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Acute kidney injury (AKI) is a pathological condition characterized by a rapid decrease in glomerular filtration rate and nitrogenous waste accumulation during hemodynamic regulation. Alisol B, from Alisma orientale, displays anti-tumor, anti-complement, and anti-inflammatory effects. However, its effect and action mechanism on AKI is still unclear. Herein, alisol B significantly attenuated cisplatin (Cis)-induced renal tubular apoptosis through decreasing expressions levels of cleaved-caspase 3 and cleaved-PARP and the ratio of Bax/Bcl-2 depended on the p53 pathway. Alisol B also alleviated Cis-induced inflammatory response (e.g. the increase of ICAM-1, MCP-1, COX-2, iNOS, IL-6, and TNF-α) and oxidative stress (e.g. the decrease of SOD and GSH, the decrease of HO-1, GCLC, GCLM, and NQO-1) through the NF-κB and Nrf2 pathways. In a target fishing experiment, alisol B bound to soluble epoxide hydrolase (sEH) as a direct cellular target through the hydrogen bond with Gln384, which was further supported by inhibition kinetics and surface plasmon resonance (equilibrium dissociation constant, K D = 1.32 μM). Notably, alisol B enhanced levels of epoxyeicosatrienoic acids and decreased levels of dihydroxyeicosatrienoic acids, indicating that alisol B reduced the sEH activity in vivo. In addition, sEH genetic deletion alleviated Cis-induced AKI and abolished the protective effect of alisol B in Cis-induced AKI as well. These findings indicated that alisol B targeted sEH to alleviate Cis-induced AKI via GSK3β-mediated p53, NF-κB, and Nrf2 signaling pathways and could be used as a potential therapeutic agent in the treatment of AKI.

Liquid-liquid phase separation (LLPS) drives the formation of extensive membrane-less compartments to regulate various cellular biological activities both physiologically and pathologically. It has been widely accepted that LLPS is closely related to amyloid diseases and increasing reports have linked this phenomenon to cancers. Mutations of tumor suppressor protein p53 exist in more than half of malignant tumors, making the protein vitally important in cancer research. Recently, p53 was reported to undergo phase separation, which may regulate the function of p53. The molecular mechanism of p53 phase separation and how this process relates to cancer remains largely unclear. Herein, we find that the disordered unstructured basic region (UBR) plays a crucial role in p53 LLPS, driven by electrostatic and hydrophobic interactions. Mutations in the tetramerization domain (TD) disrupt p53 phase separation by preventing the tetramer formation. Furthermore, our results have revealed that, in response to DNA damage in cell, the wild type (WT) p53 undergoes LLPS, while LLPS in oncogenic mutations is diminished or eliminated. The expression of the target gene of p53 decreased significantly with the mutations and cell survival increased with the mutations. Thus, we propose a novel mechanism of p53 carcinogenesis, whereby oncogenic mutations in TD impair the formation of p53 condensates, decreasing the activation of target genes and promoting cancer progression. This study helps to understand the behavior and function of p53 in a different aspect and may provide insights into cancer therapies targeting p53.

Coiled-coil domain containing 3 (CCDC3) was previously shown to regulate liver lipid metabolism as a secretory protein. Here, we report an unexpected intracellular role of CCDC3 as a tumor suppressor in breast cancer (BrC). Bioinformatics datasets analysis showed that CCDC3 is under-expressed in BrCs, while its higher levels are correlated with higher overall survival and lower relapse of cancer patients, and CCDC3 is positively correlated with p53 and its target genes. Ectopic CCDC3 markedly suppressed proliferation, colony formation, and xenograft tumor growth by augmenting p53 activity in BrC cells. Depletion of endogenous CCDC3 by CRISPR-Cas9 increased proliferation and drug resistance of BrC cells by alleviating 5-Fluorouracil (5-FU)-induced p53 level and activity. Mechanistically, CCDC3 bound to the C-termini of p53 and MDM2, consequently stabilizing p53 in the nucleus and impairing MDM2 recruitment of p53 to the 26S proteosome without inhibiting p53 ubiquitination. p53 induced CCDC3 expression by binding to its promoter in BrC cells. Our results unveil a unique mechanism underlying CCDC3 activation of p53 in a positive feedback fashion to suppress BrC growth.

Extensive protein synthesis is necessary for uncontrolled cancer cell proliferation, requiring hyperactive ribosome biogenesis. Our previous Pan-cancer study has identified EXOSC8 as a potential copy number variation (CNV)-driven rRNA metabolism-related oncogene in colorectal cancer (CRC). Herein, we further investigated proliferation-prompting functions and mechanisms of EXOSC8 in CRC by performing in silico analyses and wet-lab experiments. We uncovered that increased EXOSC8 expression and CNV levels are strongly associated with ribosome biogenesis-related factor levels in CRC, including ribosome proteins (RPs), eukaryotic translation initiation factors and RNA polymerase I/III. EXOSC8 silence decreases nucleolar protein and proliferation marker levels, as well as rRNA/DNA and global protein syntheses. Clinically, EXOSC8 is upregulated across human cancers, particularly CNV-driven upregulation in CRC was markedly associated with poor clinical outcomes. Mechanistically, EXOSC8 knockdown increased p53 levels in CRC, and the oncogenic proliferation phenotypes of EXOSC8 depended on p53 in vitro and in vivo. We discovered that EXOSC8 knockdown in CRC cells triggers ribosomal stress, nucleolar RPL5/11 being released into the nucleoplasm and "hijacking" Mdm2 to block its E3 ubiquitin ligase function, thus releasing and activating p53. Furthermore, our therapeutic experiments provided initial evidence that EXOSC8 might serve as a potential therapeutic target in CRC. Our findings revealed, for the first time, that the RNA exosome gene (EXOSC8) promotes CRC tumorigenesis by regulating cancer-related ribosome biogenesis in CRC. This study further extends our previous Pan-cancer study of the rRNA metabolism-related genes. The inhibition of EXOSC8 is a novel therapeutic strategy for the RPs-Mdm2-p53 ribosome biogenesis surveillance pathway in CRC.

Ubiquitin specific peptidase 7 (USP7) is a deubiquitinating enzyme (DUB) that removes ubiquitin tags from specific target protein substrates in order to alter their degradation rate, sub-cellular localization, interaction, and activity. The induction of apoptosis upon USP7 inhibition is well established in cancer containing wild type p53, which operates through the 'USP7-Mdm2-p53' axis. However, in cancers without functional p53, USP7-dependent apoptosis is induced through many other alternative pathways. Here, we have identified another critical p53 independent path active under USP7 to regulate apoptosis. Proteomics analysis identifies XIAP as a potential target of USP7-dependent deubiquitination. GSEA analysis revealed up-regulation of apoptosis signalling upon USP7 inhibition associated with XIAP down-regulation. Modulation of USP7 expression and activity in multiple cancer cell lines showed that USP7 deubiquitinates XIAP to inhibit apoptosis in a caspase-dependent pathway, and the combinatorial inhibition of USP7 and XIAP induces apoptosis in vitro and in vivo. Immunohistochemical staining revealed that grade-wise accumulation of USP7 correlated with an elevated level of XIAP in glioma tissue. This is the first report on the identification and validation of XIAP as a novel substrate of USP7 and together, they involve in the empowerment of the tumorigenic potential of cancer cells by inhibiting apoptosis.

The tumor suppressor p53 oscillates in response to DNA double-strand breaks, a behavior that has been suggested to be essential to its anti-cancer function. Nearly all human cancers have genetic alterations in the p53 pathway; a number of these alterations have been shown to be oncogenic by experiment. These alterations include somatic mutations and copy number variations as well as germline polymorphisms. Intriguingly, they exhibit a mixed pattern of interactions in tumors, such as co-occurrence, mutual exclusivity, and paradoxically, mutual antagonism. Using a differential equation model of p53-Mdm2 dynamics, we employ Hopf bifurcation analysis to show that these alterations have a common mode of action, to abolish the oscillatory competence of p53, thereby, we suggest, impairing its tumor suppressive function. In this analysis, diverse genetic alterations, widely associated with human cancers clinically, have a unified mechanistic explanation of their role in oncogenesis.

