Nuclear Accumulation Of P53 Research Articles

The Epstein–Barr virus small capsid protein BFRF3 disrupts the NF‐кB signaling pathway by inhibiting p65 activity

AbstractEpstein–Barr virus (EBV), a common gamma herpesvirus, establishes a life‐long latent infection in the host to defend against innate immune recognition, which is closely related to a variety of malignant tumors, but its specific mechanism is unclear. BFRF3, an EBV‐encoded small capsid protein, is mainly involved in the assembly of the viral capsid structure and the maintenance of its stability. Here, we showed that BFRF3 can inhibit TNF‐α‐mediated NF‐кB promoter activation. Moreover, BFRF3 downregulates NF‐кB‐mediated promoter activation and transcription of inflammatory cytokines, including IL‐6 and IL‐8. Dual‐luciferase reporter assay demonstrated that BFRF3 restrains NF‐кB promoter activity at or below the p65 level, and coimmunoprecipitation analysis revealed that BFRF3 not only interacts with p65 but also binds to its critical truncated Rel homology domain (RHD) and transcriptional activation domain (TAD). However, BFRF3 does not affect the dimerization of p65‐p50, but overexpression of BFRF3 reduces the nuclear accumulation of p65, and the phosphorylation of p65 (Ser536) is repressed during BFRF3 transfection and EBV lytic infection, which promotes the proliferation of EBV. Overall, our study suggested that BFRF3 may play a crucial role in antiviral immunity to defend against EBV infection by inhibiting NF‐κB activity.

Identification of TPI1 As a potential therapeutic target in pancreatic cancer with dependency of TP53 mutation using multi-omics analysis.

Mutations of KRAS, CDKN2A, TP53, and SMAD4 are the four major driver genes for pancreatic ductal adenocarcinoma (PDAC), of which mutations of KRAS and TP53 are the most frequently recognized. However, molecular-targeted therapies for mutations of KRAS and TP53 have not yet been developed. To identify novel molecular targets, we newly established organoids with the Kras mutation (KrasmuOR) and Trp53 loss of function using Cre transduction and CRISPR/Cas9 (Krasmu/p53muOR) from murine epithelia of the pancreatic duct in KrasLSL-G12D mice, and then analyzed the proteomic and metabolomic profiles in both organoids by mass spectrometry. Hyperfunction of the glycolysis pathway was recognized in Krasmu/p53muOR compared with KrasmuOR. Loss of function of triosephosphate isomerase (TPI1), which is involved in glycolysis, induced a reduction of cell proliferation in human PDAC cell lines with the TP53 mutation, but not in PDAC or in human fibroblasts without TP53 mutation. The TP53 mutation is clinically recognized in 70% of patients with PDAC. In the present study, protein expression of TPI1 and nuclear accumulation of p53 were recognized in the same patients with PDAC. TPI1 is a potential candidate therapeutic target for PDAC with the TP53 mutation.

Real-world Comparison of P53 Immunohistochemistry and TP53 Mutation Analysis Using Next-generation Sequencing.

TP53 mutation in breast cancer (BC) is associated with chemoresistance, endocrine therapy resistance, and late recurrence, resulting in poor prognosis. Nuclear accumulation of p53 in immunohistochemistry (IHC) is a surrogate marker of TP53 mutation. This study analyzed the frequency, type, and distribution of TP53 mutations in BCs and assessed the efficacy of p53 IHC as a surrogate marker of TP53 mutation. We collected data from 112 BC cases, including the results of p53 IHC and next-generation sequencing (NGS). Over-expression of p53 IHC was observed in 36 patients (32.1%), complete absence in 19 patients (17.0%), aberrant cytoplasmic staining in 1 patient (0.9%), and wild-type in 56 (50.0%) patients. The concordance rate between TP53 mutation and p53 IHC was 88.4% in all BCs, 89.9% in luminal BCs, and 86.0% in triple-negative BCs (TNBC). TNBC, abnormal p53 IHC pattern, p53 IHC over-expression, neoadjuvant chemotherapy (NAC) history, TP53 mutation, and high pre-treatment ki-67 labeling index (≥50%) were significantly associated with worse distant metastasis-free survival (DMFS) and overall survival (OS) (p<0.05). Pre-NAC clinical stage III was associated with worse DMFS but not OS. Multivariate analysis showed that NAC history, TNBC, and p53 IHC over-expression were independent predictors of worse DMFS. An abnormal p53 IHC pattern and NAC history were independent predictors of worse OS. P53 IHC is a valid surrogate marker of TP53 mutation in BC. Accumulation of abnormal p53 alone, regardless of TP53 mutation, was associated with worse DMFS and can be used as an easily accessible biomarker to predict chemoresistance.

