Copy number amplification-induced overexpression of lncRNA LOC101927668 facilitates colorectal cancer progression by recruiting hnRNPD to disrupt RBM47/p53/p21 signaling.

BACKGROUND: Somatic copy number alterations (SCNAs) are pivotal in cancer progression and patient prognosis. Dysregulated long non-coding RNAs (lncRNAs), modulated by SCNAs, significantly impact tumorigenesis, including colorectal cancer (CRC). Nonetheless, the functional significance of lncRNAs induced by SCNAs in CRC remains largely unexplored. METHODS: The dysregulated lncRNA LOC101927668, induced by copy number amplification, was identified through comprehensive bioinformatic analyses utilizing multidimensional data. Subsequent in situ hybridization was employed to ascertain the subcellular localization of LOC101927668, and gain- and loss-of-function experiments were conducted to elucidate its role in CRC progression. The downstream targets and signaling pathway influenced by LOC101927668 were identified and validated through a comprehensive approach, encompassing RNA sequencing, RT-qPCR, Western blot analysis, dual-luciferase reporter assay, evaluation of mRNA and protein degradation, and rescue experiments. Analysis of AU-rich elements (AREs) within the mRNA 3' untranslated region (UTR) of the downstream target, along with exploration of putative ARE-binding proteins, was conducted. RNA pull-down, mass spectrometry, RNA immunoprecipitation, and dual-luciferase reporter assays were employed to elucidate potential interacting proteins of LOC101927668 and further delineate the regulatory mechanism between LOC101927668 and its downstream target. Moreover, subcutaneous xenograft and orthotopic liver xenograft tumor models were utilized to evaluate the in vivo impact of LOC101927668 on CRC cells and investigate its correlation with downstream targets. RESULTS: Significantly overexpressed LOC101927668, driven by chr7p22.3-p14.3 amplification, was markedly correlated with unfavorable clinical outcomes in our CRC patient cohort, as well as in TCGA and GEO datasets. Moreover, we demonstrated that enforced expression of LOC101927668 significantly enhanced cell proliferation, migration, and invasion, while its depletion impeded these processes in a p53-dependent manner. Mechanistically, nucleus-localized LOC101927668 recruited hnRNPD and translocated to the cytoplasm, accelerating the destabilization of RBM47 mRNA, a transcription factor of p53. As a nucleocytoplasmic shuttling protein, hnRNPD mediated RBM47 destabilization by binding to the ARE motif within RBM47 3'UTR, thereby suppressing the p53 signaling pathway and facilitating CRC progression. CONCLUSIONS: The overexpression of LOC101927668, driven by SCNAs, facilitates CRC proliferation and metastasis by recruiting hnRNPD, thus perturbing the RBM47/p53/p21 signaling pathway. These findings underscore the pivotal roles of LOC101927668 and highlight its therapeutic potential in anti-CRC interventions.

Distinct landscape and clinical implications of therapy-related clonal hematopoiesis.

Therapy-related clonal hematopoiesis (t-CH) is defined as clonal hematopoiesis detected in individuals previously treated with chemotherapy and/or radiation therapy. With the increased use of genetic analysis in oncological care, the detection of t-CH among cancer patients is becoming increasingly common. t-CH arises through the selective bottleneck imposed by chemotherapies and potentially through direct mutagenesis from chemotherapies, resulting in a distinct mutational landscape enriched with mutations in DNA damage-response pathway genes such as TP53, PPM1D, and CHEK2. Emerging evidence sheds light on the mechanisms of t-CH development and potential strategies to mitigate its emergence. Due to its unique characteristics that predominantly affect cancer patients, t-CH has clinical implications distinct from those of CH in the general population. This Review discusses the potential mechanisms of t-CH development, its mutational landscape, mutant-drug relationships, and its clinical significance. We highlight the distinct nature of t-CH and call for intensified research in this field.

Negative feedback loop in the activation of non-homologous end joining DNA repair pathway in Helicobacter pylori infected patients with gastritis.

