Targeting of p53 and its homolog p73 by protoporphyrin IX

The p53 tumor suppressor is recognized as a promising target for anti-cancer therapies. We previously reported that protoporphyrin IX (PpIX) disrupts the p53/murine double minute 2 (MDM2) complex and leads to p53 accumulation and activation of apoptosis in HCT 116 cells. Here we show the direct binding of PpIX to the N-terminal domain of p53. Furthermore, we addressed the induction of apoptosis in HCT 116 p53-null cells by PpIX and revealed interactions between PpIX and p73. We propose that PpIX disrupts the p53/MDM2 or MDMX and p73/MDM2 complexes and thereby activates the p53- or p73-dependent cancer cell death.     

Novel Roles for P53 in the Genesis and Targeting of Tetraploid Cancer Cells

Tetraploid (4N) cells are considered important in cancer because they can display increased tumorigenicity, resistance to conventional therapies, and are believed to be precursors to whole chromosome aneuploidy. It is therefore important to determine how tetraploid cancer cells arise, and how to target them. P53 is a tumor suppressor protein and key regulator of tetraploidy. As part of the “tetraploidy checkpoint”, p53 inhibits tetraploid cell proliferation by promoting a G1-arrest in incipient tetraploid cells (referred to as a tetraploid G1 arrest). Nutlin-3a is a preclinical drug that stabilizes p53 by blocking the interaction between p53 and MDM2. In the current study, Nutlin-3a promoted a p53-dependent tetraploid G1 arrest in two diploid clones of the HCT116 colon cancer cell line. Both clones underwent endoreduplication after Nutlin removal, giving rise to stable tetraploid clones that showed increased resistance to ionizing radiation (IR) and cisplatin (CP)-induced apoptosis compared to their diploid precursors. These findings demonstrate that transient p53 activation by Nutlin can promote tetraploid cell formation from diploid precursors, and the resulting tetraploid cells are therapy (IR/CP) resistant. Importantly, the tetraploid clones selected after Nutlin treatment expressed approximately twice as much P53 and MDM2 mRNA as diploid precursors, expressed approximately twice as many p53-MDM2 protein complexes (by co-immunoprecipitation), and were more susceptible to p53-dependent apoptosis and growth arrest induced by Nutlin. Based on these findings, we propose that p53 plays novel roles in both the formation and targeting of tetraploid cells. Specifically, we propose that 1) transient p53 activation can promote a tetraploid-G1 arrest and, as a result, may inadvertently promote formation of therapy-resistant tetraploid cells, and 2) therapy-resistant tetraploid cells, by virtue of having higher P53 gene copy number and expressing twice as many p53-MDM2 complexes, are more sensitive to apoptosis and/or growth arrest by anti-cancer MDM2 antagonists (e.g. Nutlin).     

Overexpression of the Pituitary Tumor Transforming Gene Induces p53-dependent Senescence through Activating DNA Damage Response Pathway in Normal Human Fibroblasts

Pituitary tumor transforming gene (PTTG1, securin) is involved in cell-cycle control through inhibition of sister-chromatid separation. Elevated levels of PTTG1 were found to be associated with many different tumor types that might be involved in late stage tumor progression. However, the role of PTTG1 in early stage of tumorigenesis is unclear. Here we utilized the adenovirus expression system to deliver PTTG1 into normal human fibroblasts to evaluate the role of PTTG1 in tumorigenesis. Expressing PTTG1 in normal human fibroblasts inhibited cell proliferation. Several senescence-associated (SA) phenotypes including increased SA-β-galactosidase activities, decreased bromodeoxyuridine incorporation, and increased SA-heterochromatin foci formation were also observed in PTTG1-expressing cells, indicating that PTTG1 overexpression induced a senescent phenotype in normal cells. Significantly, the PTTG1-induced senescence is p53-dependent and telomerase-independent, which is distinctively different from that of replicative senescence. The mechanism of PTTG1-induced senescence was also analyzed. Consistent with its role in regulating sister-chromatid separation, overexpression of PTTG1 inhibited the activation of separase. Consequently, the numbers of cells with abnormal nuclei morphologies and chromosome separations were increased, which resulted in activation of the DNA damage response. Thus, we concluded that PTTG1 overexpression in normal human fibroblasts caused chromosome instability, which subsequently induced p53-dependent senescence through activation of DNA-damage response pathway.     

