MDM2 Expression Research Articles

Loss of p21ras, p53, and Mdm2 Protein Expression Is Indicative of Worse Prognosis in Myelodysplastic Syndromes.

Myelodysplastic syndromes (MDS) comprise a group of clonal disorders of the hematopoietic stem cell characterized by ineffective hematopoiesis and refractory cytopenias that can progress to acute leukemia. In order to identify molecular events implicated in the pathogenesis of MDS, we analyzed the bone marrow expression and mutational status of ras, p53 and mdm2 genes in a cohort of 25 patients with MDS. The expression of p21ras, p53, and Mdm2 proteins was studied in marrow cytopins stained by APAAP (alkaline phosphatase anti-alkaline phosphatase procedure) using monoclonal antibodies Y13-219, PAb 1801 and 2A10 to detect p21ras, p53 and mdm2 proteins, respectively. N-, K-, H-ras and p53 gene mutations were assessed by PCR-SSCP (polymerase chain reaction/single strand conformation polymorphism) and DNA sequencing to exclude the presence of gene mutations. The quantitative wild-type expression of p21ras, p53 and mdm2 proteins in patient samples (combination of number of cells and staining intensity) was compared to the values of expression of these proteins in 7 normal bone marrow samples. We found overexpression of p21ras and p53 proteins in 12/22 (55%) and 7/23 (30%) of the patients. Five of 7 patients who overexpressed wt-p53 also co-overexpressed wt-p21ras protein. We observed absence of expression of p21ras, p53 and mdm2 in 4/22, 4/23 and 9/19 cases, respectively. Overall survival of patients with absence of p21ras and p53 expression was considerably worse than that of patients with normal expression of these proteins (p=0.0006 to p21ras and p=0.004 to p53 by Log-Rank test; p=0.02 to p21ras and p=0.01 to p53 by Cox proportional hazards model after age, neutrophil and platelet count, and hemoglobin adjustment). We also observed that the patients who exhibited hypoexpression (p=0.055) and absence of mdm2 expression (p=0.07) demonstrated a tendency to have worse prognosis (Log-Rank). There was no correlation between variables p21ras, p53 and mdm2 versus hemoglobin, platelet and neutrophil counts and no association between FAB SMD subcategories and absence of p53 and mdm2 expression by Fisher test (two-tailed), but there was a strong over-representation between absence of p21ras expression and MDS with excess of blasts (p=0.0006). There was a significant correlation between p53 and mdm2 gene expression (p=0.03, Pearson). Only one patient harbored one transversion mutation in codon 12 of K-ras gene and did not correlate with p53 overexpression, but with worse prognosis. No p53 mutation was found. Overall, these findings suggest that the via of signal transduction p21ras/p53 and the auto-regulatory genes p53/mdm2 may be active and play a pivotal role in the pathogenesis of MDS. Hence, loss of bone marrow expression of p21ras, p53 and/or mdm2 proteins is an indicator of worse prognosis in MDS and may serve as early markers of leukemic transformation.

LncRNA HCG11/miR-579-3p/MDM2 axis modulates malignant biological properties in pancreatic carcinoma via Notch/Hes1 signaling pathway.

Background: Increasing reports have revealed that dysregulated expression of long non-coding RNAs (lncRNAs) is involved in pancreatic carcinoma progression. This study intends to explore the function and molecular mechanism of lncRNA HLA complex group 11 (HCG11) in pancreatic carcinoma.Methods: The expression profiles of HCG11 in pancreatic carcinoma samples were detected by qPCR. Bioinformatics analysis was applied to detect the associations among HCG11/miR-579-3p/MDM2. The malignant properties of pancreatic carcinoma cells were measured by numerous biological assays. Xenograft model was exploited to detect the effect of HCG11 on tumor growth.Results: A significant increase of HCG11 was occurred in pancreatic carcinoma samples. Knockdown of HCG11 suppressed the progression of pancreatic carcinoma cells. Bioinformatics analysis revealed that HCG11 upregulated MDM2 expression by competitively targeting miR-579-3p. The rescue assays showed that miR-579-3p reversed cell behaviors caused by HCG11, and MDM2 reversed cell properties induced by miR-579-3p. The Notch1 intracellular domain (NICD) and Hes1 protein levels were increased by overexpression of HCG11/MDM2. The tumor growth was suppressed after depletion of HCG11, followed by suppressing Ki67, PCNA and Vimentin expression, increasing TUNEL-positive cells and E-cadherin expression.Conclusions: Our observations highlighted that HCG11 contributed to the progression of pancreatic carcinoma by promoting growth and aggressiveness, and inhibiting apoptosis via miR-579-3p/MDM2/Notch/Hes1 axis.