This study aimed to investigate the activation of error-prone DNA repair pathway in response to Helicobacter pylori infection. Relative changes in the expression levels of genes involved in the non-homologous end-joining pathway (NHEJ) in H. pylori-infected (Cases) and non-infected patients (Controls) with chronic gastritis were measured. A significant increase in the relative expression level of TP53, and significant decrease in the relative transcription of lncRNA LINP1 and XRCC5 were detected in the case group. The transcription of Lig4 and XRCC6 was increased in the case group, which was not statistically significant. The Spearman's Correlation Coefficient analysis showed a significant positive-correlation between the transcriptional levels of LINP1 and XRCC4/XRCC5/Lig4, and between XRCC5 and TP53/Lig4 both in the case and control groups. Moreover, a significant positive correlation between LinP1 and XRCC6 in the case, and a significant positive correlation between XRCC4 and Lig4, and a negative correlation between TP53 and LinP1/XRCC4/XRCC5 in the control group was detected. Although a relative difference was detected in transcriptional levels of the NHEJ gene mediators, downregulation of LinP1 in H. pylori-infected patients proposed the activation of a negative feedback loop, which may interfere with the NHEJ activity at the early stages of gastritis.

Haploidy-linked cell proliferation defects limit larval growth in zebrafish.

Haploid larvae in non-mammalian vertebrates are lethal, with characteristic organ growth retardation collectively called 'haploid syndrome'. In contrast to mammals, whose haploid intolerance is attributed to imprinting misregulation, the cellular principle of haploidy-linked defects in non-mammalian vertebrates remains unknown. Here, we investigated cellular defects that disrupt the ontogeny of gynogenetic haploid zebrafish larvae. Unlike diploid control larvae, haploid larvae manifested unscheduled cell death at the organogenesis stage, attributed to haploidy-linked p53 upregulation. Moreover, we found that haploid larvae specifically suffered the gradual aggravation of mitotic spindle monopolarization during 1-3 days post-fertilization, causing spindle assembly checkpoint-mediated mitotic arrest throughout the entire body. High-resolution imaging revealed that this mitotic defect accompanied the haploidy-linked centrosome loss occurring concomitantly with the gradual decrease in larval cell size. Either resolution of mitotic arrest or depletion of p53 partially improved organ growth in haploid larvae. Based on these results, we propose that haploidy-linked mitotic defects and cell death are parts of critical cellular causes shared among vertebrates that limit the larval growth in the haploid state, contributing to an evolutionary constraint on allowable ploidy status in the vertebrate life cycle.

Estrogen receptor alpha (ERα) regulates PARN-mediated nuclear deadenylation and gene expression in breast cancer cells.

The estrogen signalling pathway is highly dynamic and primarily mediated by estrogen receptors (ERs) that transcriptionally regulate the expression of target genes. While transcriptional functions of ERs have been widely studied, their roles in RNA biology have not been extensively explored. Here, we reveal a novel biological role of ER alpha (ERα) in mRNA 3' end processing in breast cancer cells, providing an alternative mechanism in regulating gene expression at the post-transcriptional level. We show that ERα activates poly(A) specific ribonuclease (PARN) deadenylase using in vitro assays, and that this activation is further increased by tumour suppressor p53, a factor involved in mRNA processing. Consistent with this, we confirm ERα-mediated activation of nuclear deadenylation by PARN in samples from MCF7 and T47D breast cancer cells that vary in expression of ERα and p53. We further show that ERα can form complex(es) with PARN and p53. Lastly, we identify and validate expression of common mRNA targets of ERα and PARN known to be involved in cell invasion, metastasis and angiogenesis, supporting the functional overlap of these factors in regulating gene expression in a transactivation-independent manner. Together, these results show a new regulatory mechanism by which ERα regulates mRNA processing and gene expression post-transcriptionally, highlighting its contribution to unique transcriptomic profiles and breast cancer progression.

Cluster classification of a Brazilian gastric cancer cohort reveals remarkable populational differences in normal p53 rate.

