Caffeic acid phenethyl ester (CAPE) confers wild type p53 function in p53Y220C mutant: bioinformatics and experimental evidence

Mutations in the tumor suppressor protein p53 is a prevalent feature in majority of cancers resulting in inactivation of its activities related to control of cell cycle progression and proliferation. p53Y220C is one of the common hotspot mutations that causes decrease in its thermodynamic stability. Some small molecules have been shown to bind to the mutated site and restore its wild type thermodynamics and tumor suppressor function. In this study, we have explored the potential of caffeic acid phenethyl ester (CAPE—a bioactive compound from propolis) to interact with p53Y220C and restore its wild type p53 (p53wt) transcription activation and tumor suppressor activities. We recruited computational methods, viz. molecular docking, molecular dynamics simulations and free energy calculations to study the interaction of CAPE at the mutation crevice and found that it has potential to restore p53wt function of the p53Y220C mutant similar to a previously described restoration molecule PK7242. We provide cell-based experimental evidence to these predictions and suggest CAPE as a potential natural drug for treatment of p53Y220C mutant harboring cancers.

Mutant p53 elicits context-dependent pro-tumorigenic phenotypes

The tumor suppressor gene TP53 is the most frequently mutated gene in numerous cancer types, including prostate cancer (PCa). Specifically, missense mutations in TP53 are selectively enriched in PCa, and cluster to particular “hot spots” in the p53 DNA binding domain with mutation at the R273 residue occurring most frequently. While this residue is similarly mutated to R273C-p53 or R273H-p53 in all cancer types examined, in PCa selective enrichment of R273C-p53 is observed. Importantly, examination of clinical datasets indicated that TP53 heterozygosity can either be maintained or loss of heterozygosity (LOH) occurs. Thus, to mimic tumor-associated mutant p53, R273C-p53 and R273H-p53 isogenic PCa models were developed in the presence or absence of wild-type p53. In the absence of wild-type p53, both R273C-p53 and R273H-p53 exhibited similar loss of DNA binding, transcriptional profiles, and loss of canonical tumor suppressor functions associated with wild-type p53. In the presence of wild-type p53 expression, both R273C-p53 and R273H-p53 supported canonical p53 target gene expression yet elicited distinct cistromic and transcriptional profiles when compared to each other. Moreover, heterozygous modeling of R273C-p53 or R273H-p53 expression resulted in distinct phenotypic outcomes in vitro and in vivo. Thus, mutant p53 acts in a context-dependent manner to elicit pro-tumorigenic transcriptional profiles, providing critical insight into mutant p53-mediated prostate cancer progression.

Wild-type p53-induced phosphatase sensitizes acute myeloid leukemia cells to conventional chemotherapy

Recently wild-type p53-induced phosphatase was implicated in the pathogenesis of acute myeloid leukemia (AML) and “pre-leukemia” myeloproliferative conditions. Here we decided to check how the strategy directed to phosphatase inhibition affected sensitivity to conventional chemotherapy. All experiments were conducted on AML cell lines cultivated in vitro. The levels of wild-type p53-induced phosphatase vary in different AML cell lines. The chemical compound GSK2830371 reduced levels of phosphatase and diminished its activity. GSK2830371 did not significantly change the cell cycle distribution of AML cells when used alone or in combination with the anti-cancer chemotherapeutic drug Cytosar but increased caspase-dependent PARP1 cleavage. In contrast with previous studies, we did not observe the negative effect of phosphatase activity inhibition and depletion on cells when a chemical inhibitor was used as monotherapy. Using a combination of GSK2830371 with Cytosar we were able to reduce the threshold of chemotherapy-dependent cytotoxicity and more efficiently eliminate leukemic cells. We propose considering inhibition of wild-type p53-induced phosphatase as a prospective strategy in improving anti-AML therapy.