Regulation of p53 stability and function in HCT116 colon cancer cells

We have used a lentiviral vector to stably express p53 at a physiological level in p53 knockout HCT116 cells. Cells transduced with wild type p53 responded to genotoxic stress by stabilizing p53 and expressing p53 target genes. The reconstituted cells underwent G(1) arrest or apoptosis appropriately depending on the type of stress, albeit less efficiently than parental wild type cells. Compared with cells expressing exogenous wild type p53, the apoptotic response to 5-fluorouracil (5FU) was >50% reduced in cells expressing S15A or S20A mutant p53, and even more reduced by combined mutation of serines 6, 9, 15, 20, 33, and 37 (N6A). Among a panel of p53 target genes tested by quantitative PCR, the gene showing the largest defect in induction by 5FU was BBC3 (PUMA), which was induced 4-fold by wild type p53 and 2-fold by the N6A mutant. Mutation of N-terminal phosphorylation sites did not prevent p53 stabilization by doxorubicin or 5FU. MDM2 silencing by RNA interference activated p53 target gene expression in normal fibroblasts but not in HCT116 cells, and exogenous p53 could be stabilized in HCT116 knockout cells despite combined mutation of p53 phosphorylation sites and silencing of MDM2 expression. The MDM2 feedback loop is thus defective, and other mechanisms must exist to regulate p53 stability and function in this widely used tumor cell line.     

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.     

PRPF19 regulates p53-dependent cellular senescence by modulating alternative splicing of MDM4 mRNA

Alteration of RNA splicing is a hallmark of cellular senescence, which is associated with age-related disease and cancer development. However, the roles of splicing factors in cellular senescence are not fully understood. In this study, we identified the splicing factor PRPF19 as a critical regulator of cellular senescence in normal human diploid fibroblasts. PRPF19 was downregulated during replicative senescence, and PRPF19 knockdown prematurely induced senescence-like cell cycle arrest through the p53–p21 pathway. RNA-sequencing analysis revealed that PRPF19 knockdown caused a switch of the MDM4 splicing isoform from stable full-length MDM4-FL to unstable MDM4-S lacking exon 6. We also found that PRPF19 regulates MDM4 splicing by promoting the physical interaction of other splicing factors, PRPF3 and PRPF8, which are key components of the core spliceosome, U4/U6.U5 tri-snRNP. Given that MDM4 is a major negative regulator of p53, our findings imply that PRPF19 downregulation inhibits MDM4-mediated p53 inactivation, resulting in induction of cellular senescence. Thus, PRPF19 plays an important role in the induction of p53-dependent cellular senescence.     

4-Hydroxynonenal induces p53-mediated apoptosis in retinal pigment epithelial cells

4-Hydroxynonenal (4-HNE) has been suggested to be involved in stress-induced signaling for apoptosis. In present studies, we have examined the effects of 4-HNE on the intrinsic apoptotic pathway associated with p53 in human retinal pigment epithelial (RPE and ARPE-19) cells. Our results show that 4-HNE causes induction, phosphorylation, and nuclear accumulation of p53 which is accompanied with down regulation of MDM2, activation of the pro-apoptotic p53 target genes viz. p21 and Bax, JNK, caspase3, and onset of apoptosis in treated RPE cells. Reduced expression of p53 by an efficient silencing of the p53 gene resulted in a significant resistance of these cells to 4-HNE-induced cell death. The effects of 4-HNE on the expression and functions of p53 are blocked in GSTA4-4 over expressing cells indicating that 4-HNE-induced, p53-mediated signaling for apoptosis is regulated by GSTs. Our results also show that the induction of p53 in tissues of mGsta4 (−/−) mice correlate with elevated levels of 4-HNE due to its impaired metabolism. Together, these studies suggest that 4-HNE is involved in p53-mediated signaling in in vitro cell cultures as well as in vivo that can be regulated by GSTs.     