BACKGROUND: Queiroz et al. showed that the application of cluster methodology for classifying gastric cancer is suitable and efficient within a Brazilian cohort, which is known for its population heterogeneity. The study highlighted the potential utilization of this method within public health services due to its low-cost, presenting a viable means to improve the diagnosis and prognosis of gastric cancer. BACKGROUND: Our Brazilian cohort with gastric cancer has a distinct distribution between mutated and normal p53. BACKGROUND: New genetic marker-based classifications improve gastric cancer diagnosis accuracy. BACKGROUND: Machine learning integration enhances predictive value in gastric cancer diagnosis. BACKGROUND: Molecular biomarkers complement clinical decisions, advancing personalized medicine. OBJECTIVE: Gastric adenocarcinoma remains an aggressive disease with a poor prognosis, as evidenced by a 5-year survival rate of approximately 31%. The histological classifications already proposed do not accurately reflect the high biological heterogeneity of this neoplasm, particularly in diverse populations, and new classification systems using genetic markers have recently been proposed. Following these newly proposed models, we aimed to assess the cluster distribution in a Brazilian cohort. Furthermore, we evaluated whether the inclusion of other clinical and histological parameters could enhance the predictive value. METHODS: We used a previously described four-immunohistochemistry/EBER-ISH marker to classify a cohort of 30 Brazilian patients with gastric adenocarcinoma into five different clusters and compared the distribution with other genetically diverse populations. Furthermore, we used artificial intelligence methods to evaluate whether other clinical and pathological parameters could improve the results of the methodology. RESULTS: Disclosing the genetic variability between populations, we observed a more balanced distribution of the aberrant/normal p53 ratio (0.6) between patients negative for the other markers tested, unlike previous studies with Asian and North American populations. In addition, decision tree analysis reinforced the efficiency of these new classifications, as the stratification accuracy was not altered with or without additional data. CONCLUSION: Our study underscores the importance of local research in characterizing diverse populations and highlights the complementary role of molecular biomarkers in personalized medicine for gastric adenocarcinoma, enhancing diagnostic accuracy and potentially improving survival rates.

Predicting p53-dependent cell transitions from thermodynamic models.

A cell's fate involves transitions among its various states, each defined by a distinct gene expression profile governed by the topology of gene regulatory networks, which are affected by 3D genome organization. Here, we develop thermodynamic models to determine the fate of a malignant cell as governed by the tumor suppressor p53 signaling network, taking into account long-range chromatin interactions in the mean-field approximation. The tumor suppressor p53 responds to stress by selectively triggering one of the potential transcription programs that influence many layers of cell signaling. These range from p53 phosphorylation to modulation of its DNA binding affinity, phase separation phenomena, and internal connectivity among cell fate genes. We use the minimum free energy of the system as a fundamental property of biological networks that influences the connection between the gene network topology and the state of the cell. We constructed models based on network topology and equilibrium thermodynamics. Our modeling shows that the binding of phosphorylated p53 to promoters of target genes can have properties of a first order phase transition. We apply our model to cancer cell lines ranging from breast cancer (MCF-7), colon cancer (HCT116), and leukemia (K562), with each one characterized by a specific network topology that determines the cell fate. Our results clarify the biological relevance of these mechanisms and suggest that they represent flexible network designs for switching between developmental decisions.

Differential effect of plakoglobin in restoring the tumor suppressor activities of p53-R273H vs. p53-R175H mutants.

The six most common missense mutations in the DNA binding domain of p53 are known as "hot spots" and include two of the most frequently occurring p53 mutations (p53-R175H and p53-R273H). p53 stability and function are regulated by various post-translational modifications such as phosphorylation, acetylation, sumoylation, methylation, and interactions with other proteins including plakoglobin. Previously, using various carcinoma cell lines we showed that plakoglobin interacted with wild-type and several endogenous p53 mutants (e.g., R280K, R273H, S241F, S215R, R175H) and restored their tumor suppressor activities in vitro. Since mutant p53 function is both mutant-specific and cell context-dependent, we sought herein, to determine if plakoglobin tumor suppressive effects on exogenously expressed p53-R273H and p53-R175H mutants are similarly maintained under the same genetic background using the p53-null and plakoglobin-deficient H1299 cell line. Functional assays were performed to assess colony formation, migration, and invasion while immunoblotting and qPCR were used to examine the subcellular distribution and expression of specific proteins and genes that are typically regulated by or regulate p53 function and are altered in mutant p53-expressing cell lines and tumors. We show that though, plakoglobin interacted with both p53-R273H and p53-R175H mutants, it had a differential effect on the transcription and subcellular distribution of their gene targets and their overall oncogenic properties in vitro. Notably, we found that plakoglobin's tumor suppressive effects were significantly stronger in p53-R175H expressing cells compared to p53-R273H cells. Together, our results indicate that exploring plakoglobin interactions with p53-R175H may be useful for the development of cancer therapeutics focused on the restoration of p53 function.