Non-alcoholic fatty liver disease (NAFLD) is the most prevalent chronic liver disease, characterized by excessive hepatic lipid accumulation. Recently, we demonstrated that Smad ubiquitination regulatory factor 1 (Smurf1) deficiency significantly alleviates mouse hepatic steatosis. However, the mechanism of Smurf1-regulating hepatic lipid accumulation requires further exploration and clarification. Hence, this study explores the potential mechanism of Smurf1 in hepatic steatosis. In this study, hepatic Smurf1 proteins in NAFLD patients and healthy individuals were determined using immunohistochemical staining. Control and NAFLD mouse models were established by feeding Smurf1-knockout (KO) and wild-type mice with either a high-fat diet (HFD) or a chow diet (CD) for eight weeks. Oleic acid (OA)-induced steatotic hepatocytes were used as the NAFLD mode cells. Lipid content in liver tissues was analyzed. Smurf1-MDM2 interaction, MDM2 and p53 ubiquitination, and p53 target genes expression in liver tissues and hepatocytes were analyzed. We found that hepatic Smurf1 is highly expressed in NAFLD patients and HFD-induced NAFLD mice. Its deletion attenuates hepatocyte steatosis. Mechanistically, Smurf1 interacts with and stabilizes mouse double minute 2 (MDM2), promoting p53 degradation. In Smurf1-deficient hepatocytes, an increase in p53 suppresses SREBP-1c expression and elevates the expression of both malonyl-CoA decarboxylase (MCD) and lipin1 (Lpin1), two essential proteins in lipid catabolism. Contrarily, the activities of these three proteins and hepatocyte steatosis are reversed by p53 knockdown in Smurf1-deficient hepatocytes. This study shows that Smurf1 is involved in the pathogenesis of NAFLD by balancing de novo lipid synthesis and lipolysis.

Cisplatin, 5FU and docetaxel (TPF) are the most common chemotherapy regimen used for advanced OSCC. However, many cancer patients experience relapse, continued tumor growth, and spread due to drug resistance, which leads to treatment failure and metastatic disease. Here, using a CRISPR/Cas9 based kinome knockout screening, Misshapen-like kinase 1 (MINK1) is identified as an important mediator of 5FU resistance in OSCC. Analysis of clinical samples demonstrated significantly higher MINK1 expression in the tumor tissues of chemotherapy non-responder as compared to chemotherapy responders. The in-vitro and xenograft experiments indicate that knocking out MINK1 restores 5FU mediated cell death in chemoresistant OSCC. An antibody based phosphorylation array screen revealed MINK1 as a negative regulator of p53. Mechanistically, MINK1 modulates AKT phosphorylation at Ser473, which enables p-MDM2 (Ser 166) mediated degradation of p53. We also identified lestaurtinib as a potent inhibitor of MINK1 kinase activity. Lestaurtinib significantly induces 5FU mediated cell death in chemoresistant OSCC lines. The patient derived chemoresistant cell based xenograft data suggest that lestaurtinib restores 5FU sensitivity and facilitates a significant reduction of tumor burden. Overall, our study suggests that MINK1 is a major driver of 5FU resistance in OSCC. The novel combination of MINK1 inhibitor lestaurtinib and 5FU needs further clinical investigation in advanced OSCC. One sentence summary Lestaurtinib reverses 5FU sensitivity in drug resistant OSCC.

Oral colon-targeted drug delivery systems (OCDDs) are designed to deliver the therapeutic agents to colonic disease sites to improve the effectiveness of drug treatment, increase bioavailability, and reduce systemic side effects and are beneficial for the treatment of colorectal cancer (CRC) and inflammatory bowel disease (IBD). However, concerns about the biosafety of OCDDs are increasing, and changes in the physiological environment of the gastrointestinal tract can affect the therapeutic efficacy of the drug. Herein, we report about an orally administered colon-accumulating mitochondria-targeted drug delivery nanoplatform (M27-39@FA-MCNs), which was synthesized using the small peptide, M27-39, and folic acid (FA)-modified mesoporous carbon nanoparticles (FA-MCNs). The phenolic resin polymerized with phloroglucinol and formaldehyde (PF) was used for fabricating MCNs using a one-step soft-template method. Folic acid (FA) can be covalently combined with chitosan-modified MCNs to obtain FA-MCNs. The M27-39@FA-MCNs were stable with a spherical morphology and an average diameter of 129 nm. The cumulative release rate of M27-39@FA-MCNs in the artificial gastric fluid (pH = 1.2) and intestinal fluid (pH = 6.8) for 6 h was 87.77%. This nanoplatform maintains the advantages of both FA and MCNs to improve the bioactivity of M27-39 with high drug accumulation in colorectal tumor tissues and the ease of excretion, thus ameliorating its biosafety and targetability. Furthermore, M27-39@FA-MCNs induced tumor-cell apoptosis and inhibited tumor growth by disrupting mitochondrial energy metabolism and regulating the mitochondrial apoptosis signaling pathway and immune inflammatory response. Thus, such a mitochondria-targeting FA-modified nanoplatform based on mesoporous carbon and a bioactive peptide may provide a precise strategy for CRC treatment. STATEMENT OF SIGNIFICANCE: In this study, we constructed an orally administered colon-accumulating mitochondria-targeted drug delivery nanoplatform (M27-39@FA-MCNs), which was synthesized using the small peptide (M27-39) and folic acid-modified mesoporous carbon nanoparticles (FA-MCNs). M27-39@FA-MCNs increased the targeting ability of M27-39 toward mitochondria and colon based on the properties of FA-MCNs; they also increased M27-39 accumulation and residence time in colon tumors. Oral administration of M27-39@FA-MCNs remarkably alleviated colorectal cancer (CRC) by targeting tumor cell mitochondria and interfering with the mitochondrial energy metabolism process, and inducing apoptosis related P53/Caspase-3 mitochondrial pathway activation. Therefore, M27-39@FA-MCNs may provide a safe and precise therapeutic strategy for CRC.

Acute kidney injury (AKI) is increasingly identified as a crucial risk factor for progression to CKD. However, the factors governing AKI to CKD progression remain largely unknown. By high-throughput RNA sequencing, we found that Neat1_2, a transcript variant of Neat1, was upregulated in 40-min ischemia/reperfusion injury (IRI), which resulted in the development of renal fibrotic lesions. The upregulation of Neat1_2 in hypoxia-treated TECs was attributed to p53 transcriptional regulation. Gain- and loss-of-function studies, both in vitro and in vivo, demonstrated that Neat1_2 promoted apoptosis of injured TECs induced by IRI and caused tubulointerstitial inflammation and fibrosis. Mechanistically, Neat1_2 shares miRNA response elements with FADD, CASP-8, and CASP-3. Neat1_2 competitively binds to miR-129-5p and prevents miR-129-5p from decreasing the levels of FADD, CASP-8, and CASP-3, and ultimately facilitates TEC apoptosis. Increased expression of Neat1_2 associated with kidney injury and TEC apoptosis was recapitulated in human AKI, highlighting its clinical relevance. These findings suggest that preventing TEC apoptosis by hindering Neat1_2 expression may be a potential therapeutic strategy for AKI to CKD progression.

Chromosomal abnormalities and gene mutations determine the prognosis of patients with chronic lymphocytic leukemia (CLL). Genetic lesions can be acquired by clonal evolution (CE) likely correlating with clinical progression.Samples of 169 CLL patients were analyzed for cytogenetic clonal evolution (CCE) and CE affecting the genes TP53 and SF3B1. Moreover, the mutational status of IGHV and the clinical outcome was evaluated. CCE was observed in 35% of CLL patients. The most frequently gained cytogenetic aberration was a deletion of TP53. Acquired TP53 deletion was more frequent in patients with SF3B1 mutations compared to those without (19% vs. 7%). CCE showed a tendency to occur more frequently in patients with an aberrant karyotype at first investigation than in patients with a normal karyotype. In 73% of patients with CCE (p = 0.002) and 92% of patients with CE affecting the genes TP53 and SF3B1 (p 0.001) an unmutated IGHV status was present. CCE and CE affecting the genes TP53 and SF3B1 were significantly associated with each other (p 0.001). In 7% of patients, CE resulted in the co-occurrence of TP53 deletion and TP53 mutation resulting in a significantly shorter overall survival.The most frequently gained cytogenetic aberration during CCE was a deletion of TP53, which was associated with SF3B1 mutations. Moreover, CCE was associated with an unmutated IGHV status. Our results indicate the importance of re-evaluation of the TP53 status during the course of the disease to ensure correct treatment guidance.