Luteolin Inhibits Indoxyl Sulfate-Induced ICAM-1 and MCP-1 Expression by Inducing HO-1 Expression in EA.hy926 Human Endothelial Cells.

In patients with chronic kidney disease, the uremic toxin indoxyl sulfate (IS) accelerates kidney damage and the progression of cardiovascular disease. IS may contribute to vascular diseases by inducing inflammation in endothelial cells. Luteolin has documented antioxidant and anti-inflammatory properties. This study aimed to investigate the effect of luteolin on IS-mediated reactive oxygen species (ROS) production and intercellular adhesion molecule (ICAM-1) and monocyte chemoattractant protein (MCP-1) expression in EA.hy926 cells and the possible mechanisms involved. IS significantly induced ROS production (by 6.03-fold, p < 0.05), ICAM-1 (by 2.19-fold, p < 0.05) and MCP-1 protein expression (by 2.18-fold, p < 0.05), and HL-60 cell adhesion (by 31%, p < 0.05), whereas, luteolin significantly decreased IS-induced ROS production, ICAM-1 and MCP-1 protein expression, and HL-60 cell adhesion. Moreover, luteolin attenuated IS-induced nuclear accumulation of p65 and c-jun. Luteolin dose-dependently increased heme oxygenase-1 (HO-1) expression and the maximum fold induction of HO-1 by luteolin was 3.68-fold (p < 0.05), whereas, HO-1 knockdown abolished the suppression of ICAM-1 and MCP-1 expression by luteolin. Luteolin may protect against IS-induced vessel damage by inducing HO-1 expression in vascular endothelial cells, which suppresses nuclear factor kappa B (NF-κB) and activator protein 1 (AP-1) mediated ICAM-1 and MCP-1 expression.

Heterogeneous Patterns of Endothelial NF-κB p65 and MAPK c-Jun Activation, Adhesion Molecule Expression, and Leukocyte Recruitment in Lung Microvasculature of Mice with Sepsis.

Sepsis is an uncontrolled systemic inflammatory response to an infection that can result in acute failure of the function of the lung called acute respiratory distress syndrome. Leukocyte recruitment is an important hallmark of acute lung failure in patients with sepsis. Endothelial cells (EC) participate in this process by facilitating tethering, rolling, adhesion, and transmigration of leukocytes via adhesion molecules on their cell surface. In in vivo studies, endothelial nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) p65 and mitogen-activated protein kinase (MAPK) c-Jun intracellular signal transduction pathways were reported to regulate the expression of adhesion molecules. Mice underwent cecal ligation and puncture (CLP) to induce polymicrobial sepsis and were sacrificed at different time points up to 72 h after sepsis onset. Immunohistochemistry and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analyses were used to determine the kinetics of nuclear localization of p65 and c-Jun in EC, expression and location of adhesion molecules E-selectin and vascular cell adhesion molecule 1 (VCAM-1). Furthermore, the extent and location of leukocyte recruitment were assessed based on Ly6G staining of neutrophils, cluster determinant (CD) 3 staining of T lymphocytes, and CD68 staining of macrophages. In all pulmonary microvascular beds, we identified p65 and c-Jun nuclear accumulation in a subset of endothelial cells within the first 24 h after CLP-sepsis initiation. E-selectin protein was expressed in a subset of microvessels at 4 and 7 h after sepsis initiation, while VCAM-1 was expressed in a scattered pattern in alveolar tissue and microvessels, without discernible changes during sepsis development. CLP-induced sepsis predominantly promoted the accumulation of neutrophils and T lymphocytes 4 and 7 h after disease onset. Neutrophil accumulation occurred in all pulmonary microvascular beds, while T lymphocytes were present in alveolar tissue and postcapillary venules. Taken together, nuclear localization of p65 and c-Jun in EC and neutrophil recruitment could be associated with induced E-selectin expression in the pulmonary microvessels in CLP-septic mice at the early stage of the disease. In alveolar capillaries, on the other hand, activation of these molecular pathways and leukocyte accumulation occurred in the absence of E-selectin or VCAM-1. Endothelial activation and leukocyte recruitment in sepsis-induced lung injury are regulated by multiple, heterogeneously controlled mechanisms, which vary depending on the type of microvascular bed involved.