Phage libraries screening on P53: Yield improvement by zinc and a new parasites-integrating analysis.

P53 is a transcription factor that controls a variety of genes, primarily involved in cell cycle and other processes related to cell survival and death. We have isolated peptides targeting P53 (protein and domains) using the "phage display" technique. Interestingly, adding ZnCl2 at 5-10 mM in panning solutions helped to recover more plaque-forming units at least at round one of the screening. Subtractive docking analyses were designed by using a pool of common redundant peptides known as parasites. This rationale helped us differentiate between possibly specific and non-specific bindings. We found notable differences in docking characteristics between different sets of peptides either related to different targets or related to zinc-conditions. The set of zinc-related peptides shows advantageous docking profiles: sharper binding for some positions and distinct exclusive bound residues, including the relevant R248 and R273. Zinc would have modulating/helping role in the targeting of protein P53 by phage displayed peptides in addition to an enhancement action on bacterial infection.

Pre-ciliated tubal epithelial cells are prone to initiation of high-grade serous ovarian carcinoma.

The distal region of the uterine (Fallopian) tube is commonly associated with high-grade serous carcinoma (HGSC), the predominant and most aggressive form of ovarian or extra-uterine cancer. Specific cell states and lineage dynamics of the adult tubal epithelium (TE) remain insufficiently understood, hindering efforts to determine the cell of origin for HGSC. Here, we report a comprehensive census of cell types and states of the mouse uterine tube. We show that distal TE cells expressing the stem/progenitor cell marker Slc1a3 can differentiate into both secretory (Ovgp1+) and ciliated (Fam183b+) cells. Inactivation of Trp53 and Rb1, whose pathways are commonly altered in HGSC, leads to elimination of targeted Slc1a3+ cells by apoptosis, thereby preventing their malignant transformation. In contrast, pre-ciliated cells (Krt5+, Prom1+, Trp73+) remain cancer-prone and give rise to serous tubal intraepithelial carcinomas and overt HGSC. These findings identify transitional pre-ciliated cells as a cancer-prone cell state and point to pre-ciliation mechanisms as diagnostic and therapeutic targets.

FUSE: Improving the estimation and imputation of variant impacts in functional screening.

Deep mutational scanning enables high-throughput functional assessment of genetic variants. While phenotypic measurements from screening assays generally align with clinical outcomes, experimental noise may affect the accuracy of individual variant estimates. We developed the FUSE (functional substitution estimation) pipeline, which leverages measurements collectively within screening assays to improve the estimation of variant impacts. Drawing data from 115 published functional assays, FUSE assesses the mean functional effect per amino acid position and makes estimates for individual allelic variants. It enhances the correlation of variant functional effects from different assay platforms and increases the classification accuracy of missense variants in ClinVar across 29 genes (area under the receiver operating characteristic [ROC] curve [AUC] from 0.83 to 0.90). In UK Biobank patients with rare missense variants in BRCA1, LDLR, or TP53, FUSE improves the classification accuracy of associated phenotypes. FUSE can also impute variant effects for substitutions not experimentally screened. This approach improves accuracy and broadens the utility of data from functional screening.

Targeting mutant p53: a key player in breast cancer pathogenesis and beyond.