Aflatoxin B1 interferes with embryonic liver development: Involvement of p53 signaling and apoptosis in zebrafish

Aflatoxin B1 (AFB1), a naturally occurring mycotoxin, is present in human placenta and cord blood. AFB1 at concentrations found in contaminated food commodities (0.25 and 0.5 μM) did not alter the spontaneous movement, heart rate, hatchability, or morphology of embryonic zebrafish. However, around 86 % of 0.25 μM AFB1-treated embryos had livers of reduced size, and AFB1 disrupted the hepatocyte structures, according to histological analysis. Additionally, AFB1 treatment that begins at any stage before 72 h post-fertilization (hpf) effectively reduced the size of embryonic livers. In hepatic areas, AFB1 suppressed the expression of Hhex and Prox1, which are two critical transcriptional factors for initiating hepatoblast specification. KEGG analysis based on transcriptome profiling indicated that p53 signaling and apoptosis are the only observed pathways in AFB1-treated embryos. AFB1 at 0.5 μM significantly activated the expression of tp53, mdm2, puma, noxa, pidd1, and gadd45aa genes that are related to the p53 pathway and also that of baxa, casp 8 and casp 3a in the apoptotic process. TUNEL staining demonstrated that AFB1 triggered the apoptosis of embryonic hepatocytes in a dose-dependent manner. These results indicate that the deficiency of both hhex and prox1 as well as hepatocyte apoptosis via the p53-Puma/Noxa-Bax axis may contribute to the embryonic liver shrinkage that is caused by AFB1.

Folate receptor α increases chemotherapy resistance through stabilizing MDM2 in cooperation with PHB2 that is overcome by MORAb-202 in gastric cancer.

BackgroundThe main function of folate receptor α (FOLRα) has been considered to mediate intracellular folate uptake and induce tumor cell proliferation. Given the broad spectrum of expression among malignant tumors, including gastric cancer (GC) but not in normal tissue, FOLRα represents an attractive target for tumor‐selective drug delivery. However, the efficacy of anti‐FOLRα monoclonal antibodies (mAbs) has not been proved so far, with the reason for this failure remaining unclear, raising the need for a better understanding of FOLRα function.MethodsThe distribution of FOLRα in GC cells was evaluated by immunohistochemistry. The impacts of FOLRα expression on the survival of GC patients and GC cell lines were examined with the Gene Expression Omnibus database and by siRNA of FOLRα. RNA‐sequencing and Microarray analysis was conducted to identify the function of FOLRα. Proteins that interact with FOLRα were identified with shotgun LC‐MS/MS. The antitumor efficacy of the anti‐FOLRα mAb farletuzumab as well as the antibody‐drug conjugate (ADC) consists of the farletuzumab and the tublin‐depolymerizing agent eribulin (MORAb‐202) was evaluated both in vitro and in vivo.ResultsFOLRα was detected both at the cell membrane and in the cytoplasm. Shorter overall survival was associated with FOLRα expression in GC patients, whereas reduction of FOLRα attenuated cell proliferation without inducing cell death in GC cell lines. Transcriptomic and proteomic examinations revealed that the FOLRα‐expressing cancer cells possess a mechanism of chemotherapy resistance supported by MDM2, and FOLRα indirectly regulates it through a chaperone protein prohibitin2 (PHB2). Although reduction of FOLRα brought about vulnerability for oxaliplatin by diminishing MDM2 expression, farletuzumab did not suppress the MDM2‐mediated chemoresistance and cell proliferation in GC cells. On the other hand, MORAb‐202 showed significant antitumor efficacy.ConclusionsThe ADC could be a more reasonable choice than mAb as a targeting agent for the FOLRα‐expressing tumor.

Clinical utility of the imunohistochemical co-expression of p53 and MDM2 in thyroid follicular lesions

In order to investigate the possible correlation between p53 and MDM2 co-expression with clinicopathological features of differentiated thyroid cancer (DTC) and its use as diagnostic and/or prognostic markers, we used immunohistochemistry to evaluate 317 thyroid samples including 208 DTC and 94 benign nodules, in addition to 15 normal tissues. MDM2 and p53 expression were highly associated (r = 0.7161; p < 0.0001). The co-expression of p53-MDM2 was observed more frequently in malignant lesions (p < 0.0001) and helped characterize follicular patterned lesions distinguishing FVPTC from FA (p < 0.0001) and FVPTC from FTC (p < 0.0001). In addition, p53-MDM2 co-expression was associated with characteristics of less aggressiveness. It was more frequent in patients ≤45 years old (p = 0.0035), with unique tumors (p = 0.0095), tumors <2 cm (p < 0.0001), tumors without extrathyroid invasion (p = 0.0425), without metastasis at evolution (p = 0.0179), and in patients evolving free of disease after treatment (p = 0.0485). We suggest that p53-MDM2 co-expression profile analysis might help establishing diagnostic and determining prognostic of DTC patients.

