 mutations, thus providing a rationale for further exploration of p53-directed therapeutic strategies. To this end, we studied nutlin-3, an inhibitor of the p53 antagonist MDM2, and RITA, a putative p53 activator, in GIST cell lines. Nutlin-3 effectively induced p53 at therapeutically relevant levels, which resulted in moderate antiproliferative effects and cell cycle arrest in p53 wildtype GIST cell lines GIST430, GIST48 and GIST48B. P53 reactivation substantially improved the apoptotic response after effective KIT inhibition with sunitinib and 17-AAG in IM-resistant cell lines. The commonly used imatinib-sensitive cell lines GIST882 and GIST-T1 were shown to harbor defective p53 and therefore failed to respond to nutlin-3 treatment. RITA induced p53 in GIST48B, followed by antiproliferative effects and a strong induction of apoptosis. Surprisingly, GIST-T1 was also highly sensitive to RITA despite lacking functional p53. This suggested a more complex, p53-independent mechanism of action for the latter compound. No antagonistic effects from p53-activating drugs were seen with any drug combination. Our data provide first evidence that modulation of the MDM2/p53 pathway may be therapeutically useful to improve the apoptotic response of KIT-inhibitory drugs in the treatment of na?ve GIST, with p53 mutation status being a predictive factor of response.     

Weak p53 permits senescence during cell cycle arrest

Cell cycle arrest coupled with hyper-active mTOR leads to cellular senescence. While arresting cell cycle, high levels of p53 can inhibit mTOR (in some cell lines), thus causing reversible quiescence instead of senescence. Nutlin-3a-induced p53 inhibited mTOR and thus caused quiescence in WI-38 cells. In contrast, while arresting cell cycle, the DNA-damaging drug doxorubicin (DOX) did not inhibit mTOR and caused senescence. Super-induction of p53 by either nutlin-3a or high concentrations of DOX (high-DOX) prevented low-DOX-induced senescence, converting it into quiescence. This explains why in order to cause senescence, DNA damaging drugs must be used at low concentrations, which arrest cell cycle but do not induce p53 at levels sufficient to suppress mTOR. Noteworthy, very prolonged treatment with nutlin-3a also caused senescence preventable by rapamycin. In RPE cells, low concentrations of nutlin-3a caused a semi-senescent morphology. Higher concentrations of nutlin-3a inhibited mTOR and caused quiescent morphology. We conclude that low p53 levels during prolonged cell cycle arrest tend to cause senescence, whereas high levels of p53 tend to cause either quiescence or cell death.     

Cell-cycle arrest and senescence in TP53-wild type renal carcinoma by enhancer RNA-P53-bound enhancer regions 2 (p53BER2) in a p53-dependent pathway

TP53 is a classic tumor suppressor, but its role in kidney cancer remains unclear. In our study, we tried to explain the role of p53 in kidney cancer through the p53-related enhancer RNA pathway. Functional experiments were used to explore whether P53-bound enhancer regions 2 (p53BER2) has a role in the cell cycle and senescence response of TP53-wild type (WT) renal cancer cells in vitro or vivo. RNA-sequencing was used to identify the potential target of p53BER2. The results showed that the expression level of P53BER2 was downregulated in renal cancer tissues and cell lines, further dual-luciferase experiments and APR-256-reactivated experiments showed p53BER2 expresses in a p53-dependent way. Moreover, knockdown p53BER2 could reverse nutlin-3-induced cytotoxic effect in TP53-WT cell lines. Further exploration showed the downregulation of p53BER2 could reverse nutlin-3-induced G1-arrest and senescence in TP53-WT cell lines. What is more, the knockdown of p53BER2 showed resistance to nutlin-3 treatment in vivo. Additionally, we found BRCA2 could be regulated by p53BER2 in vitro and vivo; further experiment showed p53BER2 could induce cell-cycle arrest and DNA repair by mediating BRCA2. In summary, the p53-associated enhancer RNA-p53BER2 mediates the cell cycle and senescence of p53 in TP53-WT renal cancer cells.Subject terms: Tumour biomarkers, Renal cell carcinoma     

Treatment with a BH3 mimetic overcomes the resistance of latency III EBV (+) cells to p53-mediated apoptosis

