MDM2 Expression Research Articles

Transcriptomic analysis of EGFR co-expression and activation in glioblastoma reveals associations with its ligands

Abstract Background Approximately half of the IDH-wildtype glioblastomas (GBM) exhibit EGFR amplification. Additionally, genomic changes that occur in the extracellular domain of EGFR can lead to ligand-hypersensitivity (R108K/A289V/G598V) or ligand-independence (EGFRvIII). Unlike in lung adenocarcinoma (LUAD), clinical trials with EGFR inhibitors showed no survival benefit for GBM and it remains unclear why. We aimed to elucidate differences in molecular mechanisms of EGFR activation and regulation between GBM and LUAD. Methods We used RNA-sequencing data to find EGFR co-regulated genes and pathways in GBM and compare EGFR signaling patterns between GBM and LUAD. Cellular origins of expression signals were determined by analyzing single-cell RNA sequencing data. Results We identified two ligands (BTC/EREG) among the significant EGFR predictor genes (TCGA-GBM: n=169, Intellance-2: n=166). Their expression was inversely correlated with EGFR amplification and incidence of ligand-sensitive mutations. Ligands were expressed by non-malignant cells and differed in their primary source of expression (BTC: neurons, EREG: myeloid). High expression of MDM2 and CDK4 was less common in EGFR-amplified GBMs with ligand-sensitive mutations compared to those without these mutations. Our analyses revealed distinct transcriptional profiles between GBM and LUAD when comparing tumors carrying activating mutations. Conclusions BTC and EREG are negatively associated with EGFR expression in GBM. These findings emphasize the role of ligands in regulating EGFR, where EGFR activation seems to be modulated by the highly varying levels of EGFR amplification, sensitivity of the receptor towards ligands and ligand expression levels. Ligand expression levels and EGFR mutations could refine patient stratification for EGFR-targeted therapies in GBM.

Chemokine CXCL12 Activates CXC Receptor 4 Metastasis Signaling Through the Upregulation of a CXCL12/CXCR4/MDMX (MDM4) Axis.

The metastasis-promoting G-protein-coupled receptor CXC Receptor 4 (CXCR4) is activated by the chemokine CXCL12, also known as stromal cell-derived factor 1 (SDF-1). The CXCL12/CXCR4 pathway in cancer promotes metastasis but the molecular details of how this pathway cross-talks with oncogenes are understudied. An oncogene pathway known to promote breast cancer metastasis in MDA-MB-231 xenografts is that of Mouse Double Minute 2 and 4 (MDM2 and MDM4, also known as MDMX). MDM2 and MDMX promote circulating tumor cell (CTC) formation and metastasis, and positively correlate with a high expression of CXCR4. Interestingly, this MDMX-associated upregulation of CXCR4 is only observed in cells grown in the tumor microenvironment (TME), but not in MDA-MB-231 cells grown in a tissue culture dish. This suggested a cross-talk signaling factor from the TME which was predicted to be CXCL12 and, as such, we asked if the exogenous addition of the cell non-autonomous CXCL12 ligand would recapitulate the MDMX-dependent upregulation of CXCR4. We used MDA-MB-231 cells and isolated CTCs, with and without MDMX knockdown, plus the exogenous addition of CXCL12 to determine if MDMX-dependent upregulation of CXCR4 could be recapitulated outside of the TME context. We added exogenous CXCL12 to the culture medium used for growth of MDA-MB-231 cells and isogenic cell lines engineered for MDM2 or MDMX depletion. We carried out immunoblotting, and quantitative RT-PCR to compare the expression of CXCR4, MDM2, MDMX, and AKT activation. We carried out Boyden chamber and wound healing assays to assess the influence of MDMX and CXCL12 on the cells' migration capacity. The addition of the CXCL12 chemokine to the medium increased the CXCR4 cellular protein level and activated the PI3K/AKT signaling pathway. Surprisingly, we observed that the addition of CXCL12 mediated the upregulation of MDM2 and MDMX at the protein, but not at the mRNA, level. A reduction in MDMX, but not MDM2, diminished both the CXCL12-mediated CXCR4 and MDM2 upregulation. Moreover, a reduction in both MDM2 and MDMX hindered the ability of the added CXCL12 to promote Boyden chamber-assessed cell migration. The upregulation of MDMX by CXCL12 was mediated, at least in part, by a step upstream of the proteasome pathway because CXCL12 did not increase protein stability after cycloheximide treatment, or when the proteasome pathway was blocked. These data demonstrate a positive feed-forward activation loop between the CXCL12/CXCR4 pathway and the MDM2/MDMX pathway. As such, MDMX expression in tumor cells may be upregulated in the primary tumor microenvironment by CXCL12 expression. Furthermore, CXCL12/CXCR4 metastatic signaling may be upregulated by the MDM2/MDMX axis. Our findings highlight a novel positive regulatory loop between CXCL12/CXCR4 signaling and MDMX to promote metastasis.