Inhibition of the mTOR pathway and reprogramming of protein synthesis by MDM4 reduce ovarian cancer metastatic properties

Epithelial ovarian cancer (EOC) is a highly heterogeneous disease with a high death rate mainly due to the metastatic spread. The expression of MDM4, a well-known p53-inhibitor, is positively associated with chemotherapy response and overall survival (OS) in EOC. However, the basis of this association remains elusive. We show that in vivo MDM4 reduces intraperitoneal dissemination of EOC cells, independently of p53 and an immune-competent background. By 2D and 3D assays, MDM4 impairs the early steps of the metastatic process. A 3D-bioprinting system, ad hoc developed by co-culturing EOC spheroids and endothelial cells, showed reduced dissemination and intravasation into vessel-like structures of MDM4-expressing cells. Consistent with these data, high MDM4 levels protect mice from ovarian cancer-related death and, importantly, correlate with increased 15 y OS probability in large data set analysis of 1656 patients. Proteomic analysis of EOC 3D-spheroids revealed decreased protein synthesis and mTOR signaling, upon MDM4 expression. Accordingly, MDM4 does not further inhibit cell migration when its activity towards mTOR is blocked by genetic or pharmacological approaches. Importantly, high levels of MDM4 reduced the efficacy of mTOR inhibitors in constraining cell migration. Overall, these data demonstrate that MDM4 impairs EOC metastatic process by inhibiting mTOR activity and suggest the usefulness of MDM4 assessment for the tailored application of mTOR-targeted therapy.

FMR1/circCHAF1A/miR-211-5p/HOXC8 feedback loop regulates proliferation and tumorigenesis via MDM2-dependent p53 signaling in GSCs.

Glioma is the most common and fatal primary malignant brain tumor. Glioma stem cells (GSCs) may be an important factor in glioma cell proliferation, invasion, chemoradiotherapy tolerance, and recurrence. Therefore, discovering novel GSCs related circular RNAs (circRNAs) may finds out a prospective target for the treatment of glioma. A novel circRNA-CHAF1A (circCHAF1A) was first found in our study. CircCHAF1A was overexpressed in glioma and related to the low survival rate. Functionally, it was found that no matter in vitro or in vivo, circCHAF1A can facilitate the proliferation and tumorigenesis of TP53wt GSCs. Mechanistically, circCHAF1A upregulated transcription factor HOXC8 expression in GSCs through miR-211-5p sponging. Then, HOXC8 can transcriptionally upregulate MDM2 expression and inhibited the antitumor effect of p53. Furtherly, the RNA binding protein FMR1 can bind to and promoted the expression of circCHAF1A via maintaining its stability, while HOXC8 also transcribed the FMR1 expression to form a feedback loop, which may be involved in the malignant transformation of glioma. The novel feedback loop among FMR1, circCHAF1A, miR-211-5p, and HOXC8 in GSCs can facilitate the proliferation and tumorigenesis of glioma and GSCs. It also provided a helpful biomarker for diagnosis and prognostic evaluation of glioma and may be applied to molecular targeted therapy.

BOREAS: A global phase 3 study of KRT-232, a first-in-class murine double minute 2 (MDM2) inhibitor in TP53WT relapsed/refractory (R/R) myelofibrosis (MF).