P53 inactivation is often observed in Burkitt''s lymphoma (BL) cells due to mutations in the p53 gene or overexpression of its negative regulator, murine double minute-2 (MDM2). This event is now considered an essential part of the oncogenic process. Epstein–Barr virus (EBV) is strongly associated with BL and is a cofactor in its development. We previously showed that nutlin-3, an antagonist of MDM2, activates the p53 pathway in BL cell lines harboring wild-type p53. However, nutlin-3 strongly induced apoptosis in EBV (−) or latency I EBV (+) cells, whereas latency III EBV (+) cells were much more resistant. We show here that this resistance to apoptosis is also observed in latency III EBV (+) lymphoblastoid cell lines. We also show that, in latency III EBV (+) cells, B-cell lymphona 2 (Bcl-2) is selectively overproduced and interacts with Bcl-2-associated X protein (Bax), preventing its activation. The treatment of these cells with the Bcl-2-homology domain 3 mimetic ABT-737 disrupts Bax/Bcl-2 interaction and allows Bax activation by nutlin-3. Furthermore, treatment with these two compounds strongly induces apoptosis. Thus, a combination of Mdm2 and Bcl-2 inhibitors might be a useful anti-cancer strategy for diseases linked to EBV infection.     

Nutlin-3 downregulates p53 phosphorylation on serine392 and induces apoptosis in hepatocellular carcinoma cells

Drug-resistance and imbalance of apoptotic regulation limit chemotherapy clinical application for the human hepatocellular carcinoma (HCC) treatment. The reactivation of p53 is an attractive therapeutic strategy in cancer with disrupted-p53 function. Nutlin-3, a MDM2 antagonist, has antitumor activity in various cancers. The post-translational modifications of p53 are a hot topic, but there are some controversy ideas about the function of phospho-Ser³⁹²-p53 protein in cancer cell lines in response to Nutlin-3. Therefore, we investigated the relationship between Nutlin-3 and phospho-Ser³⁹²-p53 protein expression levels in SMMC-7721 (wild-type TP53) and HuH-7 cells (mutant TP53). We demonstrated that Nutlin-3 induced apoptosis through down-regulation phospho-Ser³⁹²-p53 in two HCC cells. The result suggests that inhibition of p53 phosphorylation on Ser³⁹² presents an alternative for HCC chemotherapy. [BMB Reports 2014; 47(4): 221-226]     

Persistent p21 Expression after Nutlin-3a Removal Is Associated with Senescence-like Arrest in 4N Cells

Nutlin-3a is a preclinical drug that stabilizes p53 by blocking the interaction between p53 and MDM2. In our previous study, Nutlin-3a promoted a tetraploid G₁ arrest in two p53 wild-type cell lines (HCT116 and U2OS), and both cell lines underwent endoreduplication after Nutlin-3a removal. Endoreduplication gave rise to stable tetraploid clones resistant to therapy-induced apoptosis. Prior knowledge of whether cells are susceptible to Nutlin-induced endoreduplication and therapy resistance could help direct Nutlin-3a-based therapies. In the present study, Nutlin-3a promoted a tetraploid G₁ arrest in multiple p53 wild-type cell lines. However, some cell lines underwent endoreduplication to relatively high extents after Nutlin-3a removal whereas other cell lines did not. The resistance to endoreduplication observed in some cell lines was associated with a prolonged 4N arrest after Nutlin-3a removal. Knockdown of either p53 or p21 immediately after Nutlin-3a removal could drive endoreduplication in otherwise resistant 4N cells. Finally, 4N-arrested cells retained persistent p21 expression; expressed senescence-associated β-galactosidase; displayed an enlarged, flattened phenotype; and underwent a proliferation block that lasted at least 2 weeks after Nutlin-3a removal. These findings demonstrate that transient Nutlin-3a treatment can promote an apparently permanent proliferative block in 4N cells of certain cell lines associated with persistent p21 expression and resistance to endoreduplication.     

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.     

An shRNA barcode screen provides insight into cancer cell vulnerability to MDM2 inhibitors

The identification of the cellular targets of small molecules with anticancer activity is crucial to their further development as drug candidates. Here, we present the application of a large-scale RNA interference-based short hairpin RNA (shRNA) barcode screen to gain insight in the mechanism of action of nutlin-3 (1). Nutlin-3 is a small-molecule inhibitor of MDM2, which can activate the p53 pathway. Nutlin-3 shows strong antitumor effects in mice, with surprisingly few side effects on normal tissues. Aside from p53, we here identify 53BP1 as a critical mediator of nutlin-3-induced cytotoxicity. 53BP1 is part of a signaling network induced by DNA damage that is frequently activated in cancer but not in healthy tissues. Our results suggest that nutlin-3's tumor specificity may result from its ability to turn a cancer cell-specific property (activated DNA damage signaling) into a weakness that can be exploited therapeutically.     