Fractionated alpha and mixed beam radiation promote stronger pro-inflammatory effects compared to acute exposure and trigger phagocytosis

Introduction and methodsAiming to evaluate safety aspects of a recently proposed approach to target Alzheimer’s disease, we mimicked a complex boron neutron capture therapy field using a mixed beam consisting of high- and low-linear energy transfer (LET) radiation, 241Am alpha particles (α) and/or X-ray radiation respectively, in human microglial (HMC3) cells.ResultsAcute exposure to 2 Gy X-rays induced the strongest response in the formation of γH2AX foci 30 min post irradiation, while α- and mixed beam-induced damage (α:X-ray = 3:1) sustained longer. Fractionation of the same total dose (0.4 Gy daily) induced a similar number of γH2AX foci as after acute radiation, however, α- or mixed irradiation caused a higher expression of DNA damage response genes CDKN1A and MDM2 24 h after the last fraction, as well as a stronger decrease in cell viability and clonogenic survival compared to acute exposure. Phosphorylation of STING, followed by phosphorylation of NF-κB subunit p65, was rapidly induced (1 or 3 h, respectively) after the last fraction by all radiation qualities. This led to IL-1β secretion into the medium, strongly elevated expression of pro-inflammatory cytokine genes and enhanced phagocytosis after fractionated exposure to α- and mixed beam-irradiation compared to their acute counterparts 24 h post-irradiation. Nevertheless, all inflammatory changes were returning to basal levels or below 10–14 days post irradiation.DiscussionIn conclusion, we demonstrate strong transient pro-inflammatory induction by daily high-LET radiation in a microglia model, triggering phagocytosis which may aid in clearing amyloid beta, but importantly, from a safety perspective, without long-term alterations.

Investigation of the Potential Mechanism of Compound Dragon's Blood Capsule against Myocardial Ischemia Based on Network Pharmacology.

Dragon's blood is widely consumed in China, Vietnam and Laos to promote blood circulation. A Compound Dragon's blood capsule (CDC) is a patented medicine composed of dragon's blood, notoginseng, and borneol. This combination is purported to stabilize coronary heart disease and myocardial ischemia. However, the possible mechanisms and the characterization of its drug targets' relevance at the systemic level remain unclear. The present study aims to reveal the potential mechanisms of CDC's anti-myocardial ischemia effect. The potential mechanisms were investigated by network pharmacology and qRT-PCR was used to verify the expression levels of key genes of PI3k-Akt pathway. S1PR2 and AGTR1 were the common targets, which involved 6 biological processes annotated by KEGG and GO analysis. The qRT-PCR results showed a remarkable increase in the expression of Pi3k, Pdk1, Akt, Mdm2, Bcl2, and mTOR. Results also showed a decline in the expression of P53 and Casp3 after CDC intervention. CDC has a significant anti-myocardial ischemia effect through the PI3k/Akt pathway, which demonstrates that CDC is a suitable adjuvant to treat CHD and provides a theoretical basis for its further clinical application.

Melatonin effect on breast and ovarian cancers by targeting the PI3K/Akt/mTOR pathway.

Melatonin, the hormone of the pineal gland, possesses a range of physiological functions, and recently, its anticancer effect has become more apparent. A more thorough understanding of molecular alterations in the components of several signaling pathways as new targets for cancer therapy is needed because of current innate restrictions such as drug toxicity, side effects, and acquired or de novo resistance. The PI3K/Akt/mTOR pathway is overactivated in many solid tumors, such as breast and ovarian cancers. This pathway in normal cells is essential for growth, proliferation, and survival. However, it is an undesirable characteristic in malignant cells. We have reviewed multiple studies about the effect of melatonin on breast and ovarian cancer, focusing on the PI3K/Akt/mTOR pathway. Melatonin exerts its inhibitory effects via several mechanisms. A: Downregulation of downstream or upstream components of the signaling pathway such as phosphatase and tensin homolog (PTEN), phosphatidylinositol (3,4,5)-trisphosphate kinase (PI3K), p-PI3K, Akt, p-Akt, mammalian target of rapamycin (mTOR), and mTOR complex1 (mTORC1). B: Apoptosis induction by decreasing MDM2 expression, a downstream target of Akt, and mTOR, which leads to Bad activation in addition to Bcl-XL and p53 inhibition. C: Induction of autophagy in cancer cells via activating ULK1 after mTOR inhibition, resulting in Beclin-1 phosphorylation. Beclin-1 with AMBRA1 and VPS34 promotes PI3K complex I activity and autophagy in cancer cells. The PI3K/Akt/mTOR pathway overlaps with other intracellular signaling pathways and components such as AMP-activated protein kinase (AMPK), Wnt/β-catenin, mitogen-activated protein kinase (MAPK), and other similar pathways. Cancer therapy can benefit from understanding how these pathways interact and how melatonin affects these pathways.

