P53 Molecules Research Articles

PAWI-2: A novel inhibitor for eradication of cancer

Cancer is a major worldwide public health problem and is still the leading cause of death in the United States. There are many types of cancer treatment but completely successful results are oftentimes not attained. It remains a challenge to develop efficacious clinically useful cancer therapies. Therapies targeting dysregulated signal transduction pathways in cancer can be efficacious anti-cancer therapies with minimal adverse effects. In this study, we focus on novel small molecule p53 Activator Wnt Inhibitor-2 (PAWI-2) that was developed by optimizing potency and pharmaceutical properties. PAWI-2 is a nontoxic DNA-damage pathway inhibitor that shows a broad spectrum of potency and significant efficacy in vitro and in vivo. This study focuses on the application of PAWI-2 to four major types of cancers including colorectal cancer (CRC), breast cancer (BC), prostate cancer (PCa), and pancreatic cancer (PC). PAWI-2 shows a novel mechanism of action (MOA) by modulating two mechanisms of cancer invasion. In cancer with unimpaired p53, PAWI-2 activates DNA-damage checkpoint and mitochondrial p53-dependent apoptotic signaling. Consistently observed in most cancer types, PAWI-2 induces phosphorylation of optineurin (OPTN) to cause G2/M cell cycle arrest. These two mechanisms operate regardless of p53 variants and/or KRAS mutation status and also manipulate the effect of PAWI-2 to overcome tumor stemness and drug resistance in PC stem cells (PCSCs). This study summarizes the development of PAWI-2 as an attractive targeted therapeutic for mechanism-driven anti-cancer drug discovery.

Preoperative risk stratification in endometrial cancer (ENDORISK) by a Bayesian network model: A development and validation study.

BackgroundBayesian networks (BNs) are machine-learning–based computational models that visualize causal relationships and provide insight into the processes underlying disease progression, closely resembling clinical decision-making. Preoperative identification of patients at risk for lymph node metastasis (LNM) is challenging in endometrial cancer, and although several biomarkers are related to LNM, none of them are incorporated in clinical practice. The aim of this study was to develop and externally validate a preoperative BN to predict LNM and outcome in endometrial cancer patients.Methods and findingsWithin the European Network for Individualized Treatment of Endometrial Cancer (ENITEC), we performed a retrospective multicenter cohort study including 763 patients, median age 65 years (interquartile range [IQR] 58–71), surgically treated for endometrial cancer between February 1995 and August 2013 at one of the 10 participating European hospitals. A BN was developed using score-based machine learning in addition to expert knowledge. Our main outcome measures were LNM and 5-year disease-specific survival (DSS). Preoperative clinical, histopathological, and molecular biomarkers were included in the network. External validation was performed using 2 prospective study cohorts: the Molecular Markers in Treatment in Endometrial Cancer (MoMaTEC) study cohort, including 446 Norwegian patients, median age 64 years (IQR 59–74), treated between May 2001 and 2010; and the PIpelle Prospective ENDOmetrial carcinoma (PIPENDO) study cohort, including 384 Dutch patients, median age 66 years (IQR 60–73), treated between September 2011 and December 2013. A BN called ENDORISK (preoperative risk stratification in endometrial cancer) was developed including the following predictors: preoperative tumor grade; immunohistochemical expression of estrogen receptor (ER), progesterone receptor (PR), p53, and L1 cell adhesion molecule (L1CAM); cancer antigen 125 serum level; thrombocyte count; imaging results on lymphadenopathy; and cervical cytology. In the MoMaTEC cohort, the area under the curve (AUC) was 0.82 (95% confidence interval [CI] 0.76–0.88) for LNM and 0.82 (95% CI 0.77–0.87) for 5-year DSS. In the PIPENDO cohort, the AUC for 5-year DSS was 0.84 (95% CI 0.78–0.90). The network was well-calibrated. In the MoMaTEC cohort, 249 patients (55.8%) were classified with <5% risk of LNM, with a false-negative rate of 1.6%. A limitation of the study is the use of imputation to correct for missing predictor variables in the development cohort and the retrospective study design.ConclusionsIn this study, we illustrated how BNs can be used for individualizing clinical decision-making in oncology by incorporating easily accessible and multimodal biomarkers. The network shows the complex interactions underlying the carcinogenetic process of endometrial cancer by its graphical representation. A prospective feasibility study will be needed prior to implementation in the clinic.

Computational Investigation on the p53-MDM2 Interaction Using the Potential of Mean Force Study.