Enhancement of imatinib-induced apoptosis of BCR/ABL-expressing cells by nutlin-3 through synergistic activation of the mitochondrial apoptotic pathway

The BCR/ABL tyrosine kinase inhibitor imatinib is highly effective for treatment of chronic myeloid leukemia (CML) and Philadelphia-chromosome positive (Ph+) acute lymphoblastic leukemia (ALL). However, relapses with emerging imatinib-resistance mutations in the BCR/ABL kinase domain pose a significant problem. Here, we demonstrate that nutlin-3, an inhibitor of Mdm2, inhibits proliferation and induces apoptosis more effectively in BCR/ABL-driven Ton.B210 cells than in those driven by IL-3. Moreover, nutlin-3 drastically enhanced imatinib-induced apoptosis in a p53-dependent manner in various BCR/ABL-expressing cells, which included primary leukemic cells from patients with CML blast crisis or Ph+ ALL and cells expressing the imatinib-resistant E255K BCR/ABL mutant. Nutlin-3 and imatinib synergistically induced Bax activation, mitochondrial membrane depolarization, and caspase-3 cleavage leading to caspase-dependent apoptosis, which was inhibited by overexpression of Bcl-XL. Imatinib did not significantly affect the nutlin-3-induced expression of p53 but abrogated that of p21. Furthermore, activation of Bax as well as caspase-3 induced by combined treatment with imatinib and nutlin-3 was observed preferentially in cells expressing p21 at reduced levels. The present study indicates that combined treatment with nutlin-3 and imatinib activates p53 without inducing p21 and synergistically activates Bax-mediated intrinsic mitochondrial pathway to induce apoptosis in BCR/ABL-expressing cells.     

Nutlin-3 protects kidney cells during cisplatin therapy by suppressing Bax/Bak activation

Nutlins, the newly developed small molecule antagonists of MDM2, activate p53 and induce apoptosis in cancer cells, offering a novel strategy of chemotherapy. Recent studies have further suggested synergistic effects of nutlins with other chemotherapeutic drugs. However, it is unclear whether nutlins increase or decrease the side effects of these drugs in normal non-malignant cells or tissues. Cisplatin is a widely used chemotherapy drug, which has a major side effect of kidney injury. Here we show that Nutlin-3 protected kidney cells against cisplatin-induced apoptosis. The cytoprotective effects of Nutlin-3 were not related to its regulation of p53 or consequent gene expression during cisplatin treatment. Moreover, the protective effects were shown in MDM2-, MDM4-, or p53-deficient cells. On the other hand, Nutlin-3 suppressed mitochondrial events of apoptosis during cisplatin incubation, including Bax activation and cytochrome c release. Nutlin-3 attenuated cisplatin-induced oligomerization of Bax and Bak but not their interactions with Bcl-XL. In isolated mitochondria, Nutlin-3 inhibited cytochrome c release induced by Ca2+, Bim peptide, and recombinant tBid. Importantly, it blocked both Bax and Bak oligomerization under these conditions. Together, the results have uncovered a new pharmacological function of nutlins, i.e. suppression of Bax and Bak, two critical mediators of apoptosis.     

Galectin-3 Impairment of MYCN-Dependent Apoptosis-Sensitive Phenotype Is Antagonized by Nutlin-3 in Neuroblastoma Cells

MYCN amplification occurs in about 20–25% of human neuroblastomas and characterizes the majority of the high-risk cases, which display less than 50% prolonged survival rate despite intense multimodal treatment. Somehow paradoxically, MYCN also sensitizes neuroblastoma cells to apoptosis, understanding the molecular mechanisms of which might be relevant for the therapy of MYCN amplified neuroblastoma. We recently reported that the apoptosis-sensitive phenotype induced by MYCN is linked to stabilization of p53 and its proapoptotic kinase HIPK2. In MYCN primed neuroblastoma cells, further activation of both HIPK2 and p53 by Nutlin-3 leads to massive apoptosis in vitro and to tumor shrinkage and impairment of metastasis in xenograft models. Here we report that Galectin-3 impairs MYCN-primed and HIPK2-p53-dependent apoptosis in neuroblastoma cells. Galectin-3 is broadly expressed in human neuroblastoma cell lines and tumors and is repressed by MYCN to induce the apoptosis-sensitive phenotype. Despite its reduced levels, Galectin-3 can still exert residual antiapoptotic effects in MYCN amplified neuroblastoma cells, possibly due to its specific subcellular localization. Importantly, Nutlin-3 represses Galectin-3 expression, and this is required for its potent cell killing effect on MYCN amplified cell lines. Our data further characterize the apoptosis-sensitive phenotype induced by MYCN, expand our understanding of the activity of MDM2-p53 antagonists and highlight Galectin-3 as a potential biomarker for the tailored p53 reactivation therapy in patients with high-risk neuroblastomas.     