Gain of 1q confers an MDM4-driven growth advantage to undifferentiated and differentiating hESC while altering their differentiation capacity

Gain of 1q is a highly recurrent chromosomal abnormality in human pluripotent stem cells. In this work, we show that gains of 1q impact the differentiation capacity to derivates of the three germ layers, leading to mis-specification to cranial placode and non-neural ectoderm during neuroectoderm differentiation. Also, we found a weaker expression of lineage-specific markers in hepatoblasts and cardiac progenitors. Competition assays show that the cells retain their selective advantage during differentiation, which is mediated by a higher expression of MDM4, a gene located in the common region of gain. MDM4 drives the winner phenotype of the mutant cells in both the undifferentiated and differentiating state by reducing the cells’ sensitivity to DNA damage through decreased p53-mediated apoptosis. Finally, we found that cell density in culture plays a key role in promoting the competitive advantage of the cells by increasing DNA damage.

P53 and the E3 Ubiquitin Ligase MDM2 in Glaucomatous Lamina Cribrosa Cells.

Lamina cribrosa (LC) cells play an integral role in extracellular matrix remodeling and fibrosis in human glaucoma. LC cells bear similarities to myofibroblasts that adopt an apoptotic-resistant, proliferative phenotype, a process linked to dysregulation of tumor suppressor-gene p53 pathways, including ubiquitin-proteasomal degradation via murine-double-minute-2 (MDM2). Here, we investigate p53 and MDM2 in glaucomatous LC cells. Primary human LC cells were isolated from glaucomatous donor eyes (GLC) and age-matched normal controls (NLC) (n = 3 donors/group). LC cells were cultured under standard conditions ± 48-h treatment with p53-MDM2-interaction inhibitor RG-7112. Markers of p53-MDM2, fibrosis, and apoptosis were analyzed by real-time polymerase chain reaction (qRT-PCR), western blotting, and immunofluorescence. Cellular proliferation and viability were assessed using colorimetric methyl-thiazolyl-tetrazolium salt assays (MTS/MTT). In GLC versus NLC cells, protein expression of p53 was significantly decreased (p < 0.05), MDM2 was significantly increased, and immunofluorescence showed reduced p53 and increased MDM2 expression in GLC nuclei. RG-7112 treatment significantly increased p53 and significantly decreased MDM2 gene and protein expression. GLC cells had significantly increased protein expression of αSMA, significantly decreased caspase-3 protein expression, and significantly increased proliferation after 96 h. RG-7112 treatment significantly decreased COL1A1 and αSMA, significantly increased BAX and caspase-3 gene expression, and significantly decreased proliferation in GLC cells. MTT-assay showed equivocal cellular viability in NLC/GLC cells with/without RG-7112 treatment. Our data suggests that proliferation and the ubiquitin-proteasomal pathway are dysregulated in GLC cells, with MDM2-led p53 protein degradation negatively impacting its protective role.

Omega-3 polyunsaturated fatty acids protect against cisplatin-induced nephrotoxicity by activating the Nrf2 signaling pathway

Nephrotoxicity is a prevalent side effect observed in patients undergoing chemotherapy. The pathogenesis of chemotherapy-induced nephrotoxicity involves various factors such as oxidative stress, DNA damage, inflammation, and apoptosis. Omega-3 polyunsaturated fatty acids (ω-3 PUFAs), particularly eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), possess anti-inflammatory and antioxidant properties. This study investigated the effects of EPA and DHA, either alone or in combination, on cisplatin-induced nephrotoxicity in mice, as well as their underlying mechanisms of action. The combined administration of EPA and DHA demonstrated superior efficacy in mitigating cisplatin-induced nephrotoxicity compared to administration alone, including the reduction of oxidative damage, inflammation, and apoptosis. Moreover, the combination of EPA and DHA suppressed inflammation and prevented the development of chronic kidney fibrosis during prolonged observations following repeated cisplatin administration. Mechanistically, ω-3 PUFAs enhance the expression of antioxidant genes by activating the p62-Keap1-Nrf2 signaling pathway. Furthermore, Nrf2 activation can inhibit the cisplatin-induced p53 apoptosis signal by upregulating the expression of MDM2 in renal tubular epithelial cells. Consequently, ω-3 PUFAs exert a protective effect against cisplatin-induced renal injury through activating the Nrf2 signaling pathway, suggesting that ω-3 PUFAs intake holds promise as a therapeutic strategy for combating cisplatin-induced nephrotoxicity.