Murine double minute 2 (MDM2) proteins are found to be overproduced by many human tumors in order to inhibit the functioning of p53 molecules, a tumor suppressor protein. Thus, reactivating p53 functioning in cancer cells by disrupting p53–MDM2 interactions may offer a significant approach in cancer treatment. However, the structural characterization of the p53–MDM2 complex at the atomistic level and the mechanism of binding/unbinding of the p53–MDM2 complex still remain unclear. Therefore, we demonstrate here the probable binding (unbinding) pathway of transactivation domain 1 of p53 during the formation (dissociation) of the p53–MDM2 complex in terms of free energy as a function of reaction coordinate from the potential of mean force (PMF) study using two different force fields: ff99SB and ff99SB-ILDN. From the PMF plot, we noticed the PMF to have a minimum value at a p53–MDM2 separation of 12 Å, with a dissociation energy of 30 kcal mol–1. We also analyzed the conformational dynamics and stability of p53 as a function of its distance of separation from MDM2. The secondary structure content (helix and turns) in p53 was found to vary with its distance of separation from MDM2. The p53–MDM2 complex structure with lowest potential energy was isolated from the ensemble at the reaction coordinate corresponding to the minimum PMF value and subjected to molecular dynamics simulation to identify the interface surface area, interacting residues at the interface, and the stability of the complex. The simulation results highlight the importance of hydrogen bonds and the salt bridge between Lys94 of MDM2 and Glu17 of p53 in the stability of the p53–MDM2 complex. We also carried out the binding free energy calculations and the per residue energy decomposition analyses of the interface residues of the p53–MDM2 complex. We found that the binding affinity between MDM2 and p53 is indeed high [ΔGbind = −7.29 kcal mol–1 from molecular mechanics/Poisson–Boltzmann surface area (MM/PBSA) and ΔGbind = −53.29 kcal mol–1 from molecular mechanics/generalized borne surface area]. The total binding energy obtained using the MM/PBSA method was noticed to be closer to the experimental values (−6.4 to −9.0 kcal mol–1). The p53–MDM2 complex binding profile was observed to follow the same trend even in the duplicate simulation run and also in the simulation carried out with different force fields. We found that Lys51, Leu54, Tyr100, and Tyr104 from MDM2 and the residues Phe19, Trp23, and Leu26 from p53 provide the highest energy contributions for the p53–MDM2 interaction. Our findings highlight the prominent structural and binding characteristics of the p53–MDM2 complex that may be useful in designing potential inhibitors to disrupt the p53–MDM2 interactions.

ASPP2 suppression promotes malignancy via LSR and YAP in human endometrial cancer.

Apoptosis-stimulating p53 protein 2 (ASPP2) is an apoptosis inducer that acts via binding with p53 and epithelial polarity molecule PAR3. Lipolysis-stimulated lipoprotein receptor (LSR) is an important molecule at tricellular contacts, and loss of LSR promotes cell migration and invasion via Yes-associated protein (YAP) in human endometrial cancer cells. In the present study, to find how ASPP2 suppression promotes malignancy in human endometrial cancer, we investigated its mechanisms including the relationship with LSR. In endometriosis and endometrial cancers (G1 and G2), ASPP2 was observed as well as PAR3 and LSR in the subapical region. ASPP2 decreased in G3 endometrial cancer compared to G1. In human endometrial cancer cell line Sawano, ASPP2 was colocalized with LSR and tricellulin at tricellular contacts and binding to PAR3, LSR, and tricellulin in the confluent state. ASPP2 suppression promoted cell migration and invasion, decreased LSR expression, and induced expression of phosphorylated YAP, claudin-1, -4, and -7 as effectively as the loss of LSR. Knockdown of YAP prevented the upregulation of pYAP, cell migration and invasion induced by the ASPP2 suppression. Treatment with a specific antibody against ASPP2 downregulated ASPP2 and LSR, affected F-actin at tricellular contacts, upregulated expression of pYAP and claudin-1, and induced cell migration and invasion via YAP. In normal human endometrial epithelial cells, ASPP2 was in part colocalized with LSR at tricellular contacts and knockdown of ASPP2 or LSR induced expression of claudin-1 and claudin-4. ASPP2 suppression promoted cell invasion and migration via LSR and YAP in human endometrial cancer cells.

How p53 Molecules Solve the Target DNA Search Problem: A Review.

Interactions between DNA and DNA-binding proteins play an important role in many essential cellular processes. A key function of the DNA-binding protein p53 is to search for and bind to target sites incorporated in genomic DNA, which triggers transcriptional regulation. How do p53 molecules achieve “rapid” and “accurate” target search in living cells? The search dynamics of p53 were expected to include 3D diffusion in solution, 1D diffusion along DNA, and intersegmental transfer between two different DNA strands. Single-molecule fluorescence microscopy enabled the tracking of p53 molecules on DNA and the characterization of these dynamics quantitatively. Recent intensive single-molecule studies of p53 succeeded in revealing each of these search dynamics. Here, we review these studies and discuss the target search mechanisms of p53.

Anti‐apoptotic properties of mesenchymal stem cells in mouse model of corneal injury

AbstractLimbal stem cell deficiency (LSCD) is associated with the break of immune privilege of the cornea, a harmful local inflammatory reaction and a loss of corneal epithelium cells caused not only by damage after injury but also by the apoptosis in later phase. Apoptosis is caused by various factors including damaging of cell structures and chronic inflammation and may be one of the main causes of the failure of corneal regenerative mechanisms. Mesenchymal stem cells (MSCs) turned out to be a suitable source of stem cells for the therapy of bilateral LSCD. MSCs are well known for their anti‐inflammatory, anti‐apoptotic, regenerative, migration and differentiation properties. Antiapoptotic mechanisms are still not well described but their recognition could help for a long‐term regeneration of corneal epithelium after the injury or after the corneal transplantation in cases like LSCD.MSCs were obtained from murine bone marrow. Flow cytometry was used to characterize phenotypic markers of purified MSCs. For in vitro experiments, MSCs were cultured in plates for 24 hours. Excised corneas were added to the culture alone or in inserts and were stimulated by pro inflammatory cytokines. After a 48‐hour co‐cultivation the expression of genes for pro apoptotic and anti‐apoptotic molecules in corneas was detected using qPCR. Flow cytometry was used to detect phosphatidylserine on the surface of apoptotic cells by annexin V. In in vivo experiments, MSCs were seeded onto nanofiber scaffolds (allowed to adhere for 24 hours) and transplanted on the injured cornea (alkali burn)MSCs significantly decreased the number of apoptotic cells in the cornea with simulated inflammation in vitro or in injured corneas in vivo. MSCs also supressed the expression of the pro‐apoptotic molecules Bax and p53 and change the ratio of Bcl‐2 Bax expression in the cornea.Autologous MSCs suppressed apoptosis in in vitro and in vivo models of corneal injury and inflammation.

Computational investigation on the molecular interactions between MDM2 and its photoactivatable inhibitor

The Murine Double Minute 2 (MDM2) protein is a crucial negative regulator of the tumor suppressor p53 molecule. In order to restrict p53 functioning, MDM2 molecules are overproduced in many human tumors. Thus, reactivating p53 in cancer cells using inhibitors, disrupting p53-MDM2 binding, can offer an effective approach for cancer therapy. Recently a photoactivatable MDM2 inhibitor, a photoremovable-protecting group (PPG) in complex with idasanutlin has been reported to exert no functional effect on cellular outgrowth but allows for the selective, non-invasive activation of antitumor properties due to the release of active inhibitor idasanutlin from the complex upon irradiation with 400 nm light. In this study, using molecular docking and Molecular Dynamics (MD) simulations, we have investigated the interaction of (i) PPG-idasanutlin complex and (ii) the active inhibitor idasanutlin with MDM2 at the molecular level. We noticed that the PPG-idasanutlin complex fails to fit into the binding cavity of MDM2. But the active inhibitor idasanutlin when it is free from PPG was found to fit perfectly into the binding cavity of MDM2. From the Dictionary of Secondary Structure of Proteins (DSSP) analysis, we found that the number of α-helices, which aid in the stability of protein, were found to be more in the MDM2-idasanutlin complex rather than in the MDM2-PPG-idasanutlin complex. Using the PDBsum server, we have compared the interaction profiles of MDM2-PPG-idasanutlin, MDM2-idasnautlin and MDM2-p53 complexes. From the interaction profile, we found the active inhibitor, idasanutlin free from PPG to bind to the region in MDM2 where p53 prefers to bind.. Our findings from this study would shed light on designing more potent photoactivatable MDM2 inhibitors.

Novel solid form of prothioconazole and its crystallographic analysis

Prothioconazole is a broad spectrum anti-fungal agent of triazolinthione family; it has been widely used in crop protection especially in cereals. To date, only the crystal structure of form II and p-xylene solvates were reported, as per our knowledge, there are no other crystallographic reports in the literature. Here we report the dioxane solvate of prothioconazole with improved solubility. The novel form is completely characterized by powder XRD, DSC, TGA and finally by single-crystal XRD. In the present investigation, we compared the arrangement of molecules and hydrogen bonding interactions in prothioconazole form II, p-xylene solvate and novel 1,4−dioxane solvate. The crystal structure analysis revealed that prothioconazole form II crystallized in monoclinic crystal system with P21/n space group and molecules are held together by N···Cl interactions and N–H⋯O hydrogen bonding interactions. The p-xylene solvate crystallized in P21 space group and molecules are held together by N–H⋯N and C–S⋯Cl interactions. Whereas the novel solid form crystallized in the triclinic crystal system with P−1 space group and molecules are held together by N⋯O interactions. The novel solid form has shown improved solubility compared to reference drug.

Anti-Bcma PBD MEDI2228 Combats Drug Resistance and Synergizes with Bortezomib and Inhibitors to DNA Damage Response in Multiple Myeloma

A novel anti-BCMA antibody drug conjugate (ADC) MEDI2228 preferentially binds to membrane-bound vs soluble BCMA (sBCMA) to effectively deliver the pyrrolobenzodiazepine (PBD) payload tesirine to multiple myeloma (MM) cells (Leukemia. 2019;33: 766). In preclinical models, this ADC targets both MM cells in bulk and CD19+CD138- patient MM progenitor cells. We here study the potency of MEDI2228 in MM cells in the bone marrow (BM) microenvironment and examine molecular mechanisms whereby MEDI2228 overcomes drug resistance. First, MEDI2228, more effectively than its microtubule-binding monomethyl auristatin-F (MMAF) ADC homolog, inhibits proliferation (&gt;1-2-log) and survival of all MM cell lines and MM cells from patients with multiple relapsed and refractory diseases, regardless of BCMA levels, p53 status, and the protection conferred by BM stromal cells and IL-6. Significantly, MM cells with lower BCMA expression and resistance to bortezomib or immunomodulatory drugs (IMiDs, i.e., lenalidomide, pomalidomide) are more susceptible to MEDI2228 vs its MMAF ADC homolog. MEDI2228, but not its MMAF ADC homolog, activates critical DNA damage responses (DDR) via phosphorylation of ATM/ATR kinases, checkpoint kinases (CHK)1/2, and H2AX, associated with induction of multiple DDR pathway-associated genes. Low doses of MEDI2228 and bortezomib (btz) synergistically induce apoptosis of drug-sensitive and -resistant MM cells, at least in part, through modulation of RAD51, a DNA damage and repair protein. Importantly, MEDI2228 further triggers the ATM/ATR-CHK1/2 signaling cascade, associated with increased gH2AX, p21, and apoptosis molecules in MM1S-xenografted tumors in mice. In vivo, a single sub-optimal dose of 0.4 mg/kg MEDI2228 induces superior anti-MM activity than btz, indicating that MEDI2228 is significantly more effective and selective than btz as single agent therapy in vivo. Furthermore, combined treatments with MEDI2228 and btz result in potent tumor depletion and significantly prolonged host survival via increased nuclear gH2AX-expressing microfoci, DNA damage-induced growth arrest and cell death. Significant tumor necrosis is observed earlier in mice receiving both drugs than either agent alone. At 177d, 15% mice in the combination treatment group remain alive and without any tumor. Importantly, no weight loss is noted in all groups, indicating a favorable safety profile of MEDI2228, alone or with btz, in vivo. Moreover, DDR checkpoint inhibitors, i.e., AZD0156 (ATMi), AZD6738 (ATRi), AZD1775 (WEE1i), synergize with MEDI2228 to enhance MM cell cytotoxicity (combination index &lt; 1). This study therefore further supports clinical development of MEDI2228 (NCT03489525) as an important next-generation immunotherapy to improve outcome of MM patients. Disclosures Kinneer: AstraZeneca: Employment. Munshi:Amgen: Consultancy; Abbvie: Consultancy; Oncopep: Consultancy; Takeda: Consultancy; Janssen: Consultancy; Adaptive: Consultancy; Celgene: Consultancy. Anderson:Celgene: Consultancy, Speakers Bureau; Amgen: Consultancy, Speakers Bureau; Janssen: Consultancy, Speakers Bureau; Takeda: Consultancy, Speakers Bureau; Bristol-Myers Squibb: Other: Scientific Founder; Oncopep: Other: Scientific Founder; Sanofi-Aventis: Other: Advisory Board.

Gamma Radiation-Induced Disruption of Cellular Junctions in HUVECs Is Mediated through Affecting MAPK/NF-κB Inflammatory Pathways.

Ionizing radiation-induced cardiovascular diseases (CVDs) have been well documented. However, the mechanisms of CVD genesis are still not fully understood. In this study, human umbilical vein endothelial cells (HUVECs) were exposed to gamma irradiation at different doses ranging from 0.2 Gy to 5 Gy. Cell viability, migration ability, permeability, oxidative and nitrosative stresses, inflammation, and nuclear factor kappa-light-chain-enhancer of activated B cell (NF-κB) pathway activation were evaluated postirradiation. It was found that gamma irradiation at doses ranging from 0.5 Gy to 5 Gy inhibited the migration ability of HUVECs without any significant effects on cell viability at 6 h and 24 h postirradiation. The decreased transendothelial electrical resistance (TEER), increased permeability, and disruption of cellular junctions were observed in HUVECs after gamma irradiation accompanied by the lower levels of junction-related proteins such as ZO-1, occludin, vascular endothelial- (VE-) cadherin, and connexin 40. The enhanced oxidative and nitrosative stresses, e.g., ROS and NO2− levels and inflammatory cytokines IL-6 and TNF-α were demonstrated in HUVECs after gamma irradiation. Western blot results showed that protein levels of mitogen-activated protein kinase (MAPK) pathway molecules p38, p53, p21, and p27 increased after gamma irradiation, which further induced the activation of the NF-κB pathway. BAY 11-7085, an inhibitor of NF-κB activation, was demonstrated to partially block the effects of gamma radiation in HUVECs examined by TEER and FITC-dextran permeability assay. We therefore concluded that the gamma irradiation-induced disruption of cellular junctions in HUVECs was through the inflammatory MAPK/NF-κB signaling pathway.

Abstract 1719: Copper levels and the p53 protein: A delicate balance

Abstract The tumor suppressor p53 is mutated in 50% of all cancers and in about 90% of small lung cancers. Mutations lead to loss of p53 expression or the expression of mutant p53 proteins. These mutants often lose WT function, but they have also been shown to acquire invasive and metastatic functions. p53 requires a zinc molecule in its core domain to fold properly and to function as a transcription factor and tumor suppressor. Loss of this zinc through competition with copper causes p53 to unfold and to lose its transcription function. Copper is an essential heavy metal required for a large variety of biological processes as it is required for the function of many enzymes. In cancer patients, increased serum levels of copper have been found and people with a predisposition for copper accumulation in the liver (Wilson’s disease, mutations in ATP7B) are prone to develop liver cancers. Our preliminary results have indicated that an unfolded WT p53 molecule does not only lose its transcription function, but also gains mutant p53-like functions in promoting invasion. Copper-induced unfolded p53 interacted with the mutant p53 specific interacting proteins Dicer, Ago, p63 and p73 and impaired p63 function. These results raise the question as to why a WT molecule would lose its function and whether it perhaps could act as a redox sensor. Experiments are ongoing to explore p53 mediated transcription in response to copper exposure. In addition, we are setting up mouse models to investigate p53 folding in vivo in response to mild increases in copper serum levels and we are developing methods to better determine p53 folding in patients and in mice. These results will help us assess p53 status in relation to serum metal levels in vivo and determine whether p53 is important in the development of cancers and metastasis. Citation Format: Patricia Muller, Yannick von Grabowiecki, Callum Hall. Copper levels and the p53 protein: A delicate balance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1719.

IL-29 Exhibits Anti-Tumor Effect on Pan-48 Pancreatic Cancer Cells by Up-regulation of P21 and Bax.

Pancreatic cancer is the most lethal cancer of the digestive system. IL-29 is a new member of the IFNλ family and well-known for its strong antiviral activity. However, its direct effect on pancreatic cancer is still unclear. This study was performed to investigate if IL-29 has any direct effect on Pan-48 pancreatic cancer cells. Clonogenic survival assay, cell proliferation, and caspase-3 activity kits were used to evaluate the effects of IL-29 on cell survival, proliferation, and apoptosis of Pan-48 pancreatic cancer cells. RT-PCR and IHC were subsequently performed to explore IL-29's potential molecular mechanisms. The percentage of colonies of Pan-48 cells was decreased following the addition of IL-29. This was consistent with a decreased optical density (OD) value of cancer cells. Furthermore, the relative caspase-3 activity in cancer cells was increased after the addition of IL-29, indicating increased apoptosis of cancer cells. The anti-proliferative effect of IL-29 on cancer cells correlated with increased expression of the anti-proliferative molecule p21. The pro-apoptotic effect of IL-29 on cancer cells correlated with an increased expression of the pro-apoptotic molecule Bax. IL-29 constrains Pan-48 pancreatic cell growth via up-regulation of p21 and Bax. Our study suggests a potential use of IL-29 in immunotherapy for pancreatic cancer treatment.

Porcine Parvovirus Infection Causes Pig Placenta Tissue Damage Involving Nonstructural Protein 1 (NS1)-Induced Intrinsic ROS/Mitochondria-Mediated Apoptosis.

Porcine parvovirus (PPV) is an important pathogen causing reproductive failure in pigs. PPV-induced cell apoptosis has been recently identified as being involved in PPV-induced placental tissue damages resulting in reproductive failure. However, the molecular mechanism was not fully elucidated. Here we demonstrate that PPV nonstructural protein 1 (NS1) can induce host cell apoptosis and death, thereby indicating the NS1 may play a crucial role in PPV-induced placental tissue damages and reproductive failure. We have found that NS1-induced apoptosis was significantly inhibited by caspase 9 inhibitor, but not caspase 8 inhibitor, and transfection of NS1 gene into PK-15 cells significantly inhibited mitochondria-associated antiapoptotic molecules Bcl-2 and Mcl-1 expressions and enhanced proapoptotic molecules Bax, P21, and P53 expressions, suggesting that NS1-induced apoptosis is mainly through the mitochondria-mediated intrinsic apoptosis pathway. We also found that both PPV infection and NS1 vector transfection could cause host DNA damage resulting in cell cycle arrest at the G1 and G2 phases, trigger mitochondrial ROS accumulation resulting in mitochondria damage, and therefore, induce the host cell apoptosis. This study provides a molecular basis for elucidating PPV-induced cell apoptosis and reproductive failure.

Extensive mechanical tension promotes annulus fibrosus cell senescence through suppressing cellular autophagy.

Background: Mechanical load contributes a lot to the initiation and progression of disc degeneration. Annulus fibrosus (AF) cell biology under mechanical tension remains largely unclear.Objective: The present study was aimed to investigate AF cell senescence under mechanical tension and the potential role of autophagy.Methods: Rat AF cells were cultured and experienced different magnitudes (5% elongation and 20% elongation) of mechanical tension for 12 days. Control AF cells were kept static. Cell proliferation, telomerase activity, cell cycle fraction, and expression of senescence-related molecules (p16 and p53) and matrix macromolecules (aggrecan and collagen I) were analyzed to evaluate cell senescence. In addition, expression of Beclin-1 and LC3, and the ratio of LC3-II to LC3-I were analyzed to investigate cell autophagy.Results: Compared with the control group and 5% tension group, 20% tension group significantly decreased cell proliferation potency and telomerase activity, increased G1/G0 phase fraction, and up-regulated gene/protein expression of p16 and p53, whereas down-regulated gene/protein expression of aggrecan and collagen I. In addition, autophagy-related parameters such as gene/protein expression of Beclin-1 and LC3, and the ratio of LC3-II to LC3-I, were obviously suppressed in the 20% tension group.Conclusion: High mechanical tension promotes AF cell senescence though suppressing cellular autophagy. The present study will help us to better understand AF cell biology under mechanical tension and mechanical load-related disc degeneration.

In Vivo Effect of Luteolin during Oxaliplatin Treatment for Colorectal Cancer

Phytochemicals that induce nuclear factor (erythroid‐derived 2)‐like 2 (Nrf2) can lower cellular oxidative stress and thus favor cell viability. Considering our previous in vitro study demonstrating that strong antioxidative called luteolin interfered with the anticancer effect of oxaliplatin on HCT116, we hypothesized that luteolin administration would be unadvantageous during oxaliplatin‐based chemotherapy. To test this hypothesis, HCT116 xenograft mouse model was established in BALB/c nude mice. Once palpable tumors were developed after HCT116 cells were subcutaneously transplanted on both flanks of each mouse, mice were randomly allocated into four groups that were intraperitoneally injected with (1) vehicle only (control), (2) oxaliplatin at a dose of 10 mg/kg body weight (BW), (3) luteolin at 50 mg/kg BW, or (4) combination of luteolin and oxaliplatin. Tumor size and body weight were regularly monitored (three times a week) for three weeks. Results demonstrated that oxaliplatin treatment inhibited the growth of xenografted tumors as expected, luteolin treatment had little effect on the tumor size, and combinatorial treatment of oxaliplatin and luteolin most suppressed tumor growth. In addition, oxaliplatin‐luteolin combo reduced heme oxygenase‐1 (HO‐1) protein expression compared to oxaliplatin alone. Moreover, luteolin alone treatment enhanced protein expressions of p53 and its downstream molecule p21. These findings were suggestive of in vivo synergistic effect of combinatorial treatment of oxaliplatin and luteolin at the tested doses in this study and the possible coordinated regulation via p53 and/or HO‐1‐mediated cytoprotection in HCT116 xenograft tumor growth. According to the current observations, our hypothesis ought to be reconsidered; luteolin could advantageously hamper tumor growth during oxaliplatin treatment in colon cancer.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Alpha-L-fucosidase-1 is a diagnostic marker that distinguishes mucoepidermoid carcinoma from squamous cell carcinoma.

Alpha-L-fucose is a component of glycans on the cell surface. Alpha-L-fucose is correlated with tumorigenesis and malignancy, and alpha-L-fucosidase-1 (FUCA1), the enzyme that removes terminal α-L-fucose residues from glycoproteins, is downregulated in some high malignancy cancers. The expression profile of FUCA1 in head and neck tumors remains unknown, and we analyzed the expression profiles of FUCA1 and an upstream molecule p53 in mucoepidermoid carcinoma (MEC) and oral squamous cell carcinoma (OSCC). FUCA1 was expressed in most MECs irrespective of histopathological grading, whereas expression in OSCCs was low. High immunohistochemical intensity of p53 was detected in OSCCs at high frequency, but rarely detected in MECs. Genetic mutation analysis using next-generation sequencing revealed no significant mutation of TP53 in MECs. We further analyzed the expression profiles of FUCA1 in normal major and minor salivary glands and found strong expression in the intercalated duct, moderate expression in mucous acinar cells and no expression in serous acinar cells. These contrasting immunohistochemical profiles and anatomical distribution in normal salivary glands suggest that FUCA1 is a useful marker to distinguish MEC from OSCC, and many MECs have similar immunohistochemical phenotypes to intercalated duct and mucous acinar cells.

A Novel Small Molecule p53 Stabilizer for Brain Cell Differentiation.

Brain tumor, as any type of cancer, is assumed to be sustained by a small subpopulation of stem-like cells with distinctive properties that allow them to survive conventional therapies and drive tumor recurrence. Thus, the identification of new molecules capable of controlling stemness properties may be key in developing effective therapeutic strategies for cancer by inducing stem-like cells differentiation. Spiropyrazoline oxindoles have previously been shown to induce apoptosis and cell cycle arrest, as well as upregulate p53 steady-state levels, while decreasing its main inhibitor MDM2 in the HCT116 human colorectal carcinoma cell line. In this study, we made modifications in this scaffold by including combinations of different substituents in the pyrazoline ring in order to obtain novel small molecules that could modulate p53 activity and act as differentiation inducer agents. The antiproliferative activity of the synthesized compounds was assessed using the isogenic pair of HCT116 cell lines differing in the presence or absence of the p53 gene. Among the tested spirooxindoles, spiropyrazoline oxindole 1a was selective against the cancer cell line expressing wild-type p53 and presented low cytotoxicity. This small molecule induced neural stem cell (NSC) differentiation through reduced SOX2 (marker of multipotency) and increased βIII-tubulin (marker of neural differentiation) which suggests a great potential as a non-toxic inducer of cell differentiation. More importantly, in glioma cancer cells (GL-261), compound 1a reduced stemness, by decreasing SOX2 protein levels, while also promoting chemotherapy sensitization. These results highlight the potential of p53 modulators for brain cell differentiation, with spirooxindole 1a representing a promising lead molecule for the development of new brain antitumor drugs.

Ras-Driven B-Cell Transformation Targets Developmental Rewiring of Cytokine to Pre-B Cell Receptor Signaling

Ras-pathway lesions are oncogenic drivers in ~45% of B-cell acute lymphoblastic leukemia (B-ALL) cases. Activating mutations of NRAS and KRAS are oncogenic drivers in B-ALL while the BRAFV600E mutation occurs in almost all cases of B-cell hairy cell leukemia. Less frequent lesions resulting in increased ERK-signaling in B-cell malignancies include activating mutations of RAF1, MAP2K1 and the PTPN11 phosphatase as well as deleterious mutations of the Ras-GTPase activator NF1. Interestingly, increased immunoglobulin light chain gene expression was observed in B-ALL cases with RAS-pathway lesions (COG P9906), reflecting engagement of pre-B cell receptor (pre-BCR) downstream signaling. Here we tested the hypothesis that oncogenic RAS-signaling in B-ALL mimics pre-BCR-induced developmental rewiring of signal transduction at the pro-B to pre-B cell transition and identified PTPN6 and BCL6 as therapeutic targets in RAS-driven B-ALL.During early B-cell development, pro-B cells transition from cytokine- to pre-B cell receptor (pre-BCR)-dependent survival and proliferation signals. Inducible activation of immunoglobulin (Ig) µ heavy chain (µHC) expression induced developmental progression and surface expression of Ig κ light chains. Notably, inducible activation of oncogenic NRASG12D had the same effect and resulted in increased surface expression of Ig κ light chains. Furthermore, studying genetic models for this transition revealed that both pre-BCR signaling and RAS-oncogenes suppressed cytokine receptor/STAT5-signaling and induced massive de novo expression of the proto-oncogene and transcriptional repressor BCL6. Our genetic studies revealed that the SH2-domain containing protein tyrosine phosphatase PTPN6 was activated by oncogenic RAS-signaling and essential for the switch from STAT5 to BCL6-activation. Given that oncogenic RAS activated PTPN6, we tested the role of PTPN6 in RAS-driven leukemogenesis. To this end, ablation of Ptpn6 in NRASG12D-driven B-ALL resulted in depletion of cells from cell culture in competitive-growth assays and reduced the number of colonies formed in semi-solid methylcellulose. Collectively, these findings suggest that PTPN6 represents a potential therapeutic intervention point in RAS-driven B-ALL.In addition to PTPN6, we investigated the role of BCL6 in RAS-driven B-ALL. Aberrant activation of oncogenic RAS results in oncogene-induced senescence (OIS) characterized by induction of ARF/p53 and irreversible cell cycle arrest in the G1 phase. For oncogenic Ras-signaling, BCL6 was required to oppose ERK-mediated activation of p21, p27 and p53 checkpoint molecules in B-ALL. Here we tested the hypothesis that BCL6 bypasses the RAS-mediated OIS program to facilitate transformation. To this end, increases in number of colonies formed in semi-solid methylcellulose were observed upon inducible activation of Bcl6 in NRASG12D B-ALL cells. Furthermore, loss of Bcl6 function in NRASG12D B-ALL cells resulted in depletion of cells from cell culture in competitive growth assays and reduced colony forming ability. Importantly, expression of NRASG12D in Bcl6+/+ pre-B cells resulted in transformation and fatal leukemia in transplant recipient mice. In striking contrast, Bcl6-/- pre-B cells transduced with NRASG12D failed to initiate fatal disease in vivo. Furthermore, pharmacological inhibition of BCL6 restored sensitivity to chemotherapy in patient-derived KRASG12V B-ALL cells.In conclusion, we identified oncogenic RAS-signaling as functional mimics of pre-BCR signaling. Oncogenic RAS induced expression of BCL6 at the expense of cytokine receptor/STAT5-signaling. Our genetic studies identified PTPN6 as a critical effector molecule of the switch from cytokine receptor to pre-BCR signaling. Importantly, we identified PTPN6 and BCL6 as potential therapeutic intervention points in RAS-driven B-ALL. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Acidic pH promotes nucleus pulposus cell senescence through activating the p38 MAPK pathway.

Background: Nucleus pulposus (NP) cell senescence is an important cellular feature within the degenerative disc. It is known that a very acidic niche exists in the degenerative disc, which participates in regulating disc cell viability and matrix metabolism. Objective: The present study was aimed to investigate the role and potential signaling transduction pathway of an acidic pH in regulating NP cell senescence. Methods: Rat NP cells were cultured in an acidic pH of 7.2 close to that in a healthy disc (Control group) or in an acidic pH of 6.2 close to that in a severe degenerative disc (Experiment group) for 10 days. Additionally, the experimental NP cells were incubated along with the inhibitor SB203580 to analyze the role of p38 MAPK pathway in this process. Results: Compared with the control NP cells, experimental NP cells showed a suppressed cell proliferation potency, an increased G0/G1 phase fraction whereas a decreased S-phase fraction and a declined telomerase activity, an up-regulated expression of senescence-related molecules (p16 and p53), and a down-regulated expression of matrix-related moleucles (aggrecan and collagen II). Further analysis showed that inhibition of the p38 MAPK pathway partly reversed effects of acidic pH of 6.2 on the experimental NP cells. Conclusion: The very acidic niche identified in a severe degenerative disc promotes NP cell senescence through regulating the p38 MAPK pathway. The present study provides a new mechanism that drives NP cell senescence during disc degeneration.

Alterations in Expression of Cell Surface and Cell Cycle Signaling Molecules in Recurrent Nonmuscle Invasive Bladder Cancers: A Tissue Microarray Expression Analysis

Background: Nonmuscle invasive bladder cancers (NMIBC) are one of the most common urological cancers having a high risk of recurrence and progression. Recurrent tumors may acquire certain molecular alterations responsible for progression of the tumors. Identification of these alterations is important to understand the pathobiology and guide further management. Aim: We studied the differences in expression of cell surface proteins and cell cycle signaling molecules in primary and recurrent NMIBC by immunohistochemistry (IHC) on tissue microarrays (TMA). Methods: Using FFPE tissue, TMA of 82 tumors (40 primary NMIBC and 47 recurrences) were constructed. IHC for growth factor receptors [epidermal growth factor receptor (EGFR), HER2/neu and FGFR3], cell adhesion molecules (E-cadherin and beta-catenin) and cell cycle pathway molecules (p53, p21/WAF1/Cip1 and Ki-67 proliferation index) was performed. A semiquantitative H-score (Histo-score; range 0-300) was calculated according to the intensity (0, negative; 1, weak; 2, moderate; and 3, strong) and percentage of cells stained. &lt; 10% cells showing nuclear p21 expression was considered p21-loss. The differences in expression between the primary and recurrent tumors were analyzed using paired t test. Results: The mean age at presentation was 65.3 ± 13.6 years with a male predominance (n=36). The mean time to recurrence was 33.4 months (range 3-109). Progression in grade and/or stage was seen in 30 (75%) tumors. Time to recurrence was shorter in primary tumors with ≥ 5% Ki-67 proliferation index. There was no significant difference in expression of cell surface proteins between primary and recurrent tumors. Significant p21 loss was seen in recurrent tumors ( P = 0.03) and significantly correlated with loss of surface beta-catenin and nuclear p53 positivity ( P = 0.002). Ki-67 index was higher in recurrent tumors and also correlated with p53 positivity ( P = 0.007). Conclusion: We found no significant differences in expression of cell surface molecules in primary nonmuscle invasive bladder cancers and their recurrences. However, there were significant alterations in expression of molecules of cell cycle signaling pathway and cellular proliferation in recurrent tumors suggesting the role of cell cycle regulators as promising targets in these cancers.