The p53 mutation is the most common genetic mutation associated with human neoplasia. TP53 missense mutations, which frequently arise early in breast cancer, are present in over thirty percent of breast tumors. In breast cancer, p53 mutations are linked to a more aggressive course of the disease and worse overall survival rates. TP53 mutations are mostly seen in triple-negative breast cancer, a very diverse kind of the disease. The majority of TP53 mutations originate in the replacement of individual amino acids within the p53 protein's core domain, giving rise to a variety of variations referred to as "mutant p53s." In addition to gaining carcinogenic qualities through gain-of-function pathways, these mutants lose the typical tumor-suppressive features of p53 to variable degrees. The gain-of-function impact of stabilized mutant p53 causes tumor-specific dependency and resistance to therapy. P53 is a prospective target for cancer therapy because of its tumor-suppressive qualities and the numerous alterations that it experiences in tumors. Phenotypic abnormalities in breast cancer, notably poorly differentiated basal-like tumors are frequently linked to high-grade tumors. By comparing data from cell and animal models with clinical outcomes in breast cancer, this study investigates the molecular mechanisms that convert gene alterations into the pathogenic consequences of mutant p53's tumorigenic activity. The study delves into current and novel treatment approaches aimed at targeting p53 mutations, taking into account the similarities and differences in p53 regulatory mechanisms between mutant and wild-type forms, as well.

Aerobic exercise up-regulates Klotho to improve renal fibrosis associated with aging and its mechanism.

Renal fibrosis is a major cause of renal dysfunction and is a common pathological event in almost all forms of chronic kidney disease (CKD). Currently, the pathomechanisms of renal fibrosis are not well understood. However, researchers have demonstrated that aerobic exercise can improve renal fibrosis. Klotho is considered to be a negative regulator of renal fibrosis. In this study, we aimed to investigate the role and mechanism of Klotho in the improvement of renal fibrosis through aerobic exercise. We performed a 12-week aerobic exercise intervention in 19-month-old male C57BL/6J mice. Physiological and biochemical indexes were performed to assess renal function and renal fibrosis. The roles of Klotho were further confirmed through knockdown of Klotho by small interfering RNA (siRNA) in C57BL/6J mice.Q-PCR and Western blot were performed to quantify determine the expression of relevant genes and proteins in the kidney. Results: Aging decreased Klotho expression via activated the upstream TGF-ß1/p53/miR34a signaling pathway and affected its downstream signaling pathways, ultimately leading to renal fibrosis. Exposure to aerobic exercise for 12 weeks significantly improved renal fibrosis and alleviated the intrarenal genetic alterations induced by aging. Conclusion: Our results showed that aerobic exercise increased Klotho expression by inhibiting the TGF-ß1/p53/miR34a signaling pathway and further inhibited its downstream TGF-ß1/smad3 and ß-linker protein signaling pathways. These results provide a theoretical basis supporting the feasibility of exercise in the prevention and treatment of CKD.

SURF2 is a MDM2 antagonist in triggering the nucleolar stress response.

Cancer cells rely on high ribosome production to sustain their proliferation rate. Many chemotherapies impede ribosome production which is perceived by cells as "nucleolar stress" (NS), triggering p53-dependent and independent pathways leading to cell cycle arrest and/or apoptosis. The 5S ribonucleoprotein (RNP) particle, a sub-ribosomal particle, is instrumental to NS response. Upon ribosome assembly defects, the 5S RNP accumulates as free form. This free form is able to sequester and inhibit MDM2, thus promoting p53 stabilization. To investigate how cancer cells can resist to NS, here we purify free 5S RNP and uncover an interaction partner, SURF2. Functional characterization of SURF2 shows that its depletion increases cellular sensitivity to NS, while its overexpression promotes their resistance to it. Consistently, SURF2 is overexpressed in many cancers and its expression level is an independent marker of prognosis for adrenocortical cancer. Our data demonstrate that SURF2 buffers free 5S RNP particles, and can modulate their activity, paving the way for the research of new molecules that can finely tune the response to nucleolar stress in the framework of cancer therapies.

Targeting cellular plasticity: esculetin-driven reversion of stem cell-like characteristics and EMT phenotype in transforming cells with sequential p53/p73 knockdowns.