Giant cell carcinoma of the urinary bladder

We present the clinicopathological features of 23 cases of the giant cell subtype of urothelial carcinoma, a rare subtype of bladder cancer recognized in the current World Health Organization classification of urological tumors. Histologically, the architectural pattern of the tumor varied from infiltrating to the solid expansile pleomorphic tumor with giant, bizarre, anaplastic cells. Typical or atypical mitotic figures were frequently present in all cases. Between 10 and 30% of the tumor had a giant cell component. All cases were associated with conventional high-grade urothelial carcinoma, with areas of squamous cell divergent differentiation and micropapillary carcinoma present in six and two cases, respectively. In one case each had sarcomatoid, nested, small cell, or glandular divergent differentiation. At diagnosis, 35% of patients had advanced disease and 12% had distant metastases. When comparing giant cell urothelial carcinoma with conventional urothelial carcinoma in a matched analysis, differences in overall and cancer-specific survival were observed, particularly in the T1 stage category. Immunohistochemical staining showed a similar profile of urothelial lineage with frequent positive expression of uroplakin II, GATA3, CK20, CK7, and S100P in both giant cell and conventional urothelial carcinomas. High Ki67 proliferation (range, 60–90%; mean, 71%) and nuclear p53 accumulation (mutant profile; range, 50–90%; mean, 64%) were observed. Using the 22C3 assay, the expression of PD-L1 was found to be variable in two cases, and beta-HCG was negative. In conclusion, giant cell carcinoma is a subtype of urothelial carcinoma associated with advanced clinical stage and a trend to lower survival rates.

Heme oxygenase-1 protects cells from replication stress

Heme oxygenase-1 (HO-1, HMOX1) degrades heme protecting cells from heme-induced oxidative damage. Beyond its well-established cellular functions, heme has emerged as a stabilizer of G-quadruplexes. These secondary DNA structures interfere with DNA replication. We recently revealed that nuclear HO-1 colocalizes with DNA G-quadruplexes and promotes their removal. Here, we investigate whether HO-1 safeguards cells against replication stress.Experiments were conducted in control and HMOX1-deficient HEK293T cell lines. Immunostaining unveiled that DNA G-quadruplexes accumulated in the absence of HO-1, the effect that was further enhanced in response to δ-aminolevulinic acid (ALA), a substrate in heme synthesis. This was associated with replication stress, as evidenced by an elevated proportion of stalled forks analyzed by fiber assay. We observed the same effects in hematopoietic stem cells isolated from Hmox1 knockout mice and in a lymphoblastoid cell line from an HMOX1-deficient patient. Interestingly, in the absence of HO-1, the speed of fork progression was higher, and the response to DNA conformational hindrance less stringent, indicating dysfunction of the PARP1-p53-p21 axis. PARP1 activity was not decreased in the absence of HO-1. Instead, we observed that HO-1 deficiency impairs the nuclear import and accumulation of p53, an effect dependent on the removal of excess heme. We also demonstrated that administering ALA is a more specific method for increasing intracellular free heme compared to treatment with hemin, which in turn induces strong lipid peroxidation.Our results indicate that protection against replication stress is a universal feature of HO-1, presumably contributing to its widely recognized cytoprotective activity.

LGG-32. MALIGNANT TRANSFORMATION OF PEDIATRIC LOW-GRADE GLIOMA TO SECONDARY HIGH-GRADE GLIOMA - REPORT FROM THE GERMAN BRAIN TUMOR STUDIES

Abstract BACKGROUND Malignant transformation (MT) of pediatric low-grade glioma (pLGG) to secondary high-grade glioma (sHGG) is a rare but momentous event. We explored its clinical, epidemiologic, and molecular characteristics and risk factors. METHODS Forty-eight pediatric patients with histology-based or radiological (n=4) diagnosis of primary LGG (median age 10.2 years [range, 0.7–16.9]) and subsequent sHGG were identified within registries of the HIT-network from 1996-2019. For 29 patients, tumor specimens of both pLGG and corresponding sHGG were available for neuropathological review and studied genetically. RESULTS MT occurred after median 4 years (range, 0.5-14) following LGG diagnosis. Its 15-year cumulative incidence was 1.6±0.4% (23/1586 SIOP-LGG-2004 patients), but 11.7±4.1% (9/100) for diffuse pLGG WHO-grade 2 (DG2). Clinical risk factors for MT were spinal location and prior dissemination. MT was accompanied by an increment of nuclear p53 accumulation (20/29 tumors), cytogenetic and molecular alterations. CDKN2A/B homozygous deletions (7/12 tumors), 1p/1q alterations (4/12) and ATRX loss (3/12) were the most common acquired alterations detected in sHGG. 8/16 tumors were unclassifiable by methylation profiling. DG2s (n=19), harboring IDH-1 mutation in 3/9 cases, progressed to diffuse WHO-grade 3 (n=8) or grade 4 HGG (n=9), two developed gliomatosis cerebri. Circumscribed LGG (PA, n=13; GG, n=7; PXA, n=4; DNET, n=1) typically harbored MAPK/ERK pathway alterations (BRAF-/NTRK-/FGFR-fusions, n=5/9; BRAFV600E mutation, n=4/12) and transformed to non-diffuse HGG (n=14), but also to diffuse grade 4 HGG. H3 K27M-/BRAFV600E co-mutation in two of these patients eventually resulted in MT to fatal diffuse midline glioma. Thirty-five sHGG patients received radio-chemotherapy. Overall survival at 5 and 10 years following MT was 49±8% and 31±8%. CONCLUSIONS Our findings underline the importance of careful long-term follow-up of patients with pLGG. Repeated resection/biopsy of recurrent/progressing or “atypical” tumors should be pursued and include molecular typing to identify patients at risk for MT and initiate appropriate treatment.