The impact of ERP29 on the progression of pharyngeal squamous cell carcinoma

ERP29 gene encodes a chaperone protein critical for protein folding and secretion. Previous study linked ERP29 inhibition to an elevated risk of pharynx squamous cell carcinoma (PSCC) and reduced patients’ survival. However, ERP29 role in PSCC progression remains unknown. Here, we investigated ERP29 impact on PSCC progression in cisplatin (CDDP)-sensitive (FaDu and LAU-2063), CDDP-treated (FaDu-CDDP), and CDDP-resistant (FaDu-R) cells. ERP29 silencing decreased necrosis and increased migration in CDDP-sensitive, treated, and resistant cells; and reduced E-cadherin and increased vimentin immunoexpression in CDDP-sensitive 3D-spheroids. During CDDP treatment, ERP29 silencing enhanced proliferation. In CDDP-sensitive cells, ERP29 silencing upregulated genes associated with WNT, MAPK, and PI3K/AKT signaling pathways while downregulating CASP9 expression. During CDDP treatment, ERP29 silencing downregulated MDM2 and CASP9 expression. In CDDP-resistant cells, ERP29 silencing upregulated SOS1, MAPK1, AKT1, ITGAV, and CCNE1, while downregulating KRAS, JUN, MDM2, and CASP9 expression. In addition, inhibition of microRNA miR-4421 increased ERP29 expression and decreased MAPK1, AKT1, and JUN expression in CDDP-sensitive cells, as well as SOS1, MAPK1, AKT1, and ITGAV in CDDP-resistant cells. Lower ERP29 and higher miR-4421 expressions were predictive of poor survival, suggesting a potential therapeutic use for miR-4421 inhibitors. Upon validation, these findings may contribute to targeted therapies for PSCC based on ensuring ERP29 expression.

Immunohistochemical Expression of MDM2, Bcl-2, SATB2 and Ki-67 in Histological Variants of Unicystic Ameloblastoma.

To characterize the immunohistochemical expression of MDM2, Bcl-2, SATB2 and Ki-67 in histological variants of unicystic ameloblastoma (UA). Following the ethical approval, forty (40) patients with unicystic ameloblastoma were retrieved from the archives and subjected to immunohistochemistry (IHC). Sociodemographic and clinical data were also retrieved. The results were entered into a Microsoft Excel spreadsheet and analyzed using SPSS software. Human tooth germs, which served as the control, showed moderate expression of Bcl-2 and MDM2 with slight proliferative activity in ameloblasts and moderate expression of SATB2 in ectomesenchyme and odontoblasts. Luminal UA (Type 1) showed low Ki-67 index and negative to mild Bcl-2 and MDM2 expression, whilst Type 1.2 (luminal and intraluminal), Type 1.2.3 (luminal, intraluminal and mural), and Type 1.3 (luminal and mural), including the recurrent cases, showed moderate to intense expression with high mean Ki-67 index. The difference between the study groups was statistically significant (p value < 0.001). No expression of SATB2 was noted in any histological variant of UA. Furthermore, no significant differences were noted in age, gender, site and location between the groups. In contrast to luminal variant of UA, mural±intraluminal variants and recurrent cases demonstrate higher expression of Bcl-2 and MDM2 with higher mean Ki-67 index. It may thus be prudent to provide aggressive treatment for cases, not just with mural follicles but also for the patients with intraluminal plexiform proliferation, to prevent recurrence and improve patient outcomes.

Targeting telomerase with MST-312 leads to downregulation of CCND1, MDM2, MYC, and HSP90AA1 and induce apoptosis in Jurkat cell line.

Acute lymphoblastic leukemia is a challenging disease to treat, especially in older adults who are most commonly diagnosed and have a high risk of relapse, even with current treatment options. MST-312, targets the RNA component of telomerase, inhibiting its activity and leading to growth arrest and telomere shortening in cancer cells. This study aimed to investigate the effects of MST-312 on apoptosis rates and the expression of telomerase target genes, CCND1, MDM2, MYC, and HSP90AA1, in Jurkat cell line. Jurkat cell line was cultured and treated with various concentrations of MST-312(0 µM, 0.5 µM, 1 µM, 2 µM, and 4 µM). The optimal concentration of MST-312 was determined using MTT assay. Flow cytometry was employed to evaluate the apoptosis induced by MST-312 treatment. The expression levels of the target genes were measured using real-time polymerase chain reaction before and after the treatment with MST-312. P-value < 0.05 was considered statistically significant. The percentages of apoptotic cells after 48 h, as determined by flow cytometry analysis, were 30.32%, 52.35%, 57.60%, and 68.82%, respectively, compared to the control group which was 4.6%. The expression levels of all genes, including CCND1, MDM2, MYC, and HSP90AA1, were decreased compared to the control group. The results showed that MST-312 induced dose- and time-dependent apoptosis and downregulated the expression of CCND1, MDM2, MYC, and HSP90AA1in Jurkat cell line.

Key Genes Involved in the Beneficial Mechanism of Hyperbaric Oxygen for Glioblastoma and Predictive Indicators of Hyperbaric Oxygen Prolonging Survival in Glioblastoma Patients.

The prognosis of glioblastoma is poor, and therapy-resistance is largely attributed to intratumor hypoxia. Hyperbaric oxygen (HBO) effectively alleviates hypoxia. However, the sole role of HBO in glioblastoma remains controversial. We previously reported that HBO can promote apoptosis, shorten protrusions, and delay growth of glioblastoma, but the molecular mechanism is unclear. We aimed to test candidate genes in HBO-exposed glioblastoma cells and to analyze their correlation with the survival of glioblastoma patients. Glioblastoma cell lines exposed to repetitive HBO or normobaric air (NBA) were collected for RNA isolation and microarray data analysis. GO analysis, KEGG pathway analysis and survival analysis of the differentially expressed genes (DEGs) were performed. HBO not only inhibited hypoxia-inducing genes including CA9, FGF11, PPFIA4, TCAF2 and SLC2A12, but also regulated vascularization by downregulating the expression of COL1A1, COL8A1, COL12A1, RHOJ and FILIP1L, ultimately attenuated hypoxic microenvironment of glioblastoma. HBO attenuated inflammatory microenvironment by reducing the expression of NLRP2, CARD8, MYD88 and CD180. HBO prevented metastasis by downregulating the expression of NTM, CXCL12, CXCL13, CXCR4, CXCR5, CDC42, IGFBP3, IGFBP5, GPC6, MMP19, ADAMTS1, EFEMP1, PTBP3, NF1 and PDCD1. HBO upregulated the expression of BAK1, PPIF, DDIT3, TP53I11 and FAS, whereas downregulated the expression of MDM4 and SIVA1, thus promoting apoptosis. HBO upregulated the expression of CDC25A, MCM2, PCNA, RFC33, DSCC1 and CDC14A, whereas downregulated the expression of ASNS, CDK6, CDKN1B, PTBP3 and MAD2L1, thus inhibiting cell cycle progression. Among these DEGs, 17 indicator-genes of HBO prolonging survival were detected. HBO is beneficial for glioblastoma. Glioblastoma patients with these predictive indicators may prolong survival with HBO therapy. These potential therapeutic targets especially COL1A1, ADAMTS1 and PTBP3 deserve further validation.

HADHA promotes esophageal cancer progression by activating mTOR signaling and the SP1/MDM2 axis.

Esophageal cancer (EC) is one of the most recalcitrant cancers, with a 5-year survival rate of <30%. The hydroxyacyl-CoA dehydrogenase alpha subunit (HADHA) plays an essential role in long-chain fatty acid metabolism, and dysregulation of HADHA has been demonstrated to be involved in a series of metabolic diseases and cancers. However, its role in cancers remains controversial. HADHA has seldom been investigated in EC, and little is known about how HADHA regulates the malignant progression of EC. In this study, we find that HADHA is significantly upregulated in EC tissues and is correlated with poor survival. HADHA knockdown markedly inhibits EC cell proliferation both in vitro and in vivo. The loss of HADHA also induces EC cell apoptosis, causes cell cycle arrest and inhibits cell migration. Additionally, RNA profiling reveals that mTOR signaling is significantly suppressed after HADHA knockdown. Mechanistically, HADHA interacts with SP1 and induces MDM2 expression. In conclusion, both mTOR signaling and the SP1-MDM2 axis participate in the HADHA-induced malignant behavior of EC cells.

Prognostic Importance of PTEN, p53, and MDM2 Expressions in Endometrioid and Serous-Type Endometrial Carcinomas

Aim: Endometrial carcinomas (ECs) are neoplasms with the highest rate of change in the phosphatidylinositol 3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) pathway. In this study, the relationship among PTEN, MDM2, and p53 protein expression in the PI3K/AKT/mTOR pathway with clinicopathological data in endometrioid endometrial carcinomas (EECs) and serous-type endometrial carcinomas (SECs) was evaluated. Material and Method: A hundred and twenty cases of patients who underwent hysterectomy for EC between 2009 and 2021 were included in the study. Thirty cases of SEC and 90 cases of EEC were evaluated. EEC cases consist of grades 1-3 tumors, and each group includes 30 patients. p53 was examined in two groups as normal/wild type and abnormal/mutant type. PTEN and MDM2 were examined in two groups: positive and negative. The relationship among p53, PTEN, and MDM2 immunohistochemical expression status with histological grade, myometrial invasion, cervical invasion, lymphovascular invasion (LVI), metastatic lymph nodes, presence of tumor in peritoneal fluid, tumor stage, and overall and progression-free survival was evaluated. Results: Loss of PTEN was associated with EEC compared to SEC (p

Diagnostic Accuracy of Immunohistochemical Expression of p16, MDM2, and CDK4 in Well-Differentiated and De-Differentiated Liposarcoma in MDM2 Fluorescent in situ Hybridisation Confirmed Cases.

To establish the diagnostic utility of immunohistochemistry markers p16 along with MDM2 and CDK-4 in confirming the diagnosis of well-differentiated and de-differentiated liposarcoma while taking Fluorescent in situ Hybridisation (FISH) as a gold standard. A cross-sectional study. Place and Duration of the Study: Department of Histopathology, Armed Forces Institute of Pathology, Rawalpindi, Pakistan, from 30th January 2022 to 30th June 2023. A standard panel of three immunohistochemistry markers p16, MDM2, and CDK4 were applied to 36 cases of atypical lipomatous tumours, well-differentiated liposarcoma (WDLPS), and de-differentiated liposarcoma (DDLPS), on which the gold standard FISH was already performed. The sample size was calculated with the help of a WHO calculator taking prevalence 1-2% in Pakistani population. Qualitative variables such as gender and site of tumour were presented by calculating frequency and percentages and comparison of Immunohistochemistry results with FISH was done by using a 2x2 table. The sensitivity and specificity of this triple marker panel for detecting WDLPS/DDLPS were 43.47% and 15.38%, respectively. The sensitivity and specificity of CDK4 for detecting WDLPS / DDLPS were 82.6% and 15.4%, those of MDM2 were 73.9% and 61.5 %, and those of p16 were 60.9% and 53.8%, respectively. Among all three markers, CDK4 was the most sensitive and MDM2 was the most specific marker for detecting WDLPS-DDLPS. It also showed that a combination of these three markers improves the diagnostic credibility of the immunohistochemistry in diagnosing DDLPS and WDLPS but FISH is the most reliable and confirmatory method. De-differentiated liposarcoma, Well-differentiated liposarcoma, P16, CDK4, MDM2.

Malignant transformation of craniofacial fibrous dysplasia: A clinicopathological, immunohistochemical and molecular analysis of 15 cases in one single institution

ObjectiveMalignant transformation of craniofacial fibrous dysplasia (FD) is not common and its clinicopathological as well as molecular characteristics remain largely unknown with limited literature reports. Study designPatients diagnosed with FD including McCune-Albright syndrome (MAS), polyostotic fibrous dysplasia (PFD), and monostotic fibrous dysplasia (MFD), accompanied by malignant transformation at our institution over the past 18 years (2005–2023) were retrospectively screened and analyzed to investigate the epidemiology and clinicopathological features of these tumors. ResultsThree hundred and five patients were diagnosed as FD in our hospital from 2005 to 2023, with 176 females (57.7 %) and 129 males (42.3 %). The average age at diagnosis was 28.35 years, ranging from 7 to 70 years. A total number of 15 (4. 9 %) cases of FD with malignant transformation were selected. Among these 15 patients, the age of the initial diagnosis of FD ranged from 6 to 54 years (mean age 28.87 ± 16.77), and the ages when malignant transformation occurred ranged from 18 to 57 years (mean age 38.53 ± 13.05). Among 15 patients, 12 patients were female (80 %) and 3 were male (20 %). Fifteen cases included MSA in 2 patients, PFD in 4 patients, and MFD in 9 patients. Of the anatomical sites in craniofacial bones, the most common site of the lesion was the maxilla, followed by the mandible. Malignant neoplasm arising in FD were osteosarcoma (12/15), chondrosarcoma (1/15) and high-grade sarcoma of uncertain differentiation (2/15). The 3- and 5-year overall survival rate was 33.3 % (5/15) and 20 % (3/15) respectively. In secondary osteosarcoma from FD, MDM2 and CDK4 positivity were 33.3 % and 41.7 % respectively, and only one case was MDM2-amplified and CDK4-amplified. ConclusionMalignant transformation in fibrous dysplasia was an exceedingly rare event and with a female predominance. The overall survival rate was poor. Osteosarcoma was the most common malignant neoplasm arising in FD. MDM2 and CDK4 expression may aid in the diagnosis of secondary osteosarcoma in FD.

Synthetic lethality of combined ULK1 defection and p53 restoration induce pyroptosis by directly upregulating GSDME transcription and cleavage activation through ROS/NLRP3 signaling

BackgroundHigh expression of ubiquitin ligase MDM2 is a primary cause of p53 inactivation in many tumors, making it a promising therapeutic target. However, MDM2 inhibitors have failed in clinical trials due to p53-induced feedback that enhances MDM2 expression. This underscores the urgent need to find an effective adaptive genotype or combination of targets.MethodsKinome-wide CRISPR/Cas9 knockout screen was performed to identify genes that modulate the response to MDM2 inhibitor using TP53 wild type cancer cells and found ULK1 as a candidate. The MTT cell viability assay, flow cytometry and LDH assay were conducted to evaluate the activation of pyroptosis and the synthetic lethality effects of combining ULK1 depletion with p53 activation. Dual-luciferase reporter assay and ChIP-qPCR were performed to confirm that p53 directly mediates the transcription of GSDME and to identify the binding region of p53 in the promoter of GSDME. ULK1 knockout / overexpression cells were constructed to investigate the functional role of ULK1 both in vitro and in vivo. The mechanism of ULK1 depletion to activate GSMDE was mainly investigated by qPCR, western blot and ELISA.ResultsBy using high-throughput screening, we identified ULK1 as a synthetic lethal gene for the MDM2 inhibitor APG115. It was determined that deletion of ULK1 significantly increased the sensitivity, with cells undergoing typical pyroptosis. Mechanistically, p53 promote pyroptosis initiation by directly mediating GSDME transcription that induce basal-level pyroptosis. Moreover, ULK1 depletion reduces mitophagy, resulting in the accumulation of damaged mitochondria and subsequent increasing of reactive oxygen species (ROS). This in turn cleaves and activates GSDME via the NLRP3-Caspase inflammatory signaling axis. The molecular cascade makes ULK1 act as a crucial regulator of pyroptosis initiation mediated by p53 activation cells. Besides, mitophagy is enhanced in platinum-resistant tumors, and ULK1 depletion/p53 activation has a synergistic lethal effect on these tumors, inducing pyroptosis through GSDME directly.ConclusionOur research demonstrates that ULK1 deficiency can synergize with MDM2 inhibitors to induce pyroptosis. p53 plays a direct role in activating GSDME transcription, while ULK1 deficiency triggers upregulation of the ROS-NLRP3 signaling pathway, leading to GSDME cleavage and activation. These findings underscore the pivotal role of p53 in determining pyroptosis and provide new avenues for the clinical application of p53 restoration therapies, as well as suggesting potential combination strategies.

CEP-1347 Boosts Chk2-Mediated p53 Activation by Ionizing Radiation to Inhibit the Growth of Malignant Brain Tumor Cells.

Radiation therapy continues to be the cornerstone treatment for malignant brain tumors, the majority of which express wild-type p53. Therefore, the identification of drugs that promote the ionizing radiation (IR)-induced activation of p53 is expected to increase the efficacy of radiation therapy for these tumors. The growth inhibitory effects of CEP-1347, a known inhibitor of MDM4 expression, on malignant brain tumor cell lines expressing wild-type p53 were examined, alone or in combination with IR, by dye exclusion and/or colony formation assays. The effects of CEP-1347 on the p53 pathway, alone or in combination with IR, were examined by RT-PCR and Western blot analyses. The combination of CEP-1347 and IR activated p53 in malignant brain tumor cells and inhibited their growth more effectively than either alone. Mechanistically, CEP-1347 and IR each reduced MDM4 expression, while their combination did not result in further decreases. CEP-1347 promoted IR-induced Chk2 phosphorylation and increased p53 expression in concert with IR in a Chk2-dependent manner. The present results show, for the first time, that CEP-1347 is capable of promoting Chk2-mediated p53 activation by IR in addition to inhibiting the expression of MDM4 and, thus, CEP-1347 has potential as a radiosensitizer for malignant brain tumors expressing wild-type p53.

Effects of tauroursodeoxycholate on arsenic-induced hepatic injury in mice: A comparative transcriptomic analysis

BackgroundProlonged exposure to excessive arsenic (As) and its compounds can cause damage to multiple systems, including respiratory, cardiovascular, immune, nervous, and endocrine systems. Manifestations include changes in skin pigmentation, excessive keratosis on palms and soles, gastrointestinal symptoms, and anemia. The liver as an important detoxification organ of the body, is a significant target organ for arsenic toxicity, and liver diseases are common. So far, the molecular mechanism has not been fully elucidated. Evidence suggests that taurodeoxycholic acid (TUDCA) has a protective role in arsenic-induced liver injury. This study aims to reveal potential target genes at the transcriptional level following TUDCA intervention, providing insights for the intervention of arsenic-induced liver injury. MethodsThe TUDCA intervention model of arsenic liver injury in C57BL/6 N mice was established. The experiment was divided into two phases and lasted for 24 weeks. The phase I trial (12 weeks) was divided into control, low, middle and high groups according to the dose of As. The phaseⅡtrial (12 weeks) was administered in combination with 10 mg/L sodium arsenite (the first stage high arsenic group) and TUDCA, so subsequent groups was named with H indicating high arsenic. Divide into four groups: control group(C), TUDCA solvent control group(H-Vehicle), TUDCA combined with As group(H-TUDCA), arsenic group (As). As was ingested through free water and TUDCA was administered to mice by gavage at a dose of 0.1 mL/10 g.b.w (100 mg/kg) once a day for 12 weeks. The differential expression gene (DEG) profile was obtained from the second batch of mouse liver tissues by RNA sequencing technology. Comparative transcriptomic analysis methods were used to identify co-varying DEGs between arsenic induction and TUDCA intervention, along with their associated pathways. QRT-PCR was utilized for validation. ResultsTranscriptome results showed that 487 DEGs were identified after arsenic induction. TUDCA intervention identified 231 DEGs (p-values < 0.05 and | log2(fold change) | > 1). The comparison of "AS vs C" and "H_TUDCA vs AS" identified 65 covariant DEGs, and further screened the TUDCA pathways and related genes among these genes，six pathways and 11 genes (Ccl21a, Ccr7, Mdm2, Slc2a4, Akr1b7, Pnpla3, Dusp8, Hspa1a, Cyp7a1, Cybrd1, Trpm6) were obtained. Next, we screened for covariant DEGs among the top 50 potential hub genes in arsenic-induced DEGS, and obtained 7 (Hbb-bs, Hspa1a, Mdm2, Slc2a4, Ptk6, Egr1, and Dusp8). Finally, the intersection of Hub gene and pathway gene was selected as the target genes Dusp8, Hspa1a, Mdm2 and Slc2a4. The sequencing results showed that the mRNA expressions of Dusp8, Hspa1a and Mdm2 were significantly increased after arsenic induction, while the expression of Slc2a4 was significantly decreased (P<0.05). Conversely, TUDCA intervention reversed these DEGs changes, consistent with QRT-PCR validation results. ConclusionThis study contributes to understanding the potential health effects of arsenic-induced liver injury, identifying new potential targets, and providing references for TUDCA intervention.

MDM2 Is Essential to Maintain the Homeostasis of Epithelial Cells by Targeting p53

Introduction: MDM2 is known as the primary negative regulator of p53, and MDM2 promotes lung cancer fibrosis and lung injury through p53-dependent and p53-independent pathways. However, the mechanism by which MDM2 influences the pathogenesis of asthma is unknown. In this study, we investigated the function of MDM2 in lung epithelial cells in type 2 lung inflammation. Methods: We used type II alveolar epithelial cell-specific heterozygous knockout of Mdm2 mice to validate its function. Then papain-induced asthma model was established, and changes in inflammation were observed by measuring immunohistochemistry and flow cytometry analysis. Results: In this study, we knockdown the mouse Mdm2 gene in type 2 alveolar epithelial cells. We demonstrated that heterozygous Mdm2 gene-deleted mice were highly susceptible to protease allergen papain-induced pulmonary inflammation characterized by increased ILC2 numbers, IL-5 and IL-13 cytokine levels, and lung pathology. A mechanistic study showed that following the decreased expression of Mdm2 in lung epithelial cells and A549 cell line, p53 was overactivated, and the expression of its downstream genes p21, Puma, and Noxa was elevated, which resulted in apoptosis. After Mdm2 knockdown, the mRNA expression of inflammation-related gene IL-25, HMGB1, and TNF-α were increased, which further amplified the downstream ILC2 response and lung inflammation. Conclusion: These results indicate that Mdm2 maintains the homeostasis of lung epithelial cells by targeting P53 and regulates the function of lung epithelial cells under type 2 lung inflammation.