P53-dependent Signaling Research Articles

Phosphoproteomic investigation of targets of protein phosphatases in EGFR signaling.

Receptor tyrosine kinases (RTKs) initiate cellular signaling pathways, which are regulated through a delicate balance of phosphorylation and dephosphorylation events. While many studies of RTKs have focused on downstream-activated kinases catalyzing the site-specific phosphorylation, few studies have focused on the phosphatases carrying out the dephosphorylation. In this study, we analyzed six protein phosphatase networks using chemical inhibitors in context of epidermal growth factor receptor (EGFR) signaling by mass spectrometry-based phosphoproteomics. Specifically, we focused on protein phosphatase 2C (PP2C), involved in attenuating p38-dependent signaling pathways in various cellular responses, and confirmed its effect in regulating p38 activity in EGFR signaling. Furthermore, utilizing a p38 inhibitor, we classified phosphosites whose phosphorylation status depends on PP2C inhibition into p38-dependent and p38-independent sites. This study provides a large-scale dataset of phosphatase-regulation of EGF-responsive phosphorylation sites, which serves as a useful resource to deepen our understanding of EGFR signaling.

Abstract B016: Novel curcumin analogues as therapies for endometrial cancer with TP53 mutations

Abstract The protein p53 is designated as the guardian of the genome because it is the principal tumor suppressor and functions to surveil the genome, enforce cell cycle checkpoints, and trigger apoptosis when genomic instability is beyond repair. Mutations in TP53 are found in >50% of human tumors and in 25% of the deadliest endometrial malignancies. Yet, few studies have targeted the mutant protein to restore its wild type (WT) tumor suppressor functions. Curcumin, a turmeric derivative, has been shown to help return WT function to mutant p53. However, curcumin is not bioavailable to most of the body because of its water insolubility, hydrophobic nature and rapid metabolization. Our laboratory is testing novel, more bioavailable analogs of curcumin including HO-3867 and AKT-100. In these studies, we tested these agents in preclinical models of serous endometrial cancer to assess their effects on cell growth and p53-dependent signaling. We used CyQUANT cell proliferation assay to identify IC50 values for HO-3867 and AKT-100 in ECC-1 and KLE endometrial cancer cell lines. We performed RNA sequencing to elucidate mechanisms of action and verified p53 expression changes via Western blotting. IC50 values ranged from 100 nanomolar (AKT-100) to low micromolar (HO-3867). RNA sequencing data revealed the re-enforcement of cell cycle checkpoints including the upregulation of CDKN1A (p21) and GADD45 (Gadd45). Proliferative signaling downstream of ATM, an important protein kinase stimulating cell growth, was downregulated by both agents. Western blotting confirmed the classic negative feedback loop of p53, where total p53 levels decreased after drug treatment. Taken together, our findings show that AKT-100 and HO-3867 are able to restore WT tumor suppressor function in otherwise oncogenic mutant p53 by reducing cell replication and promoting apoptosis. Thus, curcumin analogues such as AKT-100 and HO-3867 show promise in the treatment of a subset of endometrial tumors expressing mutant p53 by returning WT p53 functionality, and future studies will move to in vivo analyses of curcumin analogue function. Citation Format: Kimberly K. Leslie, Alex Goss, Jamie Padilla, Lane E. Smith. Novel curcumin analogues as therapies for endometrial cancer with TP53 mutations [abstract]. In: Proceedings of the AACR Special Conference on Endometrial Cancer: Transforming Care through Science; 2023 Nov 16-18; Boston, Massachusetts. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(5_Suppl):Abstract nr B016.

Features of Intracellular Signal Transduction in Neural Stem Cells under the Influence of Alkaloid Songorine, an Agonist of Fibroblast Growth Factor Receptors.

We performed a comparative in vitro study of the involvement of NF-κB, PI3K, cAMP, ERK1/2, p38, JAKs, STAT3, JNK, and p53-dependent intracellular signaling in the functioning of neural stem cells (NSC) under the influence of basic fibroblast growth factor (FGF) and FGF receptor agonist, diterpene alkaloid songorine. The significant differences in FGFR-mediated intracellular signaling in NSC were revealed for these ligands. In both cases, stimulation of progenitor cell proliferation occurs with the participation of NF-κB, PI3K, ERK1/2, JAKs, and STAT3, while JNK and p53, on the contrary, inhibit cell cycle progression. However, under the influence of songorin, cAMP- and p38-mediated cascades are additionally involved in the transmission of the NSC division-activating signal. In addition, unlike FGF, the alkaloid stimulates progenitor cell differentiation by activating ERK1/2, p38, JNK, p53, and STAT3.

