Buxus alkaloid compound destabilizes mutant p53 through inhibition of the HSF1 chaperone axis

BackgroundP53 is the most frequently mutated gene in most tumour types, and the mutant p53 protein accumulates at high levels in tumours to promote tumour development and progression. Thus, targeting mutant p53 for degradation is one of the therapeutic strategies used to manage tumours that depend on mutant p53 for survival. Buxus alkaloids are traditionally used in the treatment of cardiovascular diseases. We found that triterpenoid alkaloids extracted from Buxus sinica found in the Yunnan Province exhibit anticancer activity by depleting mutant p53 levels in colon cancer cells.PurposeTo explore the anticancer mechanism of action of the triterpenoid alkaloid KBA01 compound by targeting mutant p53 degradation.Study design and methodsDifferent mutant p53 cell lines were used to evaluate the anticancer activity of KBA01. MTT assay, colony formation assay and cell cycle analysis were performed to examine the effect of KBA01 on cancer cell proliferation. Western blotting and qPCR were used to investigate effects of depleting mutant p53, and a ubiquitination assay was used to determine mutant p53 ubiquitin levels after cells were treated with the compound. Co-IP and small interfering RNA assays were used to explore the effects of KBA01 on the interaction of Hsp90 with mutant p53.ResultsThe triterpenoid alkaloid KBA01 can induce G2/M cell cycle arrest and the apoptosis of HT29 colon cancer cells. KBA01 decreases the stability of DNA contact mutant p53 proteins through the proteasomal pathway with minimal effects on p53 mutant protein conformation. Moreover, KBA01 enhances the interaction of mutant p53 with Hsp70, CHIP and MDM2, and knocking down CHIP and MDM2 stabilizes mutant p53 levels in KBA01-treated cells. In addition, KBA01 disrupts the HSF1-mutant p53-Hsp90 complex and releases mutant p53 to enable its MDM2- and CHIP-mediated degradation.ConclusionOur study reveals that KBA01 depletes mutant p53 protein in a chaperone-assisted ubiquitin/proteasome degradation pathway in cancer cells, providing insights into potential strategies to target mutant p53 tumours.     

Targeting p53 by PTD-mediated transduction

p53 is a major target for tumor therapy. Attempts have been made to restore or enhance p53 activity in tumor cells, including overexpression of exogenous p53 and small molecules that can rescue mutant p53. Notably, p53 peptides corresponding to the p53 carboxyl terminus can trigger a p53 response in both wild-type or mutant p53-containing cells. The recent protein transduction domain (PTD)-mediated cell entry might solve the obstacle of efficient delivery of peptides or large molecular biological cargos into cells. PTD-mediated transfer through the cell membrane occurs through a kind of endocytosis, macropinocytosis. Destabilization of macropinocytosomes by the influenza virus hemagglutinin protein (HA2) helps the escape of the PTD-cargo from macropinocytosomes and therefore significantly enhances the functional impact of transduced cargo.     

Dissection of cell context-dependent interactions between HBx and p53 family members in regulation of apoptosis: A role for HBV-induced HCC

Chronic hepatitis B virus (HBV) infection is the major risk for hepatocellular carcinomas (HCC). HBV X protein (HBx) and p53 tumor suppressor family interactions may be crucial for HCC induction. We compared p53 and p73 interactions with HBx in normal and HCC tumor cell lines differing in their p53 status. In the latter, HBx was pro-apoptotic but exhibited opposite effects in non-tumor cells. In these normal cells, p53 and p73 were retained in the cytoplasm. In hepatoma cells, however, HBx led to nuclear translocation of p53 and p73, followed by enhanced transactivation of p53-dependent promoters. The nuclear transfer of p53, but not of p73, was abrogated by protein kinase C inhibitor Gö6976. HBx overexpression in HCC cells led to strong p53 phosphorylation at Ser15, but not in non-tumor cells. Our results define ATM kinase as mediator for HBx-induced p53 phosphorylation. While HBx promotes cell death in p53/p73-positive hepatoma cells also in presence of increased levels of the oncogenic ΔTAp73 isoform, it significantly potentiates ΔTAp73-mediated proliferation and malignant transformation of fibroblasts. Our data suggest that prevention of apoptosis in normal cells by HBx through inhibition of pro-apoptotic p53 family members via direct interaction and coaction with anti-apoptotic ΔTAp73 seems to be the key element in the decision in favor of cell survival. The complex cell context-dependent interactions between p53 family members and HBx in the regulation of apoptosis may be essential in HBV-induced HCC and anticancer therapy.     

Bacterial cupredoxin azurin hijacks cellular signaling networks: Protein–protein interactions and cancer therapy

Azurin secreted by Pseudomonas aeruginosa is an anticancer bacteriocin, which preferentially enters human cancer cells and induces apoptosis or growth inhibition. It turns out that azurin is a multi‐target anticancer agent interfering in the p53 signaling pathway and the non‐receptor tyrosine kinases signaling pathway. This suggests that azurin exerts its anticancer activity by interacting with multiple targets and interfering in multiple steps in disease progression. Therefore, azurin could overcome resistance to therapy. Besides azurin, putative bacteriocins that possess functional properties similar to those of azurin have been identified in more bacteria species. A systematic investigation on the anticancer mechanisms of azurin and the azurin‐like bacteriocins will provide more and better options in cancer therapy. In this review, we summarize how azurin and the derived peptides hijack key cellular regulators or cell surface receptors to remodel the cellular signaling networks. In particular, we highlight the necessity of determining the structure of azurin/p53 complex and investigating the influence of post‐translational modifications on interactions between azurin and p53. Therapeutic applications of azurin and derived peptides are also discussed.     

Differential recognition by the tumor suppressor protein p53 of DNA modified by the novel antitumor trinuclear platinum drug BBR3464 and cisplatin

The trinuclear platinum agent BBR3464, a representative of a new class of anticancer drugs, is more potent than conventional mononuclear cisplatin [cis-diamminedichloroplatinum(II)]. BBR3464 retains significant activity in human tumor cell lines and xenografts that are refractory or poorly responsive to cisplatin, and displays a high activity in human tumor cell lines that are characterized by both wild-type and mutant p53 gene. In contrast, on average, cells with mutant p53 are more resistant to the effect of cisplatin. It has been hypothesized that the sensitivity or resistance of tumor cells to cisplatin might be also associated with cell cycle control and repair processes that involve p53. DNA is a major pharmacological target of platinum compounds and DNA binding activity of the p53 protein is crucial for its tumor suppressor function. This study, using gel-mobility-shift assays, was undertaken to examine the interactions of active and latent p53 protein with DNA fragments and oligodeoxyribonucleotide duplexes modified by BBR3464 in a cell free medium and to compare these results with those describing the interactions of these proteins with DNA modified by cisplatin. The results indicate that structurally different DNA adducts of BBR3464 and cisplatin exhibit a different efficiency to affect the binding affinity of the modified DNA to p53 protein. It has been suggested that different structural perturbations induced in DNA by the adducts of BBR3464 and cisplatin produce a differential response to p53 protein activation and recognition and that a 'molecular approach' to control of downstream effects such as protein recognition and pathways of apoptosis induction may consist in design of structurally unique DNA adducts as cell signals.     

mRNA display selection of an optimized MDM2-binding peptide that potently inhibits MDM2-p53 interaction

p53 is a tumor suppressor protein that prevents tumorigenesis through cell cycle arrest or apoptosis of cells in response to cellular stress such as DNA damage. Because the oncoprotein MDM2 interacts with p53 and inhibits its activity, MDM2-p53 interaction has been a major target for the development of anticancer drugs. While previous studies have used phage display to identify peptides (such as DI) that inhibit the MDM2-p53 interaction, these peptides were not sufficiently optimized because the size of the phage-displayed random peptide libraries did not cover all of the possible sequences. In this study, we performed selection of MDM2-binding peptides from large random peptide libraries in two stages using mRNA display. We identified an optimal peptide named MIP that inhibited the MDM2-p53 and MDMX-p53 interactions 29- and 13-fold more effectively than DI, respectively. Expression of MIP fused to the thioredoxin scaffold protein in living cells by adenovirus caused stabilization of p53 through its interaction with MDM2, resulting in activation of the p53 pathway. Furthermore, expression of MIP also inhibited tumor cell proliferation in a p53-dependent manner more potently than DI. These results show that two-stage, mRNA-displayed peptide selection is useful for the rapid identification of potent peptides that target oncoproteins.     

Small-molecule CB002 restores p53 pathway signaling and represses colorectal cancer cell growth

Much effort is currently focused on the p53 pathway. p53 is a key tumor suppressor, which is mutated or lost in many human cancers. Restoration of the p53 pathway holds the potential to induce selective cell death in tumor cells without harming normal cells that have intact p53 pathways. Most tumor cells express mutated p53 or suppress p53 by overexpression of MDM2. In this study, a compound referred to as CB002 with one closely related compound from the Chembridge library were evaluated for tumor cytotoxicity without affecting normal cells by restoration of the p53 pathway. A decrease of mutant p53 protein expression, restoration of inactivated p53, or some activation of p73 are candidate mechanisms this agent could cause tumor cell apoptosis and growth arrest. We further show that CB002 activates p53 pathway signaling in part via p73 in p53 mutant cancer cell lines. However, it is important to note that we have not established a role for p73 in the anti-tumor effect of CB002 or R1. CB002 causes tumor cell death with synergistic effects with traditional chemotherapeutics CPT-11 and 5-FU.     

MSH2 deficiency abolishes the anticancer and pro-aging activity of short telomeres

Mutations in the mismatch repair (MMR) pathway occur in human colorectal cancers with microsatellite instability. Mounting evidence suggests that cell-cycle arrest in response to a number of cellular stresses, including telomere shortening, is a potent anticancer barrier. The telomerase-deficient mouse model illustrates the anticancer effect of cell-cycle arrest provoked by short telomeres. Here, we describe a role for the MMR protein, MSH2, in signaling cell-cycle arrest in a p21/p53-dependent manner in response to short telomeres in the context of telomerase-deficient mice. In particular, progressively shorter telomeres at successive generations of MSH2^−/–Terc^−/–- mice did not suppress cancer in these mice, indicating that MSH2 deficiency abolishes the tumor suppressor activity of short telomeres. Interestingly, MSH2 deficiency prevented degenerative pathologies in the gastrointestinal tract of MSH2^−/–Terc^−/– mice concomitant with a rescue of proliferative defects. The abolishment of the anticancer and pro-aging effects of short telomeres provoked by MSH2 abrogation was independent of changes in telomere length. These results highlight a role for MSH2 in the organismal response to dysfunctional telomeres, which in turn may be important in the pathobiology of human cancers bearing mutations in the MMR pathway.     

Questioning the role of checkpoint kinase 2 in the p53 DNA damage response

Cdc25C and p53 have been reported to be physiological targets of checkpoint kinase 2 (Chk2). Surprisingly, although Chk2 purified from DNA damage sustaining cells has dramatically increased ability to phosphorylate Cdc25C when compared with untreated cells, its ability to phosphorylate p53 is weak before treatment, and there is no increase in its activity toward p53 after DNA damage by gamma irradiation or the radiomimetic agent neocarzinostatin. Furthermore, introduction of Chk2 short interfering RNA into three different human tumor cell lines leads to marked reduction of Chk2 protein, but p53 is still stabilized and active after DNA damage. The results with Chk1 short interfering RNA indicate as well that Chk1 does not play a role in human p53 stabilization after DNA damage. Thus, Chk1 and Chk2 are unlikely to be regulators of p53 in at least some human tumor cells. We discuss our results in the context of previous findings demonstrating a requirement for Chk2 in p53 stabilization and activity.