Small molecule RITA binds to p53, blocks p53-HDM-2 interaction and activates p53 function in tumors

In tumors that retain wild-type p53, its tumor-suppressor function is often impaired as a result of the deregulation of HDM-2, which binds to p53 and targets it for proteasomal degradation. We have screened a chemical library and identified a small molecule named RITA (reactivation of p53 and induction of tumor cell apoptosis), which bound to p53 and induced its accumulation in tumor cells. RITA prevented p53-HDM-2 interaction in vitro and in vivo and affected p53 interaction with several negative regulators. RITA induced expression of p53 target genes and massive apoptosis in various tumor cells lines expressing wild-type p53. RITA suppressed the growth of human fibroblasts and lymphoblasts only upon oncogene expression and showed substantial p53-dependent antitumor effect in vivo. RITA may serve as a lead compound for the development of an anticancer drug that targets tumors with wild-type p53.     

The absence of Ser389 phosphorylation in p53 affects the basal gene expression level of many p53-dependent genes and alters the biphasic response to UV exposure in mouse embryonic fibroblasts

Phosphorylation is important in p53-mediated DNA damage responses. After UV irradiation, p53 is phosphorylated specifically at murine residue Ser389. Phosphorylation mutant p53.S389A cells and mice show reduced apoptosis and compromised tumor suppression after UV irradiation. We investigated the underlying cellular processes by time-series analysis of UV-induced gene expression responses in wild-type, p53.S389A, and p53^−/− mouse embryonic fibroblasts. The absence of p53.S389 phosphorylation already causes small endogenous gene expression changes for 2,253, mostly p53-dependent, genes. These genes showed basal gene expression levels intermediate to the wild type and p53^−/−, possibly to readjust the p53 network. Overall, the p53.S389A mutation lifts p53-dependent gene repression to a level similar to that of p53^−/− but has lesser effect on p53-dependently induced genes. In the wild type, the response of 6,058 genes to UV irradiation was strictly biphasic. The early stress response, from 0 to 3 h, results in the activation of processes to prevent the accumulation of DNA damage in cells, whereas the late response, from 12 to 24 h, relates more to reentering the cell cycle. Although the p53.S389A UV gene response was only subtly changed, many cellular processes were significantly affected. The early response was affected the most, and many cellular processes were phase-specifically lost, gained, or altered, e.g., induction of apoptosis, cell division, and DNA repair, respectively. Altogether, p53.S389 phosphorylation seems essential for many p53 target genes and p53-dependent processes.     

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.     

MDMX overexpression prevents p53 activation by the MDM2 inhibitor Nutlin

The p53 tumor suppressor plays a key role in maintaining genomic stability and protection against malignant transformation. MDM2 and MDMX are both p53-binding proteins that regulate p53 stability and activity. Recent development of the MDM2 inhibitor Nutlin 3 has greatly facilitated functional analysis of MDM2-p53 binding. We found that although MDMX is homologous to MDM2 and binds to the same region on p53 N terminus, Nutlin does not disrupt p53-MDMX interaction. The ability of Nutlin to activate p53 is compromised in tumor cells overexpressing MDMX. Combination of Nutlin with MDMX siRNA resulted in synergistic activation of p53 and growth arrest. These results suggest that MDMX is also a valid target for p53 activation in tumor cells. Development of novel compounds that are MDMX-specific or optimized for dual-inhibition of MDM2 and MDMX are necessary to achieve full activation of p53 in tumor cells.