Background Long non-coding RNAs (lncRNAs) play an essential role in biological processes. However, the expression patterns of lncRNAs that regulate the non-Mendelian inheritance feather phenotypes remain unknown. Objective This study aimed to compare the expression profiles of lncRNAs in the follicles of the late-feathering cocks (LC) and late-feathering hens (LH) that followed genetic rules and the early-feathering hen (EH) and early-feathering cock (EC) that did not conform to the genetic laws. Methods We performed RNA sequencing and investigated the differentially expressed lncRNAs (DElncRNAs) between the early- and late-feathering chickens, which function by cis-acting or participate in the competing endogenous RNA (ceRNA) network. Results A total of 53 upregulated and 43 downregulated lncRNAs were identified in EC vs. LC, and 58 upregulated and 109 downregulated lncRNAs were identified in EH vs. LH. The target mRNAs regulated by lncRNAs in cis were enriched in the pentose phosphate pathway, TGF-β signaling pathway and Jak-STAT signaling pathway in EC vs. LC and were associated with the TGF-β signaling pathway, Wnt signaling pathway, p53 signaling pathway and Jak-STAT signaling pathway in EH vs. LH. In addition, the lncRNA-mediated ceRNA regulatory pathways of hair follicle formation were mainly enriched in the TGF-β signaling pathway, Wnt signaling pathway, melanogenesis, and calcium signaling pathways. The levels of ENSGALG00000047626 were significantly higher in the late-feathering chickens than in the early-feathering chickens, which regulated the expression of SSTR2 by gga-miR-1649-5p. Conclusion This study provides a novel molecular mechanism of lncRNA’s response to the feather rate that does not conform to the genetic laws in chickens.

The TP53 gene is mutated in 80% of triple-negative breast cancers. Cells that harbor the hot-spot p53 gene mutation R273H produce an oncogenic mutant p53 (mtp53) that enhances cell proliferative and metastatic properties. The enhanced activities of mtp53 are collectively referred to as gain-of-function (GOF), and may include transcription-independent chromatin-based activities shared with wild-type p53 (wtp53) such as association with replicating DNA and DNA replication associated proteins like PARP1. However, how mtp53 upregulates cell proliferation is not well understood. wtp53 interacts with PARP1 using a portion of its C-terminus. The wtp53 oligomerization and far C-terminal domain (CTD) located within the C-terminus constitute putative GOF-associated domains, because mtp53 R273H expressing breast cancer cells lacking both domains manifest slow proliferation phenotypes. We addressed if the C-terminal region of mtp53 R273H is important for chromatin interaction and breast cancer cell proliferation using CRISPR-Cas9 mutated MDA-MB-468 cells endogenously expressing mtp53 R273H C-terminal deleted isoforms (R273HΔ381–388 and R273HΔ347–393). The mtp53 R273HΔ347–393 lacks the CTD and a portion of the oligomerization domain. We observed that cells harboring mtp53 R273HΔ347–393 (compared with mtp53 R273H full-length) manifest a significant reduction in chromatin, PARP1, poly-ADP-ribose (PAR), and replicating DNA binding. These cells also exhibited impaired response to hydroxyurea replicative stress, decreased sensitivity to the PARP-trapping drug combination temozolomide–talazoparib, and increased phosphorylated 53BP1 foci, suggesting reduced Okazaki fragment processing. Implications: The C-terminal region of mtp53 confers GOF activity that mediates mtp53–PARP1 and PAR interactions assisting DNA replication, thus implicating new biomarkers for PARP inhibitor therapy.

Close to half of human cancers harbor point mutations in the tumor-suppressor p53 gene, giving rise to the cellular accumulation of mutant p53 (mutp53) proteins with novel neomorphic gain-of-function (GOF) properties. The destruction of mutp53 proteins through either autophagic or proteasomal degradation is a viable strategy for the targeted therapy of p53-mutated cancers. Several nanomaterials, including zinc-iron and ZIF-8 nanoparticles (NPs), have been reported to induce the proteasomal degradation of mutp53 proteins. However, how autophagy, the other major cellular degradative pathway, influences NP-induced mutp53 degradation has not been investigated. This article shows that AIE-Mit-TPP, a mitochondria-targeting material with aggregation-induced emission (AIE) characteristics, elicits ubiquitination-dependent proteasomal degradation of a broad range of mutp53 proteins. Meanwhile, AIE-Mit-TPP also induces massive mitochondrial damage and autophagy. The inhibition of autophagy further increases AIE-Mit-TPP-elicited mutp53 degradation, revealing the negative impact of autophagy on AIE-Mit-TPP-induced mutp53 degradation. As expected, the degradation of mutp53 proteins by AIE-Mit-TPP abrogated mutp53-manifested GOF, leading to reductions in cell proliferation and migration and increases in cell cycle arrest and cell death. Consequently, AIE-Mit-TPP inhibited the growth of mutp53 tumors. This paper unravels the interesting interplay between the proteasomal and autophagic degradative pathways and pinpoints the modulation of autophagy as a potential strategy for optimizing NP-induced mutp53 degradation and p53-targeted cancer therapy. STATEMENT OF SIGNIFICANCE: We have designed three different types of AIE materials: non-targeting (AIE-Br), mitochondria-targeting (AIE-Mit-TPP), lysosome-targeting (AIE-Lyso). Our results proved that mitochondria-targeting AIE material induced degradation of mutp53 proteins via the proteasome degradation pathway and abrogated mutp53-conferred GOF phenotypes. Furthermore, we performed in vitro studies on the effect of the tested materials in mutp53-expressing cancer cells and demonstrated our findings via in vivo investigations in a mouse subcutaneous p53supR175H/supTOV112D ovarian cancer model. Our results confirmed the link between the proteasome pathway and autophagy and thus proposed a strategy of combining AIE-Mit-TPP with autophagy inhibitors for the targeted treatment of mutp53-associated tumors. Finally, we found that AIE-Mit-TPP could induce degradation of a wide-spectrum mutp53 proteins, which makes mitochondria-targeting AIE materials an effective therapeutic strategy for p53-mutated cancers.

Malignancy of medulloblastoma depends on its molecular classification. Sonic Hedgehog (SHH)-type medulloblastoma with p53 mutation was recognized as one of the most aggressive types of tumors. We developed a novel drug, chlorambucil-conjugated PI-polyamides (Chb-M'), which was designed to compete with the RUNX consensus DNA-binding site. Chb-M' specifically recognizes this consensus sequence and alkylates it to inhibit the RUNX transcriptional activity. In-silico analysis showed all the RUNX families were upregulated in the SHH-type medulloblastoma. Thus, we tested the anti-tumor effects of Chb-M' in vitro and in vivo using Daoy cell lines, which belong to SHH with p53 mutation. Chb-M' inhibited tumor growth of Daoy cells by inducing apoptosis. The same inhibitory effect was also observed by knocking down of RUNX1 or RUNX2, but not RUNX3. Apoptosis array analysis showed that Chb-M' treatment induced phosphorylation of p53 serine 15 residues. In a subcutaneous tumor model, intratumoral injection of Chb-M' induced tumor growth retardation. Chb-M' mediated inhibition of RUNX1 and RUNX2 can be a novel therapeutic strategy for SHH-type medulloblastoma with p53 mutation.

p53 plays a critical role in regulating diverse biological processes: DNA repair, cell cycle arrest, apoptosis and senescence. The p53 pathway has therefore served as the focus of multiple drug-discovery efforts. p53 is negatively regulated by hDMX and hDM2; prior studies have identified 14-3-3 proteins as hDMX and hDM2 client proteins. 14-3-3 proteins are adaptor proteins that modulate localization, degradation and interactions of their targets in response to phosphorylation. Thus, 14-3-3 proteins may indirectly modulate the interaction between hDMX or hDM2 and p53 and represent potential targets for modulation of the p53 pathway. In this manuscript, we report on the biophysical and structural characterization of peptide/protein interactions that are representative of the interaction between 14-3-3 and hDMX or hDM2. The data establish that proximal phosphosites spaced ~20–25 residues apart in both hDMX and hDM2 co-operate to facilitate high-affinity 14-3-3 binding and provide structural insight that can be utilized in future stabilizer/inhibitor discovery efforts.