Impact of Intravesical instillation of a Novel Biological Response Modifier (P-MAPA) on Progress of Non-Muscle Invasive Bladder Cancer Treatment in a rat model

This work describes the effects of immunotherapy with Protein Aggregate Magnesium-Ammonium Phospholinoleate-Palmitoleate Anhydride (P-MAPA) in the treatment of non-muscle invasive bladder cancer (NMIBC) in an animal model. NMIBC was induced by treating female Fischer 344 rats with N-methyl-N-nitrosourea (MNU). After treatment with MNU, the rats were distributed into four experimental groups: Control (without MNU) group, MNU (cancer) group, MNU-BCG (Bacillus Calmette-Guerin) group and MNU-P-MAPA group. P-MAPA intravesical treatment was more effective in histopathological recovery from cancer state in relation to BCG treatment. Western blot assays showed an increase in the protein levels of c-Myc, COUP-TFII and wild-type p53 in P-MAPA-treated rats in relation to BCG-treated rats. In addition, rats treated with P-MAPA intravesical immunotherapy showed the highest BAX protein levels and the lowest proliferation/apoptotic ratio in relation to BCG-treated rats, pointing out a preponderance of apoptosis. P-MAPA intravesical treatment increased the wild-type p53 levels and enhanced c-Myc/COUP-TFII-induced apoptosis mediated by p53. These alterations were fundamental for histopathological recovery from cancer and for suppress abnormal cell proliferation. This action of P-MAPA on apoptotic pathways may represent a new strategy for treating NMIBC.

MDM2, MDMX, and p73 regulate cell-cycle progression in the absence of wild-type p53

The p53 tumor suppressor protein, known to be critically important in several processes including cell-cycle arrest and apoptosis, is highly regulated by multiple mechanisms, most certifiably the Murine Double Minute 2–Murine Double Minute X (MDM2–MDMX) heterodimer. The role of MDM2–MDMX in cell-cycle regulation through inhibition of p53 has been well established. Here we report that in cells either lacking p53 or expressing certain tumor-derived mutant forms of p53, loss of endogenous MDM2 or MDMX, or inhibition of E3 ligase activity of the heterocomplex, causes cell-cycle arrest. This arrest is correlated with a reduction in E2F1, E2F3, and p73 levels. Remarkably, direct ablation of endogenous p73 produces a similar effect on the cell cycle and the expression of certain E2F family members at both protein and messenger RNA levels. These data suggest that MDM2 and MDMX, working at least in part as a heterocomplex, may play a p53-independent role in maintaining cell-cycle progression by promoting the activity of E2F family members as well as p73, making them a potential target of interest in cancers lacking wild-type p53.

Targeting mutant p53 for cancer therapy: direct and indirect strategies

TP53 is a critical tumor-suppressor gene that is mutated in more than half of all human cancers. Mutations in TP53 not only impair its antitumor activity, but also confer mutant p53 protein oncogenic properties. The p53-targeted therapy approach began with the identification of compounds capable of restoring/reactivating wild-type p53 functions or eliminating mutant p53. Treatments that directly target mutant p53 are extremely structure and drug-species-dependent. Due to the mutation of wild-type p53, multiple survival pathways that are normally maintained by wild-type p53 are disrupted, necessitating the activation of compensatory genes or pathways to promote cancer cell survival. Additionally, because the oncogenic functions of mutant p53 contribute to cancer proliferation and metastasis, targeting the signaling pathways altered by p53 mutation appears to be an attractive strategy. Synthetic lethality implies that while disruption of either gene alone is permissible among two genes with synthetic lethal interactions, complete disruption of both genes results in cell death. Thus, rather than directly targeting p53, exploiting mutant p53 synthetic lethal genes may provide additional therapeutic benefits. Additionally, research progress on the functions of noncoding RNAs has made it clear that disrupting noncoding RNA networks has a favorable antitumor effect, supporting the hypothesis that targeting noncoding RNAs may have potential synthetic lethal effects in cancers with p53 mutations. The purpose of this review is to discuss treatments for cancers with mutant p53 that focus on directly targeting mutant p53, restoring wild-type functions, and exploiting synthetic lethal interactions with mutant p53. Additionally, the possibility of noncoding RNAs acting as synthetic lethal targets for mutant p53 will be discussed.