TPS7057 Background: The prognosis for patients (pts) with MF who have primary resistance to or who have progressed after treatment with ruxolitinib (RUX) is poor (median OS is ̃13 months), highlighting the unmet need for novel treatments in this setting. MDM2 is a key negative regulator of the tumor suppressor protein p53 that is overexpressed in CD34+ cells in MF pts. Elevated MDM2 expression attenuates p53 activity, resulting in proliferation of malignant CD34+ cells. KRT-232 is a potent, selective, orally available MDM2 inhibitor that restores p53 function resulting in apoptosis of malignant stem and progenitor cells. KRT-232 has the potential to demonstrate disease-modifying effects in MF pts who are R/R to prior JAK inhibitor (JAKi) therapy. The FDA has granted KRT-232 Fast Track designation for the treatment of JAKi R/R MF. Clinical proof-of-concept for KRT-232 in R/R MF was established in phase 2 of this study. Once-daily dosing of KRT-232 at 240 mg (Day 1-7 of 28-day cycle) yielded a best spleen volume reduction (SVR) ≥35% by central review in 16% of pts, best total symptom score (TSS) response &gt; 50% in 30% of pts, and 87% reduction of CD34+ cells in peripheral blood at Week 24. Spleen responses were superior in pts who were off RUX vs those on RUX at baseline imaging (best SVR ≥35%: 29% vs 0%). KRT-232 demonstrated a tolerable safety profile that included prophylaxis for nausea/vomiting (Al-Ali. EHA 2020). Methods: BOREAS is a randomized, controlled, open-label, global phase 3 study in MF pts (primary MF/post–polycythemia vera MF/post–essential thrombocythemia MF) who are R/R to JAKi. Pts aged ≥18 y with confirmed MF per WHO criteria, intermediate-1, 2, or high-risk disease (per DIPSS), ECOG performance status ≤2, adequate hematologic function (platelets ≥50 x 109/L), and wild-type p53 will be enrolled; pts with JAKi treatment = 28 d before baseline MRI/CT will be excluded. Pts will be randomized (2:1) to KRT-232 (240 mg on Day 1-7/28-day cycle; n = 188) or best available treatment (BAT; n = 94) and stratified by MF type (primary vs secondary) and baseline TSS (≤10 vs &gt; 10). BAT options include hydroxyurea, chemotherapy, or supportive care (including; but not limited to: corticosteroids and androgens); treatment selection is at the discretion of the investigator. Pts in BAT arm with documented disease progression at any time (spleen volume increase ≥25% from baseline or confirmed leukemic transformation) or those who complete Week 24 assessments may crossover to the KRT-232 arm. Primary endpoint is rate of SVR ≥35% by MRI/CT at Week 24 (central review). Key secondary endpoints are ≥50% reduction in TSS rate at Week 24 (per MFSAF v4.0), PFS, OS, best overall SVR ≥35%, and duration of spleen response. Enrollment is planned at 137 sites in 21 countries in North and South America, Europe, and Asia-Pacific (ClinicalTrials.gov: NCT03662126). Clinical trial information: NCT03662126.

Low‐grade osteosarcoma is predominant in gnathic osteosarcomas: A report of seven cases of osteosarcoma of the jaw

ObjectivePrimary osteosarcoma of the jaw bones is very rare, and histological features of gnathic osteosarcoma remain obscure. The purpose of this study was to describe the clinicopathological features of gnathic osteosarcoma.Materials and methodsSeven cases of gnathic osteosarcoma from Japan diagnosed during the period between 2000 and 2016 were examined retrospectively. The histology of the surgical pathology materials was reviewed by two pathologists. Clinical information was obtained from the hospital's information system.ResultsOf the seven cases, two patients had secondary osteosarcomas. As for the five cases of primary osteosarcoma, their ages ranged from 26 to 58 years (mean: 36.2, median: 28). Histologically, three cases were fibrotic tumors composed of spindle‐shaped cells with mild to moderate nuclear atypia and the collagenous stroma accompanied by woven bones or mature lamellar‐like bones. Two cases had cartilage formation. MDM2 and CDK4 expression was observed in two out of three cases on immunostaining. The histopathology of these three cases was regarded as the counterpart of low‐grade osteosarcomas, namely, parosteal osteosarcoma and low‐grade central osteosarcoma, arising in long bones.ConclusionsThe surprisingly high incidence (60%, 3/5 cases) of low‐grade osteosarcoma explains the reason why gnathic osteosarcomas present a more favorable prognosis than osteosarcomas arising in long bones. Furthermore, it provides insight into the tumorigenesis mechanism of low‐grade osteosarcomas arising in the jaw and other sites.

Alteration of MDM2 by the Small Molecule YF438 Exerts Antitumor Effects in Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) exhibits a high mortality rate and is the most aggressive subtype of breast cancer. As previous studies have shown that histone deacetylases (HDAC) may represent molecular targets for TNBC treatment, we screened a small library of synthetic molecules and identified a potent HDAC inhibitor (HDACi), YF438, which exerts effective anti-TNBC activity both in vitro and in vivo. Proteomic and biochemical studies revealed that YF438 significantly downregulated mouse double minute 2 homolog (MDM2) expression. In parallel, loss of MDM2 expression or blocking MDM2 E3 ligase activity rendered TNBC cells less sensitive to YF438 treatment, revealing an essential role of MDM2 E3 ligase activity in YF438-induced inhibition of TNBC. Mechanistically, YF438 disturbed the interaction between HDAC1 and MDM2, induced the dissociation of MDM2-MDMX, and subsequently increased MDM2 self-ubiquitination to accelerate its degradation, which ultimately inhibited growth and metastasis of TNBC cells. In addition, analysis of clinical tissue samples demonstrated high expression levels of MDM2 in TNBC, and MDM2 protein levels closely correlated with TNBC progression and metastasis. Collectively, these findings show that MDM2 plays an essential role in TNBC progression and targeting the HDAC1-MDM2-MDMX signaling axis with YF438 may provide a promising therapeutic option for TNBC. Furthermore, this novel underlying mechanism of a hydroxamate-based HDACi in altering MDM2 highlights the need for further development of HDACi for TNBC treatment. SIGNIFICANCE: This study uncovers the essential role of MDM2 in TNBC progression and suggests that targeting the HDAC1-MDM2-MDMX axis with a hydroxamate-based HDACi could be a promising therapeutic strategy for TNBC.