The p53 tumor suppressor regulates cell functions either by acting as a transcription factor or by interacting with other proteins. Previously, we reported that the non-transcriptional actions of p53 can be facilitated by the binding of p53 to p21. Herein, we investigated whether p53/p21 interaction influences the transcriptional activity of p53. We observed that the expression of the p53 promoter-based reporter gene is dependent on p21 levels. Moreover, using a p21 variant that is unable to bind p53, we showed that p53 promoter activity requires p53/p21 interaction. To investigate the possible role of p21 in regulating the expression of endogenous p53 targets, we analyzed mRNA levels of Puma, Mdm2, and Gadd45a in untreated control and γ-ray-irradiated cells. We observed that while Puma expression is dependent on p53 regardless of γ-irradiation, p53 mediates the expression of Mdm2 and Gadd45a only in irradiated cells. Notably, p53/p21 interaction is required only for the p53-dependent expression of the tested genes and not Mdm2 and Gadd45a in non-irradiated cells. Moreover, chromatin immunoprecipitation assay revealed that p21 is required for the binding of p53 to the promoters of Puma, Mdm2, and Gadd45a. Collectively, our data support the view that the p53/p21 complex is involved in regulating p53-dependent gene expression. These findings provide a new foundation for understanding the transcriptional action of p53.

Thymol (2-isopropyl-5-methylphenol) is a colorless crystalline derivative of cymene, that possesses pleotropic pharmacological properties, including analgesic, antibacterial, antispasmodic, and anti-inflammatory activities. Thymol has also been recognized for its beneficial effect as an anti-tumor agent, but the precise mechanism for this has not been fully elucidated. We aimed to identifying whether thymol exerts anti-cancer activity in human U-87 malignant glioblastoma (GB) cells (U-87).Cell viability and apoptosis was evaluated in U-87 cells treated with thymol at different concentrations. Reactive oxygen species (ROS) production, mRNA expressions of apoptosis-related genes and cell cycle characteristics were assessed. The cytotoxic activity of the co-exposure of thymol and temozolomide (TMZ) was also evaluated. The half-maximal inhibitory concentration (IC50) of thymol in the U-87 cells was 230 μM assessed at 24 h after exposure. Thymol did not exhibit any cytotoxic effects on normal L929 cells at this concentration. Thymol treatment increased the expression of Bax and p53, and also increased apoptotic cell death, and excessive generation of ROS. Moreover, the cytotoxic activity of thymol on the U-87 cells may be related to the arrest of the cell cycle at the G0/G1 interface. Combination therapy showed that the cytotoxic effects of thymol synergized with TMZ, and combined treatment had more cytotoxic potential compared to either of the agents alone.Our data indicate the potential cytotoxic activities of thymol on U-87 cells. Further studies are required to evaluate the spectrum of the antitumor activity of thymol on GB cells.

Colorectal cancer (CRC) is now the third most prevalent tumor and one of the deadliest cancers worldwide, with an increasing prevalence every year. Therefore, we urgently need to understand the mechanisms regulating the progression of colorectal cancer and find potential diagnostic biomarkers. In this study, we performed an analysis using the TCGA and GEO databases to find a molecular biomarker for the diagnosis of CRC, namely CTPS1. The results of this analysis revealed that CTPS1 could promote tumor proliferation and metastasis. Furthermore, bioinformatics analysis revealed that CTPS1 promoted CRC progression through cell cycle and p53 pathways. Further investigation demonstrated that CTPS1 might be involved in the regulation of CCNB1, RRM2, GTSE1, CDK2 and CHEK2 genes. Moreover, PCR confirmed that CTPS1 regulated GTSE1 and CDK2 molecules. Then, western blot was used to verify that CTPS1 promoted the expression of GTSE1 and CDK2 by inhibiting the expression of p53. In summary, we identified an important diagnostic biomarker for CRC, namely CTPS1, and its importance was validated at the cellular level. These results suggest that CTPS1 could serve as a candidate biomarker for CRC and CTPS1 inhibitors may be a potential treatment for CRC.

Over 90% of ovarian cancer cells exhibit p53 mutations or inactivation. In addition, p53 is exported outside of the nucleus by exportin-1 (XPO1), a protein that mediates the nuclear export of several cancer suppressor proteins. Overexpression of XPO1 is associated with resistance to chemotherapy, leading to poor prognosis in various cancers. The MDM2 inhibitor, RG-7388, is a known reactivator of p53 and has been tested with high interest as a therapeutic agent for cancer treatment. In addition, Selinexor, which is a second-generation selective inhibitor of nuclear export (SINE), is known to cause an accumulation of p53 in the nucleus and is also being explored as a therapy potentiating agent in combination treatments. This study was conducted to assess the efficacy of RG-7388 in combination with Selinexor for treating ovarian cancer. A combination of Selinexor and RG-7388 treatments was able to reduce the cell viability compared to individual treatments. In addition, the combination treatment revealed significant up-regulation of several cancer suppressor proteins in the whole lysate, cytoplasm, and nucleus. Finally, our results confirm that the combination of Selinexor with RG-7388 can induce a caspase-mediated apoptotic mechanism via up-regulation of p53 and p21.

The N-terminal transactivation domain (TAD) of p53 is a disordered region with multiple phosphorylation sites. Phosphorylation at Thr18 is crucial for the release of p53 from its negative regulator, MDM2. In stressed cells, CK1δ is responsible for phosphorylating Thr18, but requires Ser15 to be phosphorylated. To understand the mechanistic underpinnings of this sequential phosphorylation, molecular modeling and molecular dynamics simulation studies of these phosphorylation events were carried out. Our models suggest that a positively charged region on CK1δ near the adenosine triphosphate (ATP) binding pocket, which is conserved across species, sequesters the negatively charged pSer15, thereby constraining the positioning of the rest of the peptide, such that the side chain of Thr18 is positioned close to the γ-phosphate of ATP. Furthermore, our studies show that the phosphorylated p53 TAD1 (p53pSer15) peptide binds more strongly to CK1δ than does p53. p53 adopts a helical structure when bound to CK1δ, which is lost upon phosphorylation at Ser15, thus gaining higher flexibility and ability to morph into the binding site. We propose that upon phosphorylation at Ser15 the p53 TAD1 peptide binds to CK1δ through an electrostatically driven induced fit mechanism resulting in a flanking fuzzy complex.

Although several studies on mutant p53 reported cancer-promoting activities via "gain-of-function", the mechanism underlying these differences in function between p53 R175H, R175P, and p53 wild-type (WT) remains unclear.Linking miniTurbo with p53 WT, R175H, and R175P, the expression of fusion and biotinylated proteins were assessed by Western blotting. The function and subcellular localization of fusion proteins were detected by apoptosis assay and immunofluorescence, respectively. Biotinylated proteins were analyzed by liquid chromatography-tandem mass spectrometry, followed by bioinformatics analysis. Small-scale pull-downs and Co-Immunoprecipitation were performed to validate the interaction between mutant or p53 WT and biotinylated proteins.The fusion protein's cellular localization and function were consistent with those of previous studies on the corresponding p53. Comparative profiles of R175H versus WT showed that most of the interacting proteins belonged to the intracellular organelle lumen, and the pathways involved were metabolism and genetic information processing. Comparative profiles of R175P versus WT suggested that the majority of the interacting proteins belonged to the intracellular organelle lumen and the extracellular membrane-bounded organelle, and the pathways involved were metabolism and genetic information processing pathways. The comparison between R175H and R175P revealed that most interacting proteins belonged to the organelle lumen, and pathways involved were genetic information processing pathways. Finally, the mutation of p53 significantly altered the interaction with the target proteins were confirmed.We verified the reliability of the miniTurbo system and obtained candidate targets of mutant p53, which provided new thoughts on the mechanism of mutant p53 gain-of-function and new potential targets for cancer therapy.

Li-Fraumeni syndrome, caused by germline pathogenic variants in TP53, results in susceptibility to multiple cancers. Variants of uncertain significance (VUS) and reclassification of variants over time pose management concerns given improved survival with cancer surveillance for LFS patients. We describe the experience of TP53 variant reclassification at a pediatric cancer center. Methods: We reviewed medical records (2010–2019) of 756 patients seen in Texas Children’s Cancer Genetics Clinic. We noted initial TP53 classification and any reclassifications. We then classified TP53 variants following ClinGen TP53 variant curation expert panel recommendations using data from ClinVar, medical literature and IARC database. Results: Of 234 patients tested for TP53, 27 (11.5%) reports contained pathogenic/likely pathogenic (P/LP) variants and 7 (3)% contained VUS. By January 2022, 4 of 6 unique VUS and 2 of 16 unique P/LP variants changed interpretations in ClinVar. Reinterpretation of these 4 VUS in ClinVar matched clinical decision at the time of initial report. Applying TP53 VCEP specifications classified 3 VUS to P/LP/benign, and one pathogenic variant to likely benign. Conclusions: Planned review of variant significance is essential, especially for patients with high probability of LFS.