Inhibition of nonsense-mediated decay rescues p53β/γ isoform expression and activates the p53 pathway in MDM2-overexpressing and select p53-mutant cancers

Inactivation of p53 is present in almost every tumor, and hence, p53-reactivation strategies are an important aspect of cancer therapy. Common mechanisms for p53 loss in cancer include expression of p53-negative regulators such as MDM2, which mediate the degradation of wildtype p53 (p53α), and inactivating mutations in the TP53 gene. Currently, approaches to overcome p53 deficiency in these cancers are limited. Here, using non–small cell lung cancer and glioblastoma multiforme cell line models, we show that two alternatively spliced, functional truncated isoforms of p53 (p53β and p53γ, comprising exons 1 to 9β or 9γ, respectively) and that lack the C-terminal MDM2-binding domain have markedly reduced susceptibility to MDM2-mediated degradation but are highly susceptible to nonsense-mediated decay (NMD), a regulator of aberrant mRNA stability. In cancer cells harboring MDM2 overexpression or TP53 mutations downstream of exon 9, NMD inhibition markedly upregulates p53β and p53γ and restores activation of the p53 pathway. Consistent with p53 pathway activation, NMD inhibition induces tumor suppressive activities such as apoptosis, reduced cell viability, and enhanced tumor radiosensitivity, in a relatively p53-dependent manner. In addition, NMD inhibition also inhibits tumor growth in a MDM2-overexpressing xenograft tumor model. These results identify NMD inhibition as a novel therapeutic strategy for restoration of p53 function in p53-deficient tumors bearing MDM2 overexpression or p53 mutations downstream of exon 9, subgroups that comprise approximately 6% of all cancers.     

Nutlin-3 induces apoptosis,disrupts viral latency and inhibits expression of angiopoietin-2 in Kaposi sarcoma tumor cells

Kaposi sarcoma (KS) tumors often contain a wild-type p53. However, the function of this tumor suppressor in KS tumor cells is inhibited by both MDM2 and latent nuclear antigen (LANA) of Kaposi sarcoma-associated herpes virus (KSHV). Here, we report that MDM2 antagonist Nutlin-3 efficiently reactivates p53 in telomerase-immortalized human umbilical vein endothelial cells (TIVE) that had been malignantly transformed by KSHV as well as in KS tumor cells. Reactivation of p53 results in a G₁ cell cycle arrest, leading to inhibition of proliferation and apoptosis. Nutlin-3 inhibits the growth of “KS-like” tumors resulting from xenografted TIVE-KSHV cells in nude mice. In addition, Nutlin-3 strongly inhibits expression of the pro-angiogenic and pro-inflammatory cytokine angiopoietin-2 (Ang-2). It also disrupts viral latency by inducing expression of KSHV lytic genes. These results suggest that Nutlin-3 might serve as a novel therapy for KS.     

(R)-roscovitine (CYC202, Seliciclib) sensitizes SH-SY5Y neuroblastoma cells to nutlin-3-induced apoptosis

In this study, we have analyzed the consequences, on several neuroblastoma cell lines, of combined treatments with (R)-roscovitine (CYC202, Seliciclib), a CDK inhibitory drug, and nutlin-3, a p53 activating drug. Both compounds were found to synergize, causing significant levels of apoptosis in cultured cells when combined at sublethal concentrations. In SH-SY5Y cells, Bcl-XL protein overexpression protected from apoptosis induced by either nutlin-3 alone or the (R)-roscovitine plus nutlin-3 association but failed to prevent apoptosis triggered by (R)-roscovitine alone. Moreover, Western blot studies showed that (R)-roscovitine increased nutlin-3-mediated p53 stabilization. Therefore, we conclude the contribution of (R)-roscovitine to the synergism is basically the sensitization of SH-SY5Y cells to the action of nutlin-3 on p53. The relevance of this pharmacological synergism with respect to the treatment of neuroblastoma is discussed.