STAT3 protects hematopoietic stem cells by preventing activation of a deleterious autocrine type-I interferon response.

Hematopoietic stem and progenitor cells (HSPCs) maintain blood-forming and immune activity, yet intrinsic regulators of HSPCs remain elusive. STAT3 function in HSPCs has been difficult to dissect as Stat3-deficiency in the hematopoietic compartment induces systemic inflammation, which can impact HSPC activity. Here, we developed mixed bone marrow (BM) chimeric mice with inducible Stat3 deletion in 20% of the hematopoietic compartment to avoid systemic inflammation. Stat3-deficient HSPCs were significantly impaired in reconstitution ability following primary or secondary bone marrow transplantation, indicating hematopoietic stem cell (HSC) defects. Single-cell RNA sequencing of Lin-ckit+Sca1+ BM cells (LSKs) revealed aberrant activation of cell cycle, p53, and interferon (IFN) pathways in Stat3-deficient HSPCs. Stat3-deficient LSKs accumulated γH2AX and showed increased expression of DNA sensors and type-I IFN (IFN-I), while treatment with A151-ODN inhibited expression of IFN-I and IFN-responsive genes. Further, the blockade of IFN-I receptor signaling suppressed aberrant cell cycling, STAT1 activation, and nuclear p53 accumulation. Collectively, our results show that STAT3 inhibits a deleterious autocrine IFN response in HSCs to maintain long-term HSC function. These data signify the importance of ensuring therapeutic STAT3 inhibitors are targeted specifically to diseased cells to avoid off-target loss of healthy HSPCs.

Altered p53/p16 expression is linked to urothelial carcinoma progression but largely unrelated to prognosis in muscle-invasive tumors

Background Most inactivating p53 mutations result in a nuclear p53 accumulation – detectable by immunohistochemistry (IHC). p53 alterations leading to a complete lack of p53 protein and absence of immunostaining do also occur – not easily detectable by IHC. p16 is upregulated in p53 inactivated cells. We hypothesized that a positive p16 IHC may help to distinguish p53 inactivation in IHC negative cases. Material and methods We investigated p53 and p16 immunostaining on 2710 urothelial bladder carcinomas in a tissue microarray format to understand their impact in relation to clinicopathological parameters of disease progression and patient outcome. Results p16 immunostaining was absent in normal urothelium but occurred in 63.5% (30.4% strong) of cancers. p16 strongly positive cases increased from pTaG2 low-grade (9.6%) to pTaG3 high-grade tumors (46.5%, p < .0001) but decreased from pTaG3 to pT4 (33.3%; p = .0030). Among pT2-4 carcinomas, p16 positivity was linked to high-grade (p = .0005) but unrelated to overall survival. p53 staining was negative in 8.4%, very weak in 15.4%, weak in 55.3%, strong in 4.7%, and very strong in 16.2% cancers. p53 negative (potentially p53 null phenotype), strong, and very strong p53 positivity increased from pTaG2 low-grade to pTaG3 high-grade tumors (p < .0001) and from pTaG3 to pT2-4 cancers (p = .0007). p53 staining was largely unrelated to histopathological parameters or patient prognosis among pT2-4 carcinomas, except of p53 strong/very strong immunostaining. p16 expression predominated in tumors with very strong, strong, and negative p53 staining and the combination of p53 negative/p16 strongly positive cancers was linked to features of tumor aggressiveness. Conclusion Aberrant p53 and p16 immunostaining increases during grade and stage progression although p53 negative and p16 positive immunostaining lack prognostic significance in pT2–4 carcinomas. Potential diagnostic features are that high level p16 expression is limited to neoplastic urothelium and p53 null phenotype to aggressive cancers (grade 3 and invasive).

Safety and Efficacy of Selinexor Sequential Azacytidine in Newly Diagnosed Patients with Myelodysplastic Syndromes EB1 or EB2: A Single-Center, Single-Arm, Phase Ib/II Trial

Background: Azacytidine is one of the hypomethylating agents (HMAs) for the treatment of higher-risk myelodysplastic syndromes (MDS). However, clinical outcomes of patients treated with azacytidine monotherapy were far from satisfactory with overall response rate (ORR) of 41%-59% and overall survival (OS) of 15-18 months. Selinexor is an oral, first-in-class selective inhibitor of nuclear export compound that inhibits exportin 1 (XPO1). Preclinical studies have shown that inhibition of XPO1 causes nuclear accumulation of p53 and disruption of NF-κB signaling, both relevant targets for myelodysplastic syndromes. Prior study reported an ORR of 26% and mOS of 8.7 months with selinexor in HMAs-refractory MDS patients. We therefore aimed to assess the safety and efficacy of selinexor sequential azacytidine in newly diagnosed patients with MDS-EB1 or EB2. Methods: The effectiveness of simultaneous and sequential treatment of azacytidine and selinexor was measured by cell death rate, cell cycle and colony formation rate in vitro. Western Blot was performed to detect the sublocation of p53. MOLM-13-luciferase xenograft murine model was established to verify the synergetic effect in vivo. Based on the experiments, we conducted a single-center, single-arm, phase Ib/II trial, which included newly diagnosed patients 18 years or older with MDS-EB1 or EB2. Eligible patients received 2-4 weeks long cycles of oral selinexor (40mg qw for 4 weeks; 40mg biw for 2 weeks; 60mg qw for 4 weeks). The primary endpoints were to find the recommended phase 2 dose (RP2D) and maximum tolerated dose (MTD). The secondary endpoint was ORR including complete remission (CR), partial remission (PR), marrow complete remission (mCR), and hematological improvement (HI). This trial was registered with the Chinese Clinical Trial Registry (#ChiCTR2200065167). Results: First we treated three AML cell lines: AML-OCI2, THP1, MV4-11 and three MDS cell lines: SKM-1, MOLM-13, MDSL with azacitidine and selinexor simultaneously or sequentially. Sequential treatment showed stronger synergetic effect (Figure 1A) and reduced colony formation by 90% in SKM-1 and MOLM-13 (Figure 1B). Cell cycle arrest were induced and the percentage of Annexin V cells obviously increased with sequential treatment (Figure 1C). Sequential treatment decreased leukemia burden and prolonged survival of MOLM-13-luciferase xenograft murine model (Figure 1D, E). Mechanistically, pretreatment of azacitidine on SKM-1 and MOLM-13 upregulated the expression level of p53, and selinexor led to massive nuclear accumulation of p53, resulted in better vulnerability lethal to MDS cells (Figure 1F). Secondly, 15 patients were enrolled in our trial with a median age of 57. 26.7% of patients were MDS-EB1, 73.3% were EB2. 93.3% of patients were high or very high risk by the Revised International Prognositic Scoring System (IPSS-R) (see the Table). At Phase Ib, the following selinexor dosages were administered: 40mg qw (n=3), 40mg biw (n=3) and 60mg qw (n=3). One patient in the 60mg qw group discontinued treatment because of nausea, vomiting, fatigue and Herpes simplex virus infection. Subsequently, in order to find RP2D, an extension cohort of 3 additional patients were treated with 40mg qw and 40mg biw, respectively. Therefore, 14 patients were evaluable for efficacy assessment. ORR was 78.6% in 11 patients including CR in 3 patients (21.4%); mCR+HI in 1 patient (7.1%); and mCR in 7 patients (50.0%) (see the swimmer plot). Especially, among the TP53 mutated patients (n=3), one achieved CR, the other two achieved mCR. The common adverse events were neutropenia (93.3%), anemia (86.7%), thrombocytopenia (80.0%), fatigue (53.3%), nausea (26.7%), dizziness (26.7%), vomiting (20.0%) and decreased appetite (20.0%). There were no treatment related deaths. Conclusion: We verified the activity of selinexor sequential azacytidine in MDS both in vitro and in vivo. The treatment showed with encouraging efficacy in newly diagnosed patients with MDS-EB1 or EB2. Adverse events were manageable with supportive care implementation. Enrollment is ongoing to assess the efficacy and safety of this sequential therapy, which may be a new treatment option for EB subjects.

P53/TP53 Status Assessment in Gastroesophageal Adenocarcinoma.

Chromosomal instability (CIN) is very frequent in gastroesophageal adenocarcinoma (GEA) and it is characterized by TP53 deletions/mutations resulting in p53 nuclear accumulation, as revealed by immunohistochemistry (IHC), which considers the cases with "high" staining levels to be positive. Aiming to improve aberrant TP53 detection, droplet digital PCR (ddPCR) was used to evaluate TP53 deletion in formalin-fixed, paraffin-embedded DNA (FFPE-DNA) and cell-free DNA (cfDNA). To further investigate the mutational TP53 profile, next-generation sequencing (NGS) was performed in a subset of FFPE samples. After combining "low" and "high" IHC staining level groups, the proportion of deletion events was significantly higher compared to the "intermediate" group (72.9% vs. 47.5%, p-value = 0.002). The ddPCR TP53 deletion assay was feasible for cfDNA but only had good agreement (72.7%, Cohen's kappa = 0.48) with the assay performed with FFPE-DNA of the "low-level" group. NGS analysis confirmed that, in the "low-level" group, a high percentage (66.7%) of cases were aberrant, with disruptive mutations that probably led to p53 loss. Data suggested that p53 IHC alone underestimates the CIN phenotype in GEA and that molecular analysis in both solid and liquid biopsies could be integrated with it; in particular, in cases of completely negative staining.

Soluble Guanylate Cyclase β1 Subunit Represses Human Glioblastoma Growth.

Malignant glioma is the most common and deadly brain tumor. A marked reduction in the levels of sGC (soluble guanylyl cyclase) transcript in the human glioma specimens has been revealed in our previous studies. In the present study, restoring the expression of sGCβ1 alone repressed the aggressive course of glioma. The antitumor effect of sGCβ1 was not associated with enzymatic activity of sGC since overexpression of sGCβ1 alone did not influence the level of cyclic GMP. Additionally, sGCβ1-induced inhibition of the growth of glioma cells was not influenced by treatment with sGC stimulators or inhibitors. The present study is the first to reveal that sGCβ1 migrated into the nucleus and interacted with the promoter of the TP53 gene. Transcriptional responses induced by sGCβ1 caused the G0 cell cycle arrest of glioblastoma cells and inhibition of tumor aggressiveness. sGCβ1 overexpression impacted signaling in glioblastoma multiforme, including the promotion of nuclear accumulation of p53, a marked reduction in CDK6, and a significant decrease in integrin α6. These anticancer targets of sGCβ1 may represent clinically important regulatory pathways that contribute to the development of a therapeutic strategy for cancer treatment.

P53 Expression in Luminal Breast Cancer Correlates With TP53 Mutation and Primary Endocrine Resistance

TP53 mutation is associated with primary endocrine resistance in luminal breast cancer (BC). Nuclear accumulation of p53, as determined by immunohistochemistry (IHC), is a surrogate marker for TP53 mutation. The immunohistochemical p53 index that defines a p53-positive status is not well established. This study determined the optimal p53 index cutoff to identify luminal BCs harboring TP53 mutations. In total, 364 luminal BCs from the West German Study Group ADAPT trial (NCT01779206) were analyzed for TP53 mutations by next-generation sequencing and for p53 expression by IHC (DO-7 antibody). P53 indices were determined by automated image analysis. All tumors were from patients treated with short-term preoperative endocrine therapy (pET; tamoxifen or aromatase inhibitor) before tumor resection. IHC evaluation included needle biopsies before therapy (baseline) and resections specimens after therapy (post-pET). Optimal p53 index cutoffs were defined with Youden statistics. TP53 mutations were detected in 16.3% of BC cases. The median p53 indices were significantly higher in TP53-mutated BCs compared to BCs harboring wild-type TP53 (baseline: 47.0% vs 6.4%, P < .001; post-pET: 50.1% vs 1.1%, P < .001). Short-term pET decreased p53 indices in BCs harboring wild-type TP53 (P < .001) but not in TP53-mutated BCs (P = .102). For baseline biopsies, the optimal p53 index cutoff was ≥34.6% (specificity 0.92, sensitivity 0.63, Youden index 0.54, accuracy: 0.87). For post-pET specimens, the optimal cutoff was ≥25.3% (specificity 0.95, sensitivity 0.65, Youden index 0.60, accuracy: 0.90). Using these cutoffs to define the p53 status, p53-positive BCs were >2-fold more common in pET nonresponders compared to pET responders (baseline: 37/162, 22.8% vs 18/162, 11.1%, P = .007; post-pET: 36/179, 20.1% vs 16/179, 8.9%, P = .004). In summary, IHC for p53 identifies TP53-mutated luminal BCs with high specificity and accuracy. Optimal cutoffs are ≥35% and ≥25% for treatment-naïve and endocrine-pretreated patients, respectively.

SPRR3 Contributes to Aggressiveness of Pancreatic Cancer Cells via NF-κB Signaling Pathway.

Pancreatic cancer remains a deadly solid tumor with worst survival, and a better understanding of the mechanisms of carcinogenesis of pancreatic cancer is critical to promote the survival of patients with pancreatic cancer. qPCR and western blot assay were used to determine the expression of SPRR3 in pancreatic cancer. Anchorage-independent growth ability, BrdU labeling, Transwell assay, and in vivo experiment were used to examine the functions of SPRR3 in aggressiveness of pancreatic cancer. Luciferase reporter assay, nucleoplasmic-separation technique, qPCR, and western blot assay were used to investigate the mechanism of SPRR3 regulating aggressiveness of pancreatic cancer. Our results showed that SPRR3 was significantly increased in pancreatic cancer, which resulted in poor survival for patients with pancreatic cancer. Further analysis showed that overexpression of SPRR3 contributed to anchorage-independent growth ability, growth rate, and invasion ability of pancreatic cancer cells. While, knockdown of SPRR3 showed the reverse results. Mechanistically, overexpression of SPRR3 can promote the transcription of NF-κB pathway, nuclear accumulation of p65, and mRNA levels of NF-κB pathway downstream genes. But, knockdown of SPRR3 induced the reverse results. The above findings clarified the important roles of SPRR3 in the progression of pancreatic cancer through NF-κB pathway. And targeting SPRR3 might be an effective strategy to therapy pancreatic cancer.

Correlation of P53 Expression with Clinicopathological Parameters, Hormone Receptors and HER 2 Neu Status in Breast Carcinoma

Background: Progesterone receptor (PR), estrogen receptor (ER) expression, human epidermal growth factor 2 (HER 2 neu) amplification/ overexpression, and p53 nuclear protein accumulation are all significant prognostic biomarkers in breast cancer (BC). This work aims to assess the co-presence of p53 overexpression with ER, PR, and HER 2 neu expression in invasive BC specimens by IHC and compare their expression with various clinicopathological features. Objectives: To evaluate the expression of ER, PR, HER-2neu and p53 in invasive breast carcinoma specimens and to correlate the expression of p53 with ER, PR, HER-2 neu and the clinicopathological parameters. Methods: Cases of Modified Radical Mastectomy received from June 2015 to May 2017 were studied. In addition to hematoxylin and eosin sections, immunohistochemical staining was done for ER, PR, HER 2 neu and p53. The expression of ER, PR, HER 2 neu and p53 was noted and the findings were correlated with other clinicopathological parameters. Results: Among 86 cases studied most of them belonged to the age group 41-60 years (54, 62.8%). A significant positive correlation of p53 over expression with ER and PR positive tumours and Triple Negative tumours was noted. Also p53 overexpression showed a positive significant correlation with tumour grade. Conclusion: P53 overexpression in breast carcinoma indicates an aggressive clinical course and can be used as a marker of poor prognosis.

DNA virus oncoprotein HPV18 E7 selectively antagonizes cGAS-STING-triggered innate immune activation.

Cellular infections by DNA viruses trigger innate immune responses mediated by DNA sensors. The cyclic GMP-AMP synthase (cGAS)-stimulator of interferon gene (STING) signaling pathway has been identified as a DNA-sensing pathway that activates interferons in response to viral infection and, thus, mediates host defense against viruses. Previous studies have identified oncogenes E7 and E1A of the DNA tumor viruses, human papillomavirus 18 (HPV18) and adenovirus, respectively, as inhibitors of the cGAS-STING pathway. However, the function of STING in infected cells and the mechanism by which HPV18 E7 antagonizes STING-induced Interferon beta production remain unknown. We report that HPV18 E7 selectively antagonizes STING-triggered nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) activation but not IRF3 activation. HPV18 E7 binds to STING in a region critical for NF-κB activation and blocks the nuclear accumulation of p65. Moreover, E7 inhibition of STING-triggered NF-κB activation is related to HPV pathogenicity but not E7-Rb binding. HPV18 E7, severe acute respiratory syndrome coronavirus-2open reading frame 3a, human immunodeficiency virus-2 viral protein X, and Kaposi's sarcoma-associated herpesvirusKSHV viral interferon regulatory factor 1 selectively inhibited STING-triggered NF-κB or IRF3 activation, suggesting a convergent evolution among these viruses toward antagonizing host innate immunity. Collectively, selective suppression of the cGAS-STING pathway by viral proteins is likely to be a key pathogenic determinant, making it a promising target for treating oncogenic virus-induced tumor diseases.

High-Grade Desmoplastic Foamy Gland Prostatic Adenocarcinoma.

It is important to recognize high-grade foamy gland prostatic adenocarcinoma with desmoplastic stroma given its aggressive clinical course with frequent metastases and death. To review the morphology, immunohistochemistry, and prognosis for this rare subtype of prostate adenocarcinoma. Twenty-four cases received for consultation from 2010 to 2021 were analyzed including needle biopsy (n = 21), transurethral resection (n = 2), and a cystoprostatectomy (n = 1). Patients ranged in age from 40 to 89 years (mean, 67 years). On average, 8 cores per case were involved (mean 67% core involvement). Extraprostatic extension and seminal vesicle invasion were observed in 6 of 21 (29%) and 3 of 21 (14%) needle biopsy cases, respectively. Twenty of the 24 cases (83%) were Grade Group (GG) 5 with 4 of 24 (17%) being GG4. Tumor necrosis as a component of Gleason pattern 5 was observed in 21 of 24 cases (88%). Associated intraductal adenocarcinoma (IDC) was observed in 22 of 24 cases (92%), with 4 of 24 cases (17%) demonstrating extensive IDC. Diagnostic challenges were as follows: (1) sparse isolated cancer glands embedded in the dense desmoplastic stroma; (2) fragmented glands; and (3) aberrant staining for high-molecular-weight cytokeratin in a nonbasal cell pattern in all cases. PTEN loss was observed in 9 cases, and p53 nuclear accumulation was observed in 8 cases. Three patients were lost to follow-up. Overall, of the 16 patients with meaningful follow-up, 12 (75%) either had metastases or died from prostate cancer. High-grade desmoplastic foamy gland adenocarcinoma is difficult to diagnose and grade and has a poor prognosis.

Rhamnus crenata leaf extracts exhibit anti-inflammatory activity via modulating the Nrf2/HO-1 and NF-κB/MAPK signaling pathways

Objective: To elucidate the potential anti-inflammatory mechanisms of Rhamnus crenata leaf extracts using RAW264.7 cells. Methods: We used 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide assay to measure cell viability. Nitric oxide (NO) production was measured using Griess reagent. Western blotting and RT-PCR assays were carried out for analyzing the protein and gene expressions of pro-inflammatory mediators, respectively. Moreover, PD98059 (ERK1/2 inhibitor), SB203580 (p38 inhibitor), SP600125 (JNK inhibitor), and BAY11-7082 (NF-κB inhibitor) were used to evaluate the anti-inflammatory mechanism of Rhamnus crenata leaf extract. Results: Rhamnus crenata leaf extracts significantly inhibited the production of the pro-inflammatory mediators such as NO, iNOS, COX-2, IL-1β, and TNF-α in lipopolysaccharide (LPS)-stimulated RAW264.7 cells. Rhamnus crenata leaf extracts also suppressed LPS-induced degradation of IκB-α and nuclear accumulation of p65, which resulted in the inhibition of NF-κB activation in RAW264.7 cells. Additionally, the extracts attenuated the phosphorylation of p38, ERK1/2, and JNK in LPS-stimulated RAW264.7 cells. Moreover, HO-1 expression induced by Rhamnus crenata leaf extracts was significantly downregulated by SB230580, PD98059, SP600125 and BAY11-7082. Conclusions: Rhamnus crenata leaf extract may upregulate HO-1 expression through inhibition of p38, ERK1/2, and NF-κB activation, which may contribute to the anti-inflammatory activity of the extracts. Rhamnus crenata leaf extracts may have great potential for the development of anti-inflammatory drugs to treat acute and chronic inflammatory diseases.

Inhibition of XPO1 with KPT-330 induces autophagy-dependent apoptosis in gallbladder cancer by activating the p53/mTOR pathway

BackgroundGallbladder cancer (GBC) is a highly aggressive malignant cancer in the biliary system with poor prognosis. XPO1 (chromosome region maintenance 1 or CRM1) mediates the nuclear export of several proteins, mainly tumor suppressors. Thus, XPO1 functions as a pro-oncogenic factor. KPT-330 (Selinexor) is a United States Food and Drug Administration approved selective inhibitor of XPO1 that demonstrates good therapeutic effects in hematologic cancers. However, the function of XPO1 and the effect of KPT-330 have not been reported in GBC.MethodsWe analyzed the correlation between XPO1 expression levels by q-PCR and clinical features of GBC patients. Cell proliferation assays were used to analyze the in vitro antitumor effects of XPO1 inhibitor KPT-330. mRNA sequencing was used to explore the underlying mechanisms. Western blot was performed to explore the relationship between apoptosis and autophagy. The in vivo antitumor effect of KPT-330 was investigated in a nude mouse model of gallbladder cancer.ResultsWe found that high expression of XPO1 was related to poor prognosis of GBC patients. We observed that XPO1 inhibitor KPT-330 inhibited the proliferation of GBC cells in vitro. Furthermore, XPO1 inhibitor KPT-330 induced apoptosis by reducing the mitochondrial membrane potential and triggering autophagy in NOZ and GBC-SD cells. Indeed, XPO1 inhibitor KPT-330 led to nuclear accumulation of p53 and activated the p53/mTOR pathway to regulate autophagy-dependent apoptosis. Importantly, KPT-330 suppressed tumor growth with no obvious toxic effects in vivo.ConclusionXPO1 may be a promising prognostic indicator for GBC, and KPT-330 appears to be a potential drug for treating GBC effectively and safely.