Urolithin A Inhibits Epithelial-Mesenchymal Transition in Lung Cancer Cells via P53-Mdm2-Snail Pathway.

PurposeThe epithelial-to-mesenchymal transition (EMT) is a fundamental process in tumor progression that endows cancer cells with migratory and invasive potential. Snail, a zinc finger transcriptional repressor, plays an important role in the induction of EMT by directly repressing the key epithelial marker E-cadherin. Here, we assessed the effect of urolithin A, a major metabolite from pomegranate ellagitannins, on Snail expression and EMT process.MethodsThe role of Snail in urolithin A-induced EMT inhibition in lung cancer cells was explored by wound healing assay and cell invasion assay. The qRT-PCR and CHX assay were performed to investigate how urolithin A regulates Snail expression. Immunoprecipitation assays were established to determine the effects of urolithin A in mdm2-Snail interaction. In addition, the expression of p53 was manipulated to explore its effect on the expression of mdm2 and Snail.ResultsThe urolithin A dose-dependently upregulated epithelial marker and decreased mesenchymal markers in lung cancer cells. In addition, exposure to urolithin A decreased cell migratory and invasive capacity. We have further demonstrated that urolithin A inhibits lung cancer cell EMT by decreasing Snail protein expression and activity. Mechanistically, urolithin A disrupts the interaction of p53 and mdm2 which leads Snail ubiquitination and degradation.ConclusionWe conclude that urolithin A could inhibit EMT process by controlling mainly Snail expression. These results highlighted the role of pomegranate in regulation of EMT program in lung cancer.

Modulation of Apoptosis and Epithelial-Mesenchymal Transition E-cadherin/TGF-β/Snail/TWIST Pathways by a New Ciprofloxacin Chalcone in Breast Cancer Cells.

This study aimed to investigate the effect of the new ciprofloxacin chalcone [7-(4-(N-substituted carbamoyl methyl) piperazin-1 yl)] on the proliferation, migration, and metastasis of MCF-7 and MDA-MB-231 breast cancer cell lines. Cell viability, colony formation and cell migration abilities were analysed. Cell cycle distribution and apoptosis were examined by flow cytometry. The molecular mechanism underlying chalcone's activity was investigated using qRT-PCR and western blotting. This new ciprofloxacin chalcone significantly inhibited proliferation, colony formation, and cell migration abilities of both cancer cell lines. Furthermore, it initiated apoptosis and caused cell cycle arrest at G2/M and S phase in MCF-7 and MDA-MB-231 cell lines, respectively. In addition, it up-regulated the expression of pro-apoptotic factors, p53, PUMA and NOXA, and down-regulated the expression of anti-apoptotic factors, MDM2 and MDM4. At the same time, it inhibited epithelial-mesenchymal transition by increasing the expression of E-cadherin and decreasing the expression of TGF-β1, SNAI1, TWIST1, MMP2, and MMP9. This new ciprofloxacin chalcone exhibited promising apoptotic and anti-metastatic activities against MCF-7 and MDA-MB-231 breast cancer cell lines, and, therefore, is an attractive molecule for drug development in the treatment of breast cancer.

Characterization of the Components and Pharmacological Effects of Mountain-Cultivated Ginseng and Garden Ginseng Based on the Integrative Pharmacology Strategy.

Panax ginseng C. A. Mey (PGCAM) is a herbaceous perennial belonging to the Araliaceae family, mainly including Mountain-Cultivated Ginseng (MCG) and Garden Ginseng (GG) on the market. We aimed to establish a rapid, accurate and effective method to distinguish 15-year-old MCG and GG using ultra-performance liquid chromatography-quadrupole time-of-flight-tandem mass spectrometry (UPLC-QTOF-MS/MS), and also explored the pharmacological mechanisms of the main components using the Integrative Pharmacology-based Network Computational Research Platform of Traditional Chinese Medicine (TCMIP V2.0; http://www.tcmip.cn/). Altogether, 23 potential quality markers were characterized to distinguish 15-year-old MCG and GG, including ginsenosides Ra2, Rg1, and Ra1, and malonyl-ginsenoside Ra3, etc. The contents of 19 constituents (mainly protopanaxadiol-type) were higher in MCG compared with that in GG, and four constituents (mainly carbohydrate compounds) were higher in GG. The 105 putative targets corresponding to 23 potential quality markers were mainly involved in 30 pathways, which could be divided into 10 models, such as immune regulation, systems (metabolic, nervous, cardiovascular, reproductive), blood-pressure regulation, as well as antitumor, antiaging, antibacterial and anti-inflammatory effects. Furthermore, the potential quality markers of MCG and GG could inhibit the proliferation of breast cancer by regulating the mRNA expression of PSA, S6K, MDM2, and P53 genes by acting on AR, MTOR, PI3K and other targets. The Integrative Pharmacology Strategy may provide an efficient way to identify chemical constituents and explore the pharmacological actions of TCM formulations.