Cadmium (Cd) is a highly toxic environmental pollutant that can severely damage the kidneys. Here, we show that Cd-induced apoptosis is promoted by the cytoplasmic polyubiquitination of p53 (polyUb-p53), which is regulated by the polyubiquitination of SQSTM1/p62 (polyUb-p62) and autophagy in mouse kidney mesangial cells (MES13E cells). p53 was detected in monomeric and different high-molecular-weight (HMW) forms after Cd exposure. Monomeric p53 levels decreased in a concentration- and time-dependent manner. HMW-p53 transiently accumulated in the cytoplasm independent of proteasome inhibition. The expression patterns of p53 were similar to those of p62 upon Cd exposure, and the interactions between polyUb-p53 and polyUb-p62 were observed using immunoprecipitation. P62 knockdown reduced polyUb-p53 and upregulated nuclear monomeric p53, whereas p53 knockdown reduced polyUb-p62. Autophagy inhibition induced by ATG5 knockdown reduced Cd-induced polyUb-p62 and polyUb-p53 but upregulated the levels of nuclear p53. Pharmacological inhibition of autophagy by bafilomycin A1 increased polyUb-p62 and polyUb-p53 in the cytoplasm, indicating that p53 protein levels and subcellular localization were regulated by polyUb-p62 and autophagy. Immunoprecipitation and immunofluorescence revealed an interaction between p53 and LC3B, indicating that p53 was taken up by autophagosomes. Cd-resistant RMES13E cells and kidney tissues from mice continuously injected with Cd had reduced polyUb-p53, polyUb-p62, and autophagy levels. Similar results were observed in renal cell carcinoma cell lines. These results indicate that cytoplasmic polyUb-p53 is a potential biomarker for Cd-induced acute toxicity in mesangial cells. In addition, upregulation of nuclear p53 may protect cells against Cd cytotoxicity, but abnormal p53 accumulation may contribute to tumor development.

Barrett’s esophagus progresses to high-grade dysplasia or cancer along the well-established metaplasia-dysplasia-adenocarcinoma sequence. The aim of this study was to evaluate the value of p53, Ki67, and toll-like receptor 5 (TLR5) in prediction of malignant progression of Barrett’s metaplasia and low-grade dysplasia. This was a retrospective matched case–control study based on Northern and Central Finland population. Patients diagnosed with esophageal high-grade dysplasia or adenocarcinoma were included. From these patients, all previous endoscopy samples were obtained along with original diagnostic HE-slides and clinical data. Age- and sex-matched patients with non-progressing Barrett’s metaplasia and low-grade dysplasia confirmed with follow-up endoscopies were used as controls. Two gastrointestinal pathologist re-reviewed all original HE-slides, and newly made sections to confirm representative tissue material blinded from clinical data. p53, Ki67, and TLR5 were immunohistochemically stained. Final cohort included 45 patients with progressive Barrett’s metaplasia (n = 21) or low-grade dysplasia (n = 24), and 92 patients with non-progressive Barrett’s metaplasia (n = 52) or low-grade dysplasia (n = 40). In Barrett’s metaplasia, aberrant p53 expression was observed in 6% of samples in progressors and 0% in non-progressors. In low-grade dysplasia, aberrant p53 was seen in 56% of samples in progressors and 17% in non-progressors (Odd’s ratio 6.7, 95% CI 1.8–24.6). Ki67 or TLR5 showed no association with disease progression. In this matched case–control study, p53 expression associated with a high risk of malignant progression in Barrett’s low-grade dysplasia. Routine staining of p53 is indicated in expert confirmed low-grade dysplasia.

RIO Kinase 1 (RIOK1) is involved in various pathologies, including cancer. However, the role of RIOK1 in radioresistance of colorectal cancer (CRC) remains largely unknown. In this study, we reported that RIOK1 was overexpressed in rectal cancer tissue with weaker tumor regression after neoadjuvant chemoradiotherapy (neoCRT). Moreover, higher RIOK1 expression predicted a poor prognosis in patients with rectal cancer. Blockade of RIOK1 using Toyocamycin, a pharmacological inhibitor of RIOK1, or by knocking down its expression, decreased the resistance of CRC cells to radiotherapy in vitro and in vivo. A mechanistic study revealed that RIOK1 regulates radioresistance by suppressing the p53 signaling pathway. Furthermore, we found that RIOK1 and Ras-GAP SH3 domain binding protein 2 (G3BP2) interact with each other. RIOK1 phosphorylates G3BP2 at Thr226, which increases the activity of G3BP2. RIOK1-mediated phosphorylation of G3BP2 facilitated ubiquitination of p53 by murine double minute 2 protein (MDM2). Altogether, our study revealed the clinical significance of RIOK1 in CRC, and therapies targeting RIOK1 might alleviate the CRC tumor burden in patients.

CHMP1A, a member of the ESCRT-III complex family, has been indicated as a brand-new inhibitor gene of tumors. Our previous research has revealed that CHMP1A plays a vital role in the development and progression of renal cell carcinoma (RCC).To investigate the potential target pathway of the regulation of the tumor cell growth by CHMP1A.The effect of CHMP1A on mTOR pathway was elucidated by western blotting. The effect of CHMP1A on the expression of p53 was evaluated, and A498 cell growth was assessed by colony formation and MTT assays. The expression of p53 was knocked down by shRNA-p53, and the effect of CHMP1A on mTOR after knockdown of p53 was evaluated. The effect of CHMP1A on apoptosis and its relationship with MDM2 pathway were detected by western blotting and FCM. Finally, the relationship between the regulation of p53 by CHMP1A and the PI3K/mTOR pathway was detected.This study showed that the mTOR pathway was suppressed significantly in CHMP1A-overexpressing A498 and 786-0 cells; moreover, the enhanced expression of p53 and the reduced proliferation were shown in CHMP1A-overexpressing A498 cells. Furthermore, CHMP1A was able to regulate the PI3K/PTEN/mTOR and MDM2/p53 pathways in order to suppress RCC. In addition, CHMP1A regulated Bax and Bcl-2 via MDM2/p53 to induce the apoptosis of tumor cells and upregulated the expression of p53 via the PI3K/mTOR pathway.The results convey that CHMP1A-related suppression of RCC is closely related to the PI3K/mTOR/p53 pathway.

This study aimed to explore the function and related mechanisms of elastin microfibril interfacer 2 (EMILIN2) in ovarian cancer. First, the expression level of EMILIN2 was detected in patient tissues and its correlation with overall survival rate was analysed. Then, EMILIN2 was overexpressed in ovarian cancer cell lines to observe its function and effect on Warburg effect. By detecting its promoter region methylation, the epigenetic regulatory role was explored. Finally, through the luciferase reporter assay and siRNA tools, the regulatory mechanism of p53 on EMILIN2 was investigated. It was detected in clinical samples that down-regulated EMILIN2 was associated with poor prognosis of ovarian cancer. It was further found that EMILIN2 regulated the metabolic phenotype of ovarian cancer cells. The expression of EMILIN2 was epigenetically regulated by its promoter methylation. Also, it was found that p53 regulated the expression of EMILIN2 and the p53/EMILIN2 axis regulated the Warburg effect in ovarian cancer cells. EMILIN2 was inhibited by methylation in ovarian cancer. In summary, p53 can promote and regulate its transcription by binding to the promoter region of EMILIN2, thereby affecting the Warburg effect and inhibiting tumours. Therefore, EMILIN2 might be a potential target for clinical diagnosis and treatment of ovarian cancer.

Inflammasome contributes to the pathogenesis of various inflammatory diseases, but the epigenetic mechanism controlling its activation remains elusive. Here, we found that the histone methyltransferase Ezh2 mediates the activation of multiple types of inflammasomes in macrophages/microglia independent of its methyltransferase activity and thus promotes inflammasome-related pathologies. Mechanistically, Ezh2 functions through its SANT2 domain to maintain the enrichment of H3K27 acetylation in the promoter region of the long noncoding RNA (lncRNA) Neat1, thereby promoting chromatin accessibility and facilitating p65-mediated transcription of Neat1, which is a critical mediator of inflammasome assembly and activation. In addition, the tumour suppressor protein p53 competes with Ezh2 for the same binding region in the Neat1 promoter and thus antagonises Ezh2-induced Neat1 transcription and inflammasome activation. Therefore, loss of Ezh2 strongly promotes the binding of p53, which recruits the deacetylase SIRT1 for H3K27 deacetylation of the Neat1 promoter and thus suppresses Neat1 transcription and inflammasome activation. Overall, our study demonstrates an epigenetic mechanism involved in modulating inflammasome activation through an Ezh2/p53 competition model and highlights a novel function of Ezh2 in maintaining H3K27 acetylation to support lncRNA Neat1 transcription.