The intricate interplay of cancer stem cell plasticity, along with the bidirectional transformation between epithelial-mesenchymal states, introduces further intricacy to offer insights into newer therapeutic approaches. Differentiation therapy, while successful in targeting leukemic stem cells, has shown limited overall success, with only a few promising instances. Using colon carcinoma cell strains with sequential p53/p73 knockdowns, our study underscores the association between p53/p73 and the maintenance of cellular plasticity. Morphological alterations corresponding with cell surface marker expressions, transcriptome analysis and functional assays were performed to access stemness and EMT (Epithelial-Mesenchymal Transition) characteristics in the spectrum of cells exhibiting sequential p53 and p73 knockdowns. Notably, our investigation explores the effectiveness of esculetin in reversing the shift from an epithelial to a mesenchymal phenotype, characterized by stem cell-like traits. Esculetin significantly induces enterocyte differentiation and promotes epithelial cell polarity by altering Wnt axes in Cancer Stem Cell-like cells characterized by high mesenchymal features. These results align with our previous findings in leukemic blast cells, establishing esculetin as an effective differentiating agent in both Acute Myeloid Leukemia (AML) and solid tumor cells.

Age-Dependent Differences in Radiation-Induced DNA Damage Responses in Intestinal Stem Cells.

Age at exposure is a critical modifier of the risk of radiation-induced cancer. However, the effects of age on radiation-induced carcinogenesis remain poorly understood. In this study, we focused on tissue stem cells using Lgr5-eGFP-ires-CreERT2 mice to compare radiation-induced DNA damage responses between Lgr5+ and Lgr5- intestinal stem cells. Three-dimensional immunostaining analyses demonstrated that radiation induced apoptosis and the mitotic index more efficiently in adult Lgr5- stem cells than in adult Lgr5+ stem cells but not in infants, regardless of Lgr5 expression. Supporting this evidence, rapid and transient p53 activation occurred after irradiation in adult intestinal crypts but not in infants. RNA sequencing revealed greater variability in gene expression in adult Lgr5+ stem cells than in infant Lgr5+ stem cells after irradiation. Notably, the cell cycle and DNA repair pathways were more enriched in adult stem cells than in infant stem cells after irradiation. Our findings suggest that radiation-induced DNA damage responses in mouse intestinal crypts differ between infants and adults, potentially contributing to the age-dependent susceptibility to radiation carcinogenesis.

Continued decitabine/all-trans retinoic acid treatment: extended complete remission in an elderly AML patient with multi-hit TP53 lesions and complex-monosomal karyotype.

DNA-hypomethylating agents (HMAs) induce notable remission rates in AML/MDS patients with TP53 mutations; however, secondary resistance often develops rapidly. In the DECIDER trial (NCT00867672), elderly AML patients (also those with adverse genetics) randomized to all-trans retinoic acid (ATRA) added to decitabine (DEC) attained significantly delayed time-to-resistance. An 82-year-old patient with a non-disruptive, in-frame TP53 mutation (p.Cys238_Asn239delinsTyr, VAF 90%) and complex-monosomal karyotype attained a complete hematologic and cytogenetic remission with DEC + ATRA, with 3.7 years survival after 30 treatment cycles that were well-tolerated. Further HMA + ATRA studies appear warranted in AML/MDS patients of different genetic risk groups ineligible for more intensive treatment.Trial registration: This trial was registered at ClinicalTrials.gov identifier: NCT00867672.

Germline variant affecting p53ß isoforms predisposes to familial cancer.

Germline and somatic TP53 variants play a crucial role during tumorigenesis. However, genetic variations that solely affect the alternatively spliced p53 isoforms, p53ß and p53γ, are not fully considered in the molecular diagnosis of Li-Fraumeni syndrome and cancer. In our search for additional cancer predisposing variants, we identify a heterozygous stop-lost variant affecting the p53ß isoforms (p.*342Serext*17) in four families suspected of an autosomal dominant cancer syndrome with colorectal, breast and papillary thyroid cancers. The stop-lost variant leads to the 17 amino-acid extension of the p53ß isoforms, which increases oligomerization to canonical p53α and dysregulates the expression of p53's transcriptional targets. Our study reveals the capacity of p53ß mutants to influence p53 signalling and contribute to the susceptibility of different cancer types. These findings underscore the significance of p53 isoforms and the necessity of comprehensive investigation into the entire TP53 gene in understanding cancer predisposition.