OCT3/4 enhances tumor immune response by upregulating the TET1-dependent NRF2/MDM2 axis in bladder cancer

This study aimed to investigate the function of OCT3/4 on tumor immune escape in bladder cancer. Initially, the expression of OCT3/4, TET1, NRF2 and MDM2 was quantified in tumor tissues and cells, followed by gain- or loss-of-function studies to define their roles in cell migration, invasion and apoptosis and tumorigenicity in nude mice. Bladder cancer presented with abundant expression levels of OCT3/4, TET1, NRF2 and MDM2. We found that OCT3/4 promoted TET1 expression via binding to its promoter and that TET1 recruited MLL protein to NRF2 promoter and upregulated its expression, while NRF2 enhanced MDM2 expression. Upregulated MDM2 accelerated tumor immune escape in bladder cancer in mice. OCT3/4 knockdown suppressed the cell migration and invasion while inducing apoptosis, and consequently prevented tumor growth and immune escape in mice. Collectively, OCT3/4 may promote the progression of tumor immune escape in bladder cancer through acting as a promoter of the TET1/NRF2/MDM2 axis.

Morphological and Molecular Characterization of Proliferative Inflammatory Atrophy in Canine Prostatic Samples.

Simple SummaryProstatic diseases are important worldwide, being the prostate cancer (PC) the most common tumor in men. Among the factors associated with PC development, the preneoplastic lesions are well-recognized. Preneoplastic lesions are cellular morphological alterations, induced by different factors and present a potential to progression for PC. In this scenario, dogs are considered spontaneous models. Dogs naturally develops prostatic hyperplasia, preneoplastic lesions and PC. Among the preneoplastic lesions, the proliferative inflammatory atrophy (PIA) develops spontaneously in dogs. PIA is an epithelial lesion induced by prostatic chronic inflammation, leading to a proliferative atrophy of the prostate gland. Thus, this study aimed to perform a full PIA morphological, phenotypical and molecular characterization in dogs. After reviewing the archives of the veterinary pathology service, it was identified 171 dogs containing PIA in the prostate gland, and among the PC cases (N = 84), it was identified PIA lesions surrounding 60.7% of PC cases. Besides that, we identified loss of genes related to the maintenance of prostatic tissue and can predispose to malignant transformation. Moreover, mutations in androgen receptor gene were identified, demonstration alteration in DNA in PIA. Overall, these results support the hypothesis that PIA can be considered a preneoplastic lesion in canine prostate.Proliferative inflammatory atrophy (PIA) is an atrophic lesion of the prostate gland that occurs in men and dogs and is associated with a chronic inflammatory infiltrate. In this study, we retrospectively reviewed canine prostatic samples from intact dogs, identifying 50 normal prostates, 140 cases of prostatic hyperplasia, 171 cases of PIA, 84 with prostate cancer (PC), 14 with prostatic intraepithelial neoplasia (PIN) and 10 with bacterial prostatitis. PIA samples were then selected and classified according to the human classification. The presence of PIA lesions surrounding neoplastic areas was then evaluated to establish a morphological transition from normal to preneoplastic and neoplastic tissue. In addition, the expression of PTEN, P53, MDM2 and nuclear androgen receptor (AR) were analyzed in 20 normal samples and 20 PIA lesions by immunohistochemistry and qPCR. All PIA lesions showed variable degrees of mononuclear cell infiltration around the glands and simple atrophy was the most common histopathological feature. PIA was identified between normal glands and PC in 51 (61%) out of the 84 PC samples. PIA lesions were diffusely positive for molecular weight cytokeratin (HMWC). Decreased PTEN and AR gene and protein expression was found in PIA compared to normal samples. Overall, our results strongly suggest that PIA is a frequent lesion associated with PC. Additionally, this finding corroborates the hypothesis that in dogs, as is the case in humans, PIA is a pre neoplastic lesion that has the potential to progress into PC, indicating an alternative mechanism of prostate cancer development in dogs.

Prognostic and therapeutic value of the Hippo pathway, RABL6A, and p53-MDM2 axes in sarcomas.

Additional prognostic and therapeutic biomarkers effective across different histological types of sarcoma are needed. Herein we evaluate expression of TAZ and YAP, the p53-MDM2 axis, and RABL6A, a novel oncoprotein with potential ties to both pathways, in sarcomas of different histological types. Immunohistochemical staining of a tissue microarray including 163 sarcomas and correlation with clinical data showed that elevated YAP and TAZ independently predict worse overall and progression-free survival, respectively. In the absence of p53 expression, combined TAZ and YAP expression adversely affect overall, progression free, and metastasis free survival more than TAZ or YAP activation alone. RABL6A independently predicted shorter time to metastasis and was positively correlated with p53, MDM2 and YAP expression, supporting a possible functional relationship between the biomarkers. Network analysis further showed that TAZ is positively correlated with MDM2 expression. The data implicate all five proteins as clinically relevant downstream players in the Hippo pathway. Finally, a novel inhibitor of MDM2 (MA242), effectively suppressed the survival of sarcoma cell lines from different histological types regardless of p53 status. These findings suggest both independent and cooperative roles for all five biomarkers across different histological types of sarcoma in predicting patient outcomes and potentially guiding future therapeutic approaches.

Integrative Statistics, Machine Learning and Artificial Intelligence Neural Network Analysis Correlated CSF1R with the Prognosis of Diffuse Large B-Cell Lymphoma

Tumor-associated macrophages (TAMs) of the immune microenvironment play an important role in the Diffuse Large B-cell Lymphoma (DLBCL) pathogenesis. This research aimed to characterize the expression of macrophage colony-stimulating factor 1 receptor (CSF1R) at the gene and protein level in correlation with survival. First, the immunohistochemical expression of CSF1R was analyzed in a series of 198 cases from Tokai University Hospital and two patterns of histological expression were found, a TAMs, and a diffuse B-lymphocytes pattern. The clinicopathological correlations showed that the CSF1R + TAMs pattern associated with a poor progression-free survival of the patients, disease progression, higher MYC proto-oncogene expression, lower MDM2 expression, BCL2 translocation, and a MYD88 L265P mutation. Conversely, a diffuse CSF1R + B-cells pattern was associated with a favorable progression-free survival. Second, the histological expression of CSF1R was also correlated with 10 CSF1R-related markers including CSF1, STAT3, NFKB1, Ki67, MYC, PD-L1, TNFAIP8, IKAROS, CD163, and CD68. CSF1R moderately correlated with STAT3, TNFAIP8, CD68, and CD163 in the cases with the CSF1R + TAMs pattern. In addition, machine learning modeling predicted the CSF1R immunohistochemical expression with high accuracy using regression, generalized linear, an artificial intelligence neural network (multilayer perceptron), and support vector machine (SVM) analyses. Finally, a multilayer perceptron analysis predicted the genes associated with the CSF1R gene expression using the GEO GSE10846 DLBCL series of the Lymphoma/Leukemia Molecular Profiling Project (LLMPP), with correlation to the whole set of 20,683 genes as well as with an immuno-oncology cancer panel of 1790 genes. In addition, CSF1R positively correlated with SIRPA and inversely with CD47. In conclusion, the CSF1R histological pattern correlated with the progression-free survival of the patients of the Tokai series, and predictive analytics is a feasible strategy in DLBCL.

Susceptibility to DNA damage caused by abrogation of Rad54 homolog B: A putative mechanism for chemically induced cleft palate

Exposure to environmental toxicants such as all-trans retinoic acid (atRA) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) may cause cleft palate (CP), which process is related to DNA damage. Rad54B, an important DNA damage repaired protein, has been proved to be associated with non-syndromic cleft lip with palate (NSCLP). In the present study, we sought to clarify the role of Rad54B in palatal development and environment-induced CP. atRA (100 mg/kg) and TCDD (40 μg/kg) were used to induce CP in mice (C57BL/6 J mice). In this study, mouse embryonic heads were collected on embryonic day (E) 13.5∼16.5. The expression level of DNA repair protein Rad54 homolog B (Rad54B) was significantly decreased while those of the DNA double-strand breaks (DSBs) marker γ-H2A.X, apoptosis marker caspase-3 and p53 were significantly increased in the palatal shelves upon exposure to atRA and TCDD relative to the control. Primary mouse embryonic palatal mesenchymal cells (MEPMs) were cultured and transfected with siRNA or adenovirus in vitro to knock down or increase the level of Rad54B. Rad54B knockdown resulted in increased cellular S-phase arrest and apoptosis as well as decreased cell proliferation. Rad54B overexpression also increased apoptosis and reduced cell proliferation. Western blotting was used to detect the level of γ-H2A.X in transfected cells stimulated with etoposide (ETO, a DSBs inducer), and after 5 μM ETO stimulation of transfected MEPMs, the expression of γ-H2A.X was increased in Rad54B-knockdown cells. The expression of Mdm2, Mdmx and p53 with changes in Rad54B was also detected and coimmunoprecipitation was performed to analyze the combination of Mdm2 and p53 when Rad54B was changed in MEPMs. Knockdown of Rad54B inhibited the expression of Mdm2 and Mdmx, while the level of p53 increased. The coimmunoprecipitation results showed a decreased combination of Mdm2 and p53 when Rad54B was knocked down. Therefore, Rad54B can regulate the cell cycle, proliferation, and apoptosis of MEPMs. The loss of Rad54B increased the sensitivity of MEPMs to DSBs inducers, promoted apoptosis, and suppressed the proliferation of MEPMs by inhibiting the degradation of p53. Taken together, these findings suggest that Rad54B may play a key regulatory role in environment-induced CP.

MicroRNA-302s Might Regulate ARL4C-Mediated Gastric Cancer Progression via p53 Signaling: Bioinformatics Analysis and Experiments Validation.

BackgroundOur previous studies demonstrate that ARL4C is the most critical clinical biomarker for gastric cancer (GC) patients among ARL family members (ARLs) and functions as an oncogene in GC. However, its underlying mechanisms in GC need to be further illustrated. In this study, we aim to explore the upstream and downstream molecular mechanisms of ARL4C in GC cells.MethodsThe genetic alteration of ARL4C in GC is analyzed by cBioPortal database. Potential ARL4C-targeted microRNAs (miRs) are predicted by three databases. The high-throughput RNA sequencing is performed to explore the underlying mechanisms of ARL4C in GC cells. The effects of predicted microRNAs on ARL4C, the RNA-sequencing results validation and the biological functions of ARL4C in GC cells are illustrated by in vitro experiments.ResultsGenetic analyses indicate that ARL4C is significantly upregulated in GC, which is not caused by gene amplification. MicroRNAs prediction shows the high relevance between ARL4C and miR-302 members. Moreover, miR-302c or miR-302d transfection reduces ARL4C protein expression in GC cells. Based on the high-throughput RNA sequencing of ARL4C-knockdown cells, enrichment analyses demonstrate that ARL4C is closely related to cell growth and involved in p53 signaling. Moreover, there are strong gene–gene interactions between ARL4C and genes in p53 signaling, and ARL4C downregulation could inhibit the protein expression of MDM2, a critical gene in p53 pathway. Further functional experiments demonstrate that ARL4C silencing leads to cell cycle arrest and increased cell apoptosis in AGS and MKN45 cells.ConclusionOur data suggest that miR-302c and miR-302d may function as the upstream regulators of ARL4C. And, ARL4C might promote GC cell cycle progression via regulating p53 signaling. Our findings provide novel insights into the key role of ARL4C and the underlying mechanisms in GC progression, thus facilitating the development of ARL4C-targeted therapy.

Molecular characterization, expression, and H2O2 induction of p53 and mdm2 in the ricefield eel, Monopterus albus

The proteins P53 and Mdm2 (murine double minute 2) play a prominent role in preventing the induction of uncontrolled apoptosis in numerous cell types. To better understand their potential roles during gonadal development in Monopterus albus, p53 and mdm2 homologues were isolated and characterized, and their mRNA expression patterns in developing gonadal and ovarian tissue incubated with H2O2in vitro were analysed. The open reading frames (ORFs) of p53 and mdm2 consisted of 1125 and 1497 base pairs (bp), respectively. A multiple amino acid residue alignment analysis showed that p53 and Mdm2 harboured the typical conserved domains of their gene families. Phylogenetic analysis also demonstrated that the Mdm2 of M. albus exhibited high sequence similarity with Lates calcarifer; however, the p53 sequences of the Neoteleostei, including M. albus, were distinct from those of the Euteleostei and other vertebrates. Quantitative real-time PCR revealed that p53 and mdm2 were mainly expressed in the ovary; their expression was significantly increased in the previtellogenic stage, was maintained at the increased level thereafter, showed a second increase in the early intersexual stage, and then decreased with the natural sex change from female to male. After incubation with H2O2, the expression patterns of p53 and mdm2 in ovarian fragments were similar to that of the apoptotic molecular marker caspase3, showing a significant increase at 1 h post incubation (hpi), a decrease after 2 hpi, and then a return to significantly upregulated levels at 4 hpi and 8 hpi. In addition, p53, mdm2 and caspase3 were strongly correlated. These results will be useful for further studies on the function and regulation of p53 and Mdm2 during gonadal development in Monopterus albus, particularly in the ovary.