Spinal muscular atrophy (SMA) is a childhood motor neuron disease caused by anomalies in the SMN1 gene. Although therapeutics have been approved for the treatment of SMA, there is a therapeutic time window, after which efficacy is reduced. Hallmarks of motor unit pathology in SMA include loss of motor-neurons and neuromuscular junction (NMJs). Following an increase in Smn levels, it is unclear how much damage can be repaired and the degree to which normal connections are re-established. Here, we perform a detailed analysis of motor unit pathology before and after restoration of Smn levels. Using a Smn-inducible mouse model of SMA, we show that genetic restoration of Smn results in a dramatic reduction in NMJ pathology, with restoration of innervation patterns, preservation of axon and endplate number and normalized expression of P53-associated transcripts. Notably, presynaptic swelling and elevated Pmaip levels remained. We analysed the effect of either early or delayed treated of an antisense oligonucleotide (ASO) targeting SMN2 on a range of differentially vulnerable muscles. Following ASO administration, the majority of endplates appeared fully occupied. However, there was an underlying loss of axons and endplates, which was more prevalent following a delay in treatment. There was an increase in average motor unit size following both early and delayed treatment. Together this work demonstrates the remarkably regenerative capacity of the motor neuron following Smn restoration, but highlights that recovery is incomplete. This work suggests that there is an opportunity to enhance neuromuscular junction recovery following administration of Smn-enhancing therapeutics.

The Apoptosis Stimulating Protein of p53 2 (ASPP2) is a heterozygous insufficient tumor suppressor; however, its molecular mechanism(s) in tumor suppression is not completely understood. ASPP2 plays an essential role in cell growth, as shown by liver hepatocellular carcinoma (LIHC) RNA-seq assay using the Cancer Genome Atlas (TCGA) and High-Throughput-PCR assay using ASPP2 knockdown cells. These observations were further confirmed by in vivo and in vitro experiments. Mechanistically, N-terminus ASPP2 interacted with Keratin 18 (k18) in vivo and in vitro. Interestingly, the RDIVpSGP motif of ASPP2 associates with 14-3-3 and promotes ASPP2/k18/14-3-3 ternary-complex formation which promotes MEK/ERK signal activation by impairing 14-3-3 and BRAF association. Additionally, ASPP2-rAd injection promotes paclitaxel-suppressed tumor growth by suppressing cell proliferation in the BALB/c nude mice model. ASPP2 and k18 were preferentially downregulated in Hepatocellular Carcinoma (HCC), which predicted poor prognosis in HCC patients. Overall, these findings suggested that ASPP2 promoted BRAF/MEK/ERK signal activation by promoting the formation of an ASPP2/k18/14-3-3 ternary complex via the RDIVpSGP motif at the N terminus. Moreover, this study provides novel insights into the molecular mechanism of tumor suppression in HCC patients.

p73 belongs to p53 family transcription factor activating more than 50% of cell fate p53 target genes involved in cell cycle, apoptosis, DNA damage response alongside neuronal system development and differentiation by binding to 20-bp response elements (REs) having sequence motif (PPPC-A/T-T/A-GYYY) where P-purines and Y-pyrimidines with each 10-bp separated by minimum 0 to 13-bp spacer. The promiscuous nature of recognizing both cell fate and development genes and the underlying RE selectivity mechanism by p73 is not well understood. Here, we report the molecular details of p73 recognizing the REs using the crystal structure of p73 DNA binding domain (DBD) in complex with 12 base pair DNA sequence 5'-cAGGCATGCCTg-3' and molecular dynamics simulations with six different p53 natural promoter sequences. Each 20-base pair natural promoter forms a different major/minor groove due to the presence of nucleotides A/T, A/C, G/G, T/T and G/T at positions 3, 8, 13, 18 uniquely recognized by p73 key residues Lys138 and Arg268. The loops L1 and L3 bearing these residues influence inter-and intra-dimer interfaces interactions and hence p73 forms a unique tetramer with each natural promoter sequence. Structural features of the DNA and the spacing between half-sites influence p73 tetramerization and its transactivation function.

We examined the apoptotic response of two glioblastoma cells, p53 wild type U87 and p53 mutated T98G, to doxorubicin, bortezomib, and vorinostat, which respectively target DNA, 26S proteasome and histone deacetylase, to clarify p53ʹs function in apoptosis. We demonstrated that doxorubicin induced apoptosis in U87 cells but not in T98G cells. The level of p53 was definitively correlated to the extent of DNA damage and apoptosis initiation. Dominant-negative p53 reduced p21 expression, but did not affect doxorubicin-induced apoptosis, so the transcriptional activity of p53 seemed not to participate in doxorubicin-induced apoptosis. However, p53 concentrated into the nucleus during heavy apoptosis. Bortezomib could induce apoptosis in U87 with high sensitivity and T98G cells with low sensitivity. In contrast, vorinostat promoted apoptosis in both U87 and T98G cells and reduced the basal level of p53 in U87 cells, indicating that p53 played no role in the vorinostat-induced apoptosis. To clearly define the role of p53 in bortezomib- and doxorubicin-induced apoptosis, we combined doxorubicin with bortezomib to treat U87 cells to assess this combination’s effect on apoptosis and p53 status. Interestingly, the combination of doxorubicin with bortezomib engendered compound stress, resulting in a synergistic outcome for apoptosis in U87 cells. However, the amounts of p53 in the total count and in the nucleus were much lower with the combination than with doxorubicin alone, suggesting that p53 played no role in either the compound stress, doxorubicin-only or bortezomib-induced apoptosis.

Graves’ disease (GD) is a kind of autoimmune diseases. The development of GD is closely related to the imbalance of Th1/Th2 generated by the differentiation of CD4(+) T cells. This study was sought to clarify the role of lncRNA RUNX1-IT1 and explore the mechanism of its function. The expressions of RUNX1-IT1 and Neural cell adhesion molecule (NrCAM) in the peripheral blood of GD patients were detected by qRT-PCR and Western blot. We performed RNA pull down, RIP, and ChIP experiments to verify the correlation between p53 and RUNX1-IT1, p53 and NrCAM. The levels of Th1 cells differentiation markers were detected by Flow cytometry assay and ELISA. The expressions of lncRNA RUNX1-IT1 and NrCAM were most significantly up-regulated in CD4(+) T cells of GD patients, and NrCAM expression was significantly positively correlated with RUNX1-IT1 expression. Furthermore, p53 was a potential transcription factor of NrCAM, which could interact with NrCAM. NrCAM level was up-regulated after the overexpression of p53 in CD4(+) T cells, while knockdown of RUNX1-IT1 reversed this effect. Down-regulation of NrCAM and RUNX1-IT1 could decrease the mRNA and protein levels of transcriptional regulator T-bet and CXC chemokine ligand 10 (CXCL10) in CD4(+) T cells. Our results suggested that RUNX1-IT1 regulated the expressions of the important Th1 factor T-bet, CXCL10, and interferon γ (IFN-γ) by regulating NrCAM transcription, thus participating in the occurrence and development of specific autoimmune disease GD.

Although it is well-established that p53-mediated tumor suppression mainly acts through its ability in transcriptional regulation, the molecular mechanisms of this regulation are not completely understood. Among a number of regulatory modes, acetylation of p53 attracts great interests. p53 was one of the first non-histone proteins found to be functionally regulated by acetylation and deacetylation, and subsequent work has established that reversible acetylation is a general mechanism for regulation of non-histone proteins. Unlike other types of posttranslational modifications occurred during stress responses, the role of p53 acetylation has been recently validated in vivo by using the knock-in mice with both acetylation-defective and acetylation-mimicking p53 mutants. Here, we review the role of acetylation in p53-mediated activities, with a focus on which specific acetylation sites are critical for p53-dependent transcription regulation during tumor suppression and how acetylation of p53 recruits specific "readers" to execute its promoter-specific regulation of different targets. We also discuss the role of p53 acetylation in differentially regulating its classic activities in cell cycle arrest, senescence and apoptosis as well as newly identified unconventional functions such as cell metabolism and ferroptosis.