Hepatitis B doubly spliced protein (HBDSP) promotes hepatocellular carcinoma cell apoptosis via ETS1/GATA2/YY1-mediated p53 transcription.

Hepatitis B virus (HBV) spliced variants are associated with viral persistence or pathogenicity. Hepatitis B doubly spliced protein (HBDSP), which has been previously reported as a pleiotropic transactivator protein, can potentially serve as an HBV virulence factor. However, the underlying mechanisms of HBDSP in HBV-associated liver diseases remain to be elucidated. In this study, we revealed that HBDSP promotes cellular apoptosis and induces wt-p53-dependent apoptotic signaling pathway in wt-p53 hepatocellular cells by transactivating p53 transcription, and increases the release of HBV progeny. Therefore, HBDSP may promote the HBV particles release through wt-p53-dependent hepatocellular apoptosis. Our findings suggest that blocking HBDSP-induced wt-p53-dependent apoptosis might have therapeutic values for chronic hepatitis B.

Abstract PO-076: Adrenergic regulation of the anti-tumor immune response in p53-deficient tumors

Abstract Several previous studies have shown that β2-adrenergic signaling pathway promotes tumor progression, whereas others have shown the opposite effect. The role of β2-adrenergic activation in oral squamous cell carcinoma (OSCC) remains largely unknown. Our previous study showed that the loss of p53 leads to increased adrenergic tumor innervation in OSCC, resulting in transcriptional programs that establish new neuronal identity. We hypothesized that tumor immunity in OSCC is regulated by adrenergic signaling. The aim of the present study was to determine the influences of adrenergic signaling in immune microenvironment of OSCC. We first performed multiplex immunofluorescence to analyze the immune cell infiltration and adrenergic nerve density in OSCC; and analyzed the Cancer Genome Atlas (TCGA) datasets to establish correlations between immune-related and adrenergic pathway genes. Subsequently, we performed T cell killing assay with p53 knockout (KO) and p53 wild-type (WT) using antigen-specific HLA-matched patient-derived T-cells with adrenergic agonists or antagonists. To confirm p53-dependent adrenergic signaling in OSCC, a genetically engineered p53MDM2 overexpression (OE) cell line was generated and treated with adrenergic agonists or antagonists. To test our hypothesis that p53 regulates adrenergic-dependent paracrine signaling between cancer and T cells we performed a Luminex assay on the conditioned medium from p53 KO and p53 WT coculture system. Finally, in vivo, adrenergic ablation (TH flox/flox ) and novel murine models of OSCC were used to investigate the effect of adrenergic nerve signaling on immune infiltration and responses to immunotherapy. Our data showed that the increase in adrenergic nerve density was inversely proportional to the infiltration of exhausted T cells. Analysis of the TCGA data revealed that ADRB2 strongly correlated with tumor immune infiltrate genes. Adrenergic agonists treatment induced significantly more apoptosis in the p53 KO but not in the p53 WT cells; and overexpression of MDM2 rescued the resistance to apoptosis. Conditioned media experiments showed that the coculture of tumor cells with T cells acts through paracrine signaling and that the secreted factor is CXCL10. We then knocked out CXCL10 and ADRB2 in p53KO cells and confirmed that apoptosis was significantly reduced in the absence of CXCL10 or ADRB2. Genetic ablation of adrenergic signaling in vivo undermined response to anti-programmed cell death protein 1 (anti-PD1) compared to controls. Finally, our mouse model of OSCC showed that isoprenaline (β-adrenergic agonist) treatment in combination with PD-1 therapy significantly enhanced response to immunotherapy and improved survival. Our study provides new evidence that adrenergic signaling induces immune-mediated apoptosis and response to immunotherapy. Further studies are needed to assess the role of isoprenaline combined with anti-PD-1 therapy as a novel therapeutic regimen for patients with p53-deficient OSCC tumors. Citation Format: Deborah A. Silverman, Shamima Akhter, Tongxin Xie, Shajedul Islam, Simone Anfossi, Jennifer D. Covello, Frederico O. Gleber-Netto, Elien Doorduijn, Yunfei Wang, Emily L. Ashkin, