CP-31398 restored the functional condensates of R175H p53 by stabilizing the zinc-binding domain and 251-258 segment.

Nicotine exerts a substantial influence on cervical carcinogenesis by affecting the malignant progression of human papillomavirus (HPV)-18 positive and HPV-negative cervical cancer cells, as well as HPV-immortalized cervical epithelial cells. Further research is needed to fully elucidate the impact of nicotine on HPV-16 positive cervical cancer. This study investigated the molecular mechanisms of nicotine in HPV-16-positive cervical (SiHa) cells. First, we conducted cell counting kit-8, flow cytometry, wound healing, transwell assays to evaluate cellular proliferation, migration, and invasion capabilities. The data illustrated that nicotine-treated SiHa cells displayed stronger malignant transformation capability compared to the control (p < 0.05). Furthermore, western blot analysis was used to evaluate the expression changes of cellular proteins in SiHa cells. The results revealed that nicotine induces a significant upregulation of PI3K, AKT, phosphorylated AKT (Ser473) (p-AKT), the p-AKT/AKT ratio, and matrix metalloproteinase-2 (MMP-2), along with a marked increase in its secretion. Also, it was accompanied by the suppression of tumor suppressor p53 and decreased levels of p21 and Caspase-3, as well as the active form of Caspase-3 (cleaved Caspase-3), indicating suppression of apoptosis. Critically, the use of a PI3K inhibitor (LY294002) demonstrated that the nicotine-induced downregulation of p53 and upregulation of MMP-2, as well as the enhancement of cellular invasion are dependent on PI3K/AKT pathway activation. These findings conclusively demonstrate that nicotine promotes the malignant transformation of HPV-16 positive cervical cancer cells by altering the expressions of MMP-2, p53, Caspase-3, and p21 via the activation of the PI3K/AKT pathway. This highlights the therapeutic potential of targeting this pathway in cervical cancer treatment.

Making decisions is a core task for living matter, and a task that can only be carried out through processes out of equilibrium. While important elements of nonequilibrium processes occur in the spatial distribution of resources and within genetic heterogeneity, temporal dynamics of protein concentrations play a critical yet underappreciated role in cellular decision-making. This review explores our current understanding of how cells use dynamic signaling patterns, such as oscillations, to regulate processes and cell-fate decisions across multiple biological systems. We focus on examples including the tumor suppressor p53, immune signaling via NF- κ B, and oscillatory networks in development. We further highlight theoretical frameworks and the current horizon in our understanding of this fundamental interplay. • Temporal heterogeneity increases adaptability in cells • Non-equilibrium as a key information pathway in biology • Interplay between theoretical and experimental methods • Oscillations provide functional advantages across scale and fields

Cisplatin remains a cornerstone chemotherapeutic agent for a broad spectrum of solid malignancies; however, the emergence of intrinsic and acquired resistance severely limits its clinical efficacy. Central to the cellular decision between DNA damage repair and apoptosis following cisplatin exposure is the tumor suppressor p53, whose function is tightly governed by its primary negative regulator, Murine Double Minute 2 (MDM2). This review provides a critical synthesis of the molecular interplay within the MDM2/p53 axis and its direct impact on cisplatin sensitivity. We discussed how deregulation of this axis via TP53 mutational inactivation or MDM2 overexpression enables tumor cells to tolerate cisplatin-induced genotoxic stress. Specifically, we analyzed the dual role of MDM2: its E3 ubiquitin ligase activity that targets p53 for proteasomal degradation, and its emerging p53-independent functions in promoting DNA repair fidelity. Furthermore, we evaluated the therapeutic implications of targeting this pathway with small-molecule MDM2 antagonists. We assessed preclinical and clinical evidence supporting the use of MDM2 inhibition to lower the apoptotic threshold and re-sensitize resistant tumors to cisplatin. Therefore, an understanding of the MDM2/p53 feedback loop is essential for optimizing cisplatin-based regimens and developing rational combination therapies to circumvent chemoresistance.

Colorectal cancer (CRC) frequently exhibits hypoxic regions due to poor vascularization, leading to the stabilization of hypoxia-inducible factor 1 alpha (HIF-1α). Moreover, mutations in the tumour suppressor p53 occur in approximately half of all CRCs. While the individual roles of both transcription factors in tumour cell survival are well characterized, their interaction and its influence on the metabolic adaptation of CRC cells under hypoxic stress remain unclear. Using HCT116 CRC cells with targeted deletions of TP53 and HIF1A, we examined the effects of p53 loss on HIF-1 signalling and the respective consequences for metabolic adaptation as well as the survival of CRC cells under moderate (1% O₂) and severe (0.1% O₂) hypoxia. Severe hypoxia stabilized p53 protein levels despite the transcriptional repression of TP53, possibly through posttranslational mechanisms and dependent on nutrient availability. In contrast to the assumption that p53 is transcriptionally inactive under hypoxia, we observed stable expression of p53 target genes (P21, BAX) under severe hypoxia, indicating functional transactivation. Loss of p53 impaired the early induction of HIF-1 target genes (VEGF, PHD2), although HIF-1α protein levels and DNA binding were unaffected, suggesting a coactivator role for p53. Furthermore, compared with wild-type cells, p53-deficient cells presented delayed but exaggerated expression of glycolytic genes, including Glucose Uptake Transporter 1 (GLUT1), Phosphofructokinase Liver-Type (PFKL) and Lactate Dehydrogenase A (LDHA), under hypoxia, with no impairment of glycolytic function or cell viability. Remarkably, even HIF1A knockout cells preserved glycolysis, whereas glycolytic genes were significantly downregulated, indicating HIF-1-independent metabolic compensation. Our findings position p53 as a temporal gatekeeper and key regulator of hypoxic adaptation in CRC cells, coordinating early gene induction and metabolic responses. The ability of CRC cells to maintain glycolysis despite the loss of p53, respectively, HIF-1α underscores the existence of compensatory HIF-independent pathways. Targeting these alternative circuits may represent a promising strategy in hypoxic, p53-deficient CRC.

The p53 tumor suppressor binds DNA cooperatively as a tetramer, mediated by salt-bridge interactions between p53 residues E180 and R181 from two different p53 monomers. Variants at the R181 residue are one of the most identified TP53 pathogenic variants by germline genetic testing. We show that families with TP53 p.R181H and p.R181C variants have an attenuated cancer risk phenotype compared to patients with hotspot dominant negative loss of function TP53 variants. Despite this phenotype, we find that p53 R181H and R181C variants have significantly reduced ability to bind to p53 promoter/enhancer target sequences and transactivate p53 target genes, similar to null variants. However, p53 R181H and R181C retain wild-type p53 structure and tetramerization. In addition, R181-mutant cells undergo apoptosis through wild-type p53 activity at the mitochondria. These results suggest that retention of transcription-independent p53 tumor suppressor function results in a reduced penetrance cancer risk syndrome in humans. Implications: We report the first separation of function DNA binding domain p53 mutation that results in retention of transcription-independent p53 functions despite loss of p53 transactivation activity, resulting in a reduced penetrance phenotype.

Tumor suppressor p53 is uniquely prone to misfolding and aggregation, a property tightly linked to oncogenic mutations and absent in its paralogs, p63 and p73. Yet, the biophysical origins underlying divergent aggregation propensity remain incompletely understood. Here, we integrate fluorescence spectroscopy, high-pressure and chemical denaturation NMR, molecular dynamics simulations, and energetic frustration analysis to dissect the structural and dynamic discrepancies among p53 and its paralogs. Our results reveal that p53 harbors distinct surface-exposed solvation patches and cavity-prone regions promoting local conformational plasticity. Residue-level NMR analysis uncovers a broad, asymmetric distribution of signal intensity changes with pressure, correlating with discrete solvation patches and frustration-prone regions. These features coincide with highly frustrated contact networks and altered hydrophobic core gating, collectively facilitating aggregation-prone states. In contrast, p63 and p73 exhibit more uniform solvation, reduced frustration, and tighter core packing, conferring greater structural stability. These mechanistic differences directly link uneven hydration and compressibility to the heightened amyloid aggregation propensity of p53C. These contrasting energetic landscapes illustrate how evolutionary sequence divergence fine-tunes p53 for functional flexibility at the expense of stability, predisposing it to pathological aggregation. Our findings illuminate the structural determinants that distinguish pathological folding in p53 and may guide therapeutic stabilization strategies.

Diamond-Blackfan anemia gene product RPS19 counteracts SET to maintain p53 transcriptional activity and tumor suppressor function.

Ubiquitination, a key post-translational modification, is responsible for regulating protein stability, activity, subcellular localization, and interactions via the ubiquitin-proteasome system (UPS). The UPS plays a role in non-lysosomal protein degradation, involved in the coordinated action of ubiquitin-activating enzymes (E1s), ubiquitin-conjugating enzymes (E2s), and ubiquitin ligases (E3s). The review elaborates on the functions of key E3 ligases of MDM2, β-TrCP, and SCF complexes, which modulate cell cycle progression, apoptosis, metabolic reprogramming, and drug resistance in hepatocellular carcinoma (HCC). For instance, MDM2 promotes HCC development by degrading the tumor suppressor p53, while β-TrCP influences the Wnt/β-catenin signaling axis by changing β-catenin levels. Deubiquitinating enzymes (DUBs), including the USP family and CYLD, also play significant roles in HCC by stabilizing or destabilizing critical proteins involved in oncogenesis. The clinical potential of ubiquitination-related molecules as biomarkers for predicting HCC prognosis and as therapeutic targets is also discussed. This review comprehensively examines the role of ubiquitination modification in HCC and its clinical relevance. Future research directions include the exploration of novel ubiquitination modifications, their interactions with other post-translational modifications, and the development of precision medicine strategies based on multi-omics technologies to improve HCC treatment outcomes.

Lipid Transfer Proteins and PI4KIIα Initiate Nuclear p53-Phosphoinositide Signaling.

Therapeutic potential of quercetin in hepatocellular carcinoma: Mechanisms, challenges, and clinical insights

MDM2 is an E3 ubiquitin ligase that promotes p53 tumor suppressor degradation and has emerged as a therapeutic target in the treatment of wild-type (wt) TP53 tumors. In acute myeloid leukemia (AML), TP53 mutations are infrequent (15-20%), but wt-p53 is often inactivated through overexpression of MDM2. Thus, MDM2 inhibitors are currently in clinical trials for AML. However, p53 stabilization with inhibitors upregulates MDM2, which limits their clinical efficacy. Proteolysis-targeting chimeric (PROTAC) molecules that degrade MDM2 may overcome this feedback. MD-265 is a PROTAC that recruits CRBN, degrades MDM2, restores p53 and induces apoptosis. We tested MD-265 in ex vivo cultures of 105 primary leukemic stem cells (LSCs). The median cytotoxic IC50 for MD-265 was 16 nM, median IC50 for MI-1061 was 150-fold higher. LSCs with IC50 > 1 µM were classified as MD-265 resistant and harbored mutations in TP53. Normal hematopoietic stem cells showed 100-fold higher IC50 (818 nM) than LSCs. AML patient-derived xenograft (PDX) models in NSG-SGM3 mice were treated with MD-265 or an oral MDM2 inhibitor. In PDX models, MD-265 was not toxic and prolonged survival. MD-265 is a potent and specific MDM2 degrader with broad pre-clinical activity and a promising drug candidate for the treatment of leukemias.

The development of analytical approaches capable of detecting subtle protein conformational changes is of significant interest in biomedicine, particularly for disease diagnostics and biomolecular characterization. In this work, the tumor suppressor p53 protein was selected as a model system to evaluate an analytical platform based on Raman spectroscopy combined with surface-enhanced Raman spectroscopy (SERS) and supervised machine learning for the classification of closely related protein variants. Label-free Raman and SERS spectra of recombinant wild-type p53 and its hotspot mutants R175H and R273H were acquired on bare Al and Al coated with gold nanospheres, gold nanorods, and silver nanoparticles. Principal Component Analysis (PCA) revealed distinct spectral fingerprints among the p53 variants, with relevant contributions in the amide III and CH stretching regions, indicating subtle conformational differences. Supervised classification was performed using several machine learning algorithms under a nested, group-disjoint cross-validation protocol (holding out entire acquisition lines) to account for within-line spectral correlation. Among tested models, a linear Support Vector Machine (Linear-SVM) achieved the highest performance, reaching an accuracy of 92.9±6.9% on AuNS@Al substrates. These results demonstrate that the integration of optimized nanostructured SERS substrates, Raman spectroscopy, and machine learning constitutes a robust analytical platform for the structural classification of wild-type and mutant p53 proteins. The proposed approach provides a versatile framework for studying conformational changes in biomolecules of biomedical relevance and supports its potential application in cancer biomarker detection and protein misfolding studies.

The transcription factor E2F plays crucial roles in cell proliferation and tumor suppression. In the resting state of normal cells, E2F activity is suppressed by binding of the tumor suppressor pRB and its family members. E2F activated by growth stimulation under the control of pRB (physiological E2F) activates growth-related genes and facilitates cell proliferation. In contrast, E2F activated by loss of pRB control, such as forced inactivation of pRB (distinct E2F), activates tumor suppressor genes such as ARF and TAp73 to suppress tumorigenesis. We previously reported that these genes are specifically activated by distinct E2F but not by physiological E2F. In almost all cancers, pRB function is disabled, generating distinct E2F activity, which is tolerated due to concomitant dysfunction of the tumor suppressor p53. Hence, distinct E2F activity is a characteristic feature of cancer cells. Mouse embryonic stem cells (mESCs) proliferate rapidly with shortened gap phases, enhanced cyclin dependent kinase (CDK) activity and hyper-phosphorylated (inactive) pRB. This observation prompted us to examine whether mESCs possess distinct E2F activity. We show here that, like cancer cell lines, mESCs exhibit distinct E2F activity. Moreover, introduction of a small amount of CDK inhibitors enhanced deregulated E2F activity, suggesting that it is suppressed by CDK activity in mESCs.

UV radiation (UVR), a known skin-stressor causing inflammation, aging and carcinogenesis, activates the arylhydrocarbon receptor (AhR) and downstream molecules which are crucially involved in photo-induced skin damage. In this study, the potent UV protector melatonin was investigated regarding UVR-mediated activation of AhR and downstream molecules including tumor suppressor p27, DNA double-strand break marker pH2AX, cyclooxygenase-2 (COX-2), mitogen-activated protein kinase-14 (MAPK14)/p38α, matrix metalloproteinase-2 (MMP2) and tissue inhibitor of matrix metalloproteinase-1 (TIMP1). They were studied in exvivo human full-thickness skin irradiated with UVA/B light (0, 300 mJ/cm2) 0 h and 24 h post UV exposure, comparing skin pre-incubated with or without melatonin. Protein expression was analysed by immunofluorescence staining, gene expression by real-time qPCR. UV exposure significantly up-regulated AhR (p < 0.0001), p27 (p < 0.001) and pH2AX (p < 0.0001) protein expression 0 h and 24 h post-irradiation which was significantly counteracted by melatonin (10-3 M) at both time points. Further, melatonin significantly reduced gene expression of AhR by 21.2% (p < 0.01), p27 by 24.8% (p < 0.01), COX-2 by 42.9% (p < 0.001), MAPK14 by 6.6% (p < 0.05) and MMP2 by 8.2% (p < 0.05), and caused a 10.2% (n.s.) TIMP1 reduction tendency 24 h post-irradiation. Thus, melatonin prevented UV-dependent expression of AhR and downstream regulators of AhR-mediated processes possibly related to inflammation, cellular aging and carcinogenesis on protein and gene level in UV-irradiated skin.

PurposeColorectal cancer (CRC) remains a leading cause of cancer-related mortality, necessitating the identification of novel therapeutic targets. The E3 ubiquitin ligase WW domain-containing E3 ubiquitin protein ligase 2 (WWP2) has been implicated in various cancers, yet its specific role and underlying molecular mechanisms in CRC are poorly understood. This study aimed to investigate the functional role of WWP2 in CRC progression and to elucidate its regulatory mechanisms.MethodsWWP2 expression was evaluated in CRC patient tissues and cell lines using immunohistochemistry, quantitative real-time polymerase chain reaction, and western blotting. The biological functions of WWP2 were assessed using in vitro assays for cell proliferation, migration, and invasion following adenovirus-mediated overexpression. The molecular mechanism was investigated by analyzing the protein expression levels of p53 and its downstream target, p21, via western blot. An in vivo xenograft mouse model was used to confirm the oncogenic role of WWP2.ResultsWWP2 expression was significantly upregulated in CRC tissues. Overexpression of WWP2 promoted CRC cell proliferation, migration, and invasion. Mechanistically, increased WWP2 expression led to a marked reduction in the protein levels of the tumor suppressor p53. Consequently, the expression of the p53 downstream target, the cell cycle inhibitor p21, was also suppressed. In the xenograft model, WWP2 overexpression significantly enhanced tumor growth.ConclusionOur findings demonstrate that WWP2 functions as an oncogene in CRC. It promotes cancer progression by destabilizing the tumor suppressor p53 and downregulating p21. This study highlights the WWP2-p53-p21 axis as a potential novel therapeutic target for CRC.

ReDisulphID: A discovery platform for thiol redox sensors identifies a druggable site regulating p53 activation.

The tumor suppressor p53 is the most frequently mutated protein in tumors and a target for drug development. More than 2000 cancer-associated p53 missense mutations have been reported, most of them located in the DNA-binding domain (DBD). Due to the low intrinsic thermostability of the latter, they often lead to unfolding at physiological temperature. Stabilizing the DBD with small molecules has been shown to be effective in reactivating the cavity-creating cancer mutant Y220C. Unfortunately, the majority of p53 mutants seem to lack druggable binding pockets for small molecules. Here we show that a designed ankyrin repeat protein (DARPin) that binds to the p53 DBD stabilizes temperature-sensitive (TS) p53 cancer mutants, thereby compensating for mutation-induced loss of stability. We determined high-resolution crystal structures of multiple DARPin-mutant p53 complexes, providing mechanistic insights into this mode of stabilization. Reporter gene assays across a comprehensive panel of cancer-associated mutants revealed reactivation of the majority of TS mutants, whereas DNA-contact mutants and those with local misfolding of the DNA-binding surface remained inactive, as expected. We demonstrate that this reactivation induces the transcription of canonical p53 target genes and elicits antiproliferative effects in cancer cell lines. A combination of this DARPin with an mRNA/lipid nanoparticle-based transfection approach may have the potential to reactivate most TS p53 mutants and resensitize cancer cells to chemotherapy.

Immunoglobulin class switch recombination (CSR) plays an important role in humoral immune response enabling B cells to replace the initial IgM by another antibody class (IgG, IgE or IgA), thus changing the effector functions of antibodies. CSR occurs between highly repetitive switch sequences located upstream of constant gene exons, and is initiated by the Activation-Induced cytidine Deaminase via transcription-dependent deamination of single-stranded DNA targets at switch regions. CSR is preceded by germline transcription and is controlled by the super-enhancer 3' regulatory region (3'RR) in an activation-specific manner. The 3'RR is composed of four enhancers (hs3a, hs1-2, hs3b, and hs4) which act in synergy, and its long-range activity correlates with the enhancers' transcription. In addition to its function as a tumor suppressor, the p53 transcription factor controls various developmental and cellular processes. This multifaceted function is largely due to its context-dependent transcriptional activity, involving both activation and repression of a myriad of target genes. Despite its potential importance for CSR, which implies multiple layers of transcriptional, epigenetic and DNA break/repair regulations, the role of p53 in CSR is still unclear. Here, by using a mouse line devoid of p53, we show that p53 regulates CSR in an isotype-specific manner. Moreover, we provide evidence that p53 has a dual role in the control of germline transcription as it acts both as an activator and a repressor. Finally, we show that p53 is required for hs4 transcription, suggesting a role for p53 in the regulation of the 3'RR's transcriptional activity.

Human papillomavirus type 16 (HPV16) is the most prevalent carcinogenic HPV type. Many HPV16 sequence variants have been described, some of which cause protein-coding mutations. However, most experiments have characterized the viral proteins encoded by the HPV16 prototype clone, and some researchers have proposed that variants identified in high-grade lesions and cancers may have higher oncogenic potential than the prototype. To determine whether oncoproteins encoded by such variants have greater oncogenic potential than the prototype version, we performed a comparative biochemical analysis of selected naturally occurring HPV16 E6 and E7 variant alleles identified in cervical lesions and/or control samples. We examined interactions of prototype and HPV16 E7 variant alleles with the retinoblastoma tumor suppressor pRB, the ubiquitin ligase UBR4 (p600), and the tumor suppressor PTPN14. We also compared HPV16 E6 prototype and variant protein interactions with the p53 tumor suppressor, the UBE3A (E6AP) ubiquitin ligase, and the PDZ-domain protein SCRIB. Several E6 or E7 variant proteins exhibited reduced binding to these cellular targets, suggestive of partial or complete loss of function. Our findings reveal that protein-protein interactions that enable the oncogenic activities of HPV16 E6 and E7 can be missing from naturally occurring HPV16 variants and that bona fide loss-of-function variants can be detected in high-grade cervical lesions or cancers. We conclude that clinical association alone is insufficient to predict the pathogenic potential of HPV16 E6 or E7 variant proteins.IMPORTANCEHuman papillomavirus type 16 (HPV16) is the leading cause of HPV-associated cancers, and most functional studies have been performed with the prototype viral clone. Numerous naturally occurring HPV16 variants have been detected in cervical lesions and cancers, raising the possibility that some variants may possess enhanced transforming activity. E6 and E7 are the major oncoproteins encoded by HPV16. By comparing key molecular interactions of prototype and naturally occurring E6 and E7 variant proteins, we found that several variant proteins display reduced engagement with tumor suppressors and ubiquitin ligases that are important for oncogenic activities. Notably, some variants found in high-grade lesions and cancers show partial or complete loss of engaging these critical host proteins. These findings indicate that the presence of an HPV16 variant in a cancer does not necessarily imply increased oncogenic potency. Functional characterization is therefore essential to interpret the biological and clinical significance of HPV16 genetic diversity.