P53 mutation is an important cause of chemoresistance in colorectal cancer (CRC). The investigation and identification of the downstream targets and underlying molecular mechanism of chemoresistance induced by P53 abnormalities are therefore of great clinical significance. In this study, we demonstrated and reported for the first time that leucine-rich pentatricopeptide repeat-containing protein (LRPPRC) is a key functional downstream factor and therapeutic target for P53 mutation-induced chemoresistance. Due to its RNA binding function, LRPPRC specifically bound to the mRNA of multidrug resistance 1 (MDR1), increasing MDR1 mRNA stability and protein expression. In normal cells, P53 induced by chemotherapy inhibited the expression of LRPPRC via miR-34a and in turn reduced the expression of MDR1. However, chemotherapy-induced P53/miR-34a/LRPPRC/MDR1 signalling pathway activation was lost when P53 was mutated. Additionally, the accumulated LRPPRC and MDR1 promoted drug resistance. Most importantly, gossypol-acetic acid (GAA) was recently reported by our team as the first specific inhibitor of LRPPRC. In CRC cells with P53 mutation, GAA effectively induced degradation of the LRPPRC protein and reduced chemoresistance. Both in vivo and in vitro experiments revealed that combination chemotherapy with GAA and 5-fluorouracil (5FU) yielded improved treatment outcomes. In this study, we reported a novel mechanism and target related to P53-induced drug resistance and provided corresponding interventional strategies for the precision treatment of CRC.

Exosomes from senescence cells play pivotal roles in endothelium dysfunction. We investigated the exosomal angiogenic cargo of endothelial cells (ECs) in a model of senescence in vitro. After inducing aging by H

The tumour suppressor TP53 is a master regulator of several cellular processes that collectively suppress tumorigenesis. The TP53 gene is mutated in ~50% of human cancers and these defects usually confer poor responses to therapy. The TP53 protein functions as a homo-tetrameric transcription factor, directly regulating the expression of ~500 target genes, some of them involved in cell death, cell cycling, cell senescence, DNA repair and metabolism. Originally, it was thought that the induction of apoptotic cell death was the principal mechanism by which TP53 prevents the development of tumours. However, gene targeted mice lacking the critical effectors of TP53-induced apoptosis (PUMA and NOXA) do not spontaneously develop tumours. Indeed, even mice lacking the critical mediators for TP53-induced apoptosis, G1/S cell cycle arrest and cell senescence, namely PUMA, NOXA and p21, do not spontaneously develop tumours. This suggests that TP53 must activate additional cellular responses to mediate tumour suppression. In this review, we will discuss the processes by which TP53 regulates cell death, cell cycling/cell senescence, DNA damage repair and metabolic adaptation, and place this in context of current understanding of TP53-mediated tumour suppression.

The p53 protein is structurally and functionally divided into five domains. The proline-rich domain is localized at amino acids 55–100. 319 missense mutations were identified solely in the proline domain from human cancers. Six hotspot mutations were identified at amino acids 72, 73, 82, 84, 89, and 98. Codon 72 contains a polymorphism that changes from proline (and African descent) to arginine (with Caucasian descent) with increasing latitudes northward and is under natural selection for pigmentation and protection from UV light exposure. Cancers associated with mutations in the proline domain were considerably enriched for melanomas and skin cancers compared to mutations in other p53 domains. These hotspot mutations are enriched at UV mutational signatures disrupting amino acid signals for binding SH-3-containing proteins important for p53 function. Among the protein–protein interaction sites identified by hotspot mutations were MDM-2, a negative regulator of p53, XAF-1, promoting p53 mediated apoptosis, and PIN-1, a proline isomerase essential for structural folding of this domain.

Background: Copper complex has been gaining much attention in anticancer research as a targeted agent since cancer cells uptake more copper than non-cancerous cells. Our group synthesised a ternary copper complex, which is composed of 1,10-phenanthroline and tyrosine [Cu(phen)(L-tyr)Cl].3H20. These two payloads have been designed to cleave DNA and inhibit protein degradation system (proteasome) concurrently in cancer cells, making this copper complex a dual-target compound. Objective: The current study was carried out to investigate the mode of cell death and the role of autophagy induced by [Cu(phen)(L-tyr)Cl].3H20 in MCF-7 and MDA-MB-231 breast cancer cells. Methods: Growth inhibition of [Cu(phen)(L-tyr)Cl].3H20 towards MDA-MB-231 and human non-cancerous MCF10A breast cells was determined by MTT assay. Annexin-V-FITC/PI and cell cycle analysis were evaluated by flow cytometry. The expression of p53, Bax, caspase-9, caspase-7, caspase-3 and LC3 was determined using western blot analysis. The cells were then co-treated with hydroxychloroquine to ascertain the role of autophagy induced by [Cu(phen)(L-tyr)Cl].3H20. Results: [Cu(phen)(L-tyr)Cl].3H20 inhibited the growth of cancer cells dose-dependently with less toxicity towards MCF10A cells. Additionally, [Cu(phen)(L-tyr)Cl].3H20 induced apoptosis and cell cycle arrest towards MCF-7 and MDA-MB-231 breast cancer cells possibly via regulation of p53, Bax, caspase-9, caspase-3 and capase-7. The expression of LC3II was upregulated in both cancer cell lines upon treatment with [Cu(phen)(L-tyr) Cl].3H20, indicating the induction of autophagy. Co-treatment with autophagy inhibitor hydroxychloroquine significantly enhanced growth inhibition of both cell lines, suggesting that autophagy induced by [Cu(phen)(L-tyr) Cl].3H20 in both breast cancer cells promoted cell survival. Conclusion: [Cu(phen)(L-tyr)Cl].3H20 holds great potential to be developed for breast cancer treatment.

Background: Despite emerging research on new treatment strategies, chemotherapy remains one of the most important therapeutic modalities for cancers. Imidazopyridines are important targets in organic chemistry and, given their numerous applications, they are worthy of attention. Objective: The objective of this study was to design and synthesize a novel series of imidazo[1,2-a]pyridine-derived compounds and investigate their antitumor effects and the underlying mechanisms. Methods: Imidazo[1,2-a]pyridine-derived compounds were synthesized with new strategies and conventional methods. The antitumor activities of the new compounds were evaluated by MTT assay. Flow cytometry and immunofluorescence were performed to examine the effects of the most effective antiproliferative compound on cell apoptosis. Western blot analysis was used to assess the expression of apoptotic proteins. Results: Fifty-two new imidazo[1,2-a]pyridine compounds were designed and successfully synthesized. The compound, 1-(imidazo[1,2-a]pyridin-3-yl)-2-(naphthalen-2-yl)ethane-1,2-dione, named La23, showed high potential for suppressing the viability of HeLa cells (IC50 15.32 μM). La23 inhibited cell proliferation by inducing cell apoptosis, and it reduced the mitochondrial membrane potential of HeLa cells. Moreover, treatment with La23 appeared to increase the expression of apoptotic-related protein P53, Bax, cleaved caspase-3, and cytochrome c at a low concentration range. Conclusion: The novel imidazo[1,2-a]pyridine compound, La23, was synthesized and it suppressed cell growth by inducing cell apoptosis via the p53/Bax mitochondrial apoptotic pathway.

Mutations in genes encoding general transcription factors cause neurological disorders. Despite clinical prominence, the consequences of defects in the basal transcription machinery during brain development are unclear. We found that loss of the TATA-box binding protein-associated factor TAF8, a component of the general transcription factor TFIID, in the developing central nervous system affected the expression of many, but notably not all genes. Taf8 deletion caused apoptosis, unexpectedly restricted to forebrain regions. Nuclear levels of the transcription factor p53 were elevated in the absence of TAF8, as were the mRNAs of the pro-apoptotic p53 target genes Noxa, Puma and Bax. The cell death in Taf8 forebrain regions was completely rescued by additional loss of p53, but Taf8 and p53 brains failed to initiate a neuronal expression program. Taf8 deletion caused aberrant transcription of promoter regions and splicing anomalies. We propose that TAF8 supports the directionality of transcription and co-transcriptional splicing, and that failure of these processes causes p53-induced apoptosis of neuronal cells in the developing mouse embryo. [Image: see text]