Wild-Type p53-Induced Phosphatase 1 Plays a Positive Role in Hematopoiesis in the Mouse Embryonic Head

The first adult repopulating hematopoietic stem cells (HSCs) are found in the aorta-gonad-mesonephros (AGM) region, which are produced from hemogenic endothelial cells. Embryonic head is the other site for HSC development. Wild-type p53-induced phosphatase 1 (Wip1) is a type-2Cδ family serine/threonine phosphatase involved in various cellular processes such as lymphoid development and differentiation of adult HSCs. Most recently, we have shown that Wip1 modulates the pre-HSC maturation in the AGM region. However, it is not clear whether Wip1 regulates hematopoiesis in the embryonic head. Here we reported that disruption of Wip1 resulted in a decrease of hematopoietic progenitor cell number in the embryonic head. In vivo transplantation assays showed a reduction of HSC function after Wip1 ablation. We established that Wip1 deletion reduced the frequency and cell number of microglia in the embryonic head. Further observations revealed that Wip1 absence enhanced the gene expression of microglia-derived pro-inflammatory factors. Thus, it is likely that Wip1 functions as a positive regulator in HSC development by regulating the function of microglia in the embryonic head.

The Effect of Different Ester Chain Modifications of Two Guaianolides for Inhibition of Colorectal Cancer Cell Growth

Several sesquiterpene lactones (STLs) have been tested as lead drugs in cancer clinical trials. Salograviolide-A (Sal-A) and salograviolide-B (Sal-B) are two STLs that have been isolated from Centaurea ainetensis, an indigenous medicinal plant of the Middle Eastern region. The parent compounds Sal-A and Sal-B were modified and successfully prepared into eight novel guaianolide-type STLs (compounds 1–8) bearing ester groups of different geometries. Sal-A, Sal-B, and compounds 1–8 were tested against a human colorectal cancer cell line model with differing p53 status; HCT116 with wild-type p53 and HCT116 p53−/− null for p53, and the normal-like human colon mucosa cells with wild-type p53, NCM460. IC50 values indicated that derivatization of Sal-A and Sal-B resulted in potentiation of HCT116 cell growth inhibition by 97% and 66%, respectively. The effects of the different molecules on cancer cell growth were independent of p53 status. Interestingly, the derivatization of Sal-A and Sal-B molecules enhanced their anti-growth properties versus 5-Fluorouracil (5-FU), which is the drug of choice in colorectal cancer. Structure-activity analysis revealed that the enhanced molecule potencies were mainly attributed to the position and number of the hydroxy groups, the lipophilicity, and the superiority of ester groups over hydroxy substituents in terms of their branching and chain lengths. The favorable cytotoxicity and selectivity of the potent molecules, to cancer cells versus their normal counterparts, pointed them out as promising leads for anti-cancer drug design.

The Function of the Mutant p53-R175H in Cancer

Wild-type p53 is known as “the guardian of the genome” because of its function of inducing DNA repair, cell-cycle arrest, and apoptosis, preventing the accumulation of gene mutations. TP53 is highly mutated in cancer cells and most TP53 hotspot mutations are missense mutations. Mutant p53 proteins, encoded by these hotspot mutations, lose canonical wild-type p53 functions and gain functions that promote cancer development, including promoting cancer cell proliferation, migration, invasion, initiation, metabolic reprogramming, angiogenesis, and conferring drug resistance to cancer cells. Among these hotspot mutations, p53-R175H has the highest occurrence. Although losing the transactivating function of the wild-type p53 and prone to aggregation, p53-R175H gains oncogenic functions by interacting with many proteins. In this review, we summarize the gain of functions of p53-R175H in different cancer types, the interacting proteins of p53-R175H, and the downstream signaling pathways affected by p53-R175H to depict a comprehensive role of p53-R175H in cancer development. We also summarize treatments that target p53-R175H, including reactivating p53-R175H with small molecules that can bind to p53-R175H and alter it into a wild-type-like structure, promoting the degradation of p53-R175H by targeting heat-shock proteins that maintain the stability of p53-R175H, and developing immunotherapies that target the p53-R175H–HLA complex presented by tumor cells.