The retinoblastoma protein RB and the transcription factor p53 are central tumor suppressors. They are often found inactivated in various tumor types. Both proteins play central roles in regulating the cell division cycle. RB forms complexes with the E2F family of transcription factors and downregulates numerous genes. Among the RB-E2F target genes, a large number code for key cell cycle regulators. Their transcriptional repression by the RB-E2F complex is released through phosphorylation of RB, leading to expression of the cell cycle regulators. The release from repression can be prevented by the cyclin-dependent kinase inhibitor p21/CDKN1A. The CDKN1A gene is transcriptionally activated by p53. Taken together, these elements constitute the p53-p21-RB signaling pathway. Following activation of p53, for example by viral infection or induction of DNA damage, p21 expression is upregulated. High levels of p21 then result in RB-E2F complex formation and downregulation of a large number of cell cycle genes. Thus, p53-dependent transcriptional repression is indirect. The reduced expression of the many regulators leads to cell cycle arrest. Examination of the p53-p21-RB targets and genes controlled by the related p53-p21-DREAM signaling pathway reveals that there is a large overlap of the two groups. Mechanistically this can be explained by replacing RB-E2F complexes with the DREAM transcriptional repressor complex at E2F sites in target promoters. In contrast to RB-E2F, DREAM can downregulate genes also through CHR transcription factor binding sites. This results in a distinct gene set controlled by p53-p21-DREAM signaling independent of RB-E2F. Furthermore, RB has non-canonical functions without binding to E2F and DNA. Such a role of RB supporting DREAM formation may be exerted by the RB-SKP2-p27-cyclin A/E-CDK2-p130-DREAM link. In the current synopsis, the mechanism of regulation by p53-p21-RB signaling is assessed and the overlap with p53-p21-DREAM signaling is examined.

Statins trigger apoptotic cell death in some types of growing tumor cells in a cholesterol-lowering-independent manner. Self-nanoemulsifying delivery systems (SNEDs) are potentially effective for the suppression of breast cancer development. This study aims to investigate the potential anticancer activity of fluvastatin (FLV)-SNEDs in breast cancer while comparing it with FLV in vitro as well as in vivo exploiting/using MDA-MB-231 and Erhlich ascites carcinoma (EAC)-bearing mice, respectively. Biochemical analysis of liver and kidney functions, oxidative stress markers, and histopathological examinations of such tumor tissues were performed showing the potentiality of SNEDs as a nanocarrier for antitumor agents. FLV-SNEDs demonstrated more potent anticancer activity compared to FLV on MDA-MB-231 and hepatocellular carcinoma (HepG2) cells. In vivo experiments on the EAC-bearing mice model indicated that FLV and-to a greater extent-FLV-SNEDs ameliorated EAC-induced hepatotoxicity and nephrotoxicity. FLV or FLV-SNEDs evidently reduced the percent of Ki-67 +ve EAC cells by 57.5% and 86.5% in comparison to the vehicle-treated EAC group. In addition, FLV or FLV-SNEDs decreased Bcl-2 levels in serum and liver specimens. In contrast, FLV or FLV-SNEDs significantly activated the executioner caspase-3. Simultaneously, both FLV and FLV-SNEDs stimulated p53 signaling and modulated Bcl-2 protein levels in treated cells. Collectively, these results support the contribution of apoptotic cell death in mediating the anticancer activities of FLV and FLV-SNEDs against murine EAC model in vivo. This study provides new understandings of how FLV and FLV-SNEDs regulate EAC cell viability via upregulation of p53 signaling, and through modulation of cleaved caspase-3 as well as antiapoptotic Bcl-2 marker.

Cell death, cell cycle arrest and cellular senescence are three distinct cellular responses that can be induced by oncogene activation and diverse anti-cancer agents, and this often requires the action of the tumour suppressor TP53. Within a cell population, or even within an individual cell, these processes are not necessarily mutually exclusive. It is therefore important to measure all these processes simultaneously. However, current assays generally visualise only one or at best two responses, often only detecting the dominant one. Here, we present a novel flow cytometric assay that allows simultaneous assessment of cell viability and cell cycling through measurement of DNA content and DNA synthesis, and markers of cell senescence at the single cell level. We demonstrate that this assay can be performed on both human and murine cells, that are either cancerous or non-transformed, and can help to dissect complex cell fate decisions. We believe that this experimental tool will be useful for the study of diverse biological processes.

Chikusetsusaponin IV and V (CsIV and CsV), two typical oleanolic acid saponins, are mainly derived from the rhizome of Panax japonicus C.A. Mey. To reveal the anti-cancer effect of CsIV and CsV on liver cancer cells, human hepatic cancer cell lines (HepG2) were exposed to these saponins, and various physiological responses of HepG2 were investigated.HepG2 cells were treated with CsIV, CsV and 5-fluorouracil (5-FU). Cell proliferation was measured by CCK-8 assay. The cell cycle arrest, cell apoptosis and intracellular CaOur data demonstrated that CsIV and CsV exerted significant cytotoxic effects on HepG2 cells without affecting normal liver cells. And, these chikusetsusaponins, especially for CsIV, showed a potent effect on promoting cell apoptosis in HepG2 cells, which was associated with the activation of p53-mediated apoptosis pathway.

Head and neck squamous cell carcinomas (HNSCCs) arising in the mucosal linings of the upper aerodigestive tract are highly heterogeneous, aggressive, and multifactorial tumors affecting more than half a million patients worldwide each year. Classical etiological factors for HNSCC include alcohol, tobacco, and human papillomavirus (HPV) infection. Current treatment options for HNSCCs encompass surgery, radiotherapy, chemotherapy, or combinatorial remedies. Comprehensive integrative genomic analysis of HNSCC has identified mutations in TP53 gene as the most frequent of all somatic genomic alterations. TP53 mutations are associated with either loss of wild-type p53 function or gain of functions that promote invasion, metastasis, genomic instability, and cancer cell proliferation. Interestingly, disruptive TP53 mutations in tumor DNA are associated with aggressiveness and reduced survival after surgical treatment of HNSCC. This review summarizes the current evidence and impact of TP53 mutations in HNSCC.

Age-related macular degeneration (AMD) is a leading cause of vision loss with recent evidence indicating an important role for macroautophagy/autophagy in disease progression. In this study we investigate the efficacy of targeting autophagy for slowing dysfunction in a mouse model with features of early AMD. Mice lacking APOE (apolipoprotein E; B6.129P2-Apoesupitm1Unc/i/supJ/Arc) and C57BL/6 J- (wild-type, WT) mice were treated with metformin or trehalose in the drinking water from 5 months of age and the ocular phenotype investigated at 13 months. Control mice received normal drinking water. APOE-control mice had reduced retinal function and thickening of Bruch's membrane consistent with an early AMD phenotype. Immunohistochemical labeling showed reductions in MAP1LC3B/LC3 (microtubule-associated protein 1 light chain 3 beta) and LAMP1 (lysosomal-associated membrane protein 1) labeling in the photoreceptors and retinal pigment epithelium (RPE). This correlated with increased LC3-II:LC3-I ratio and alterations in protein expression in multiple autophagy pathways measured by reverse phase protein array, suggesting autophagy was slowed. Treatment of APOE-mice with metformin or trehalose ameliorated the loss of retinal function and reduced Bruch's membrane thickening, enhancing LC3 and LAMP1 labeling in the ocular tissues and restoring LC3-II:LC3-I ratio to WT levels. Protein analysis indicated that both treatments boost ATM-AMPK driven autophagy. Additionally, trehalose increased p-MAPK14/p38 to enhance autophagy. Our study shows that treatments targeting pathways to enhance autophagy have the potential for treating early AMD and provide support for the use of metformin, which has been found to reduce the risk of AMD development in human patients.bAbbreviations:/bAMD: age-related macular degeneration; AMPK: 5' adenosine monophosphate-activated protein kinase APOE: apolipoprotein E; ATM: ataxia telangiectasia mutated; BCL2L1/Bcl-xL: BCL2-like 1; DAPI: 4'-6-diamidino-2-phenylindole; ERG: electroretinogram; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GCL: ganglion cell layer; INL: inner nuclear layer; IPL: inner plexiform layer; IS/OS: inner and outer photoreceptor segments; LAMP1: lysosomal-associated membrane protein 1; MAP1LC3B/LC3: microtubule-associated protein 1 light chain 3 beta; MTOR: mechanistic target of rapamycin kinase; OCT: optical coherence tomography; ONL: outer nuclear layer; OPs: oscillatory potentials; p-EIF4EBP1: phosphorylated eukaryotic translation initiation factor 4E binding protein 1; p-MAPK14/p38: phosphorylated mitogen-activated protein kinase 14; RPE: retinal pigment epithelium; RPS6KB/p70 S6 kinase: ribosomal protein S6 kinase; SQSTM1/p62: sequestosome 1; TP53/TRP53/p53: tumor related protein 53; TSC2: TSC complex subunit 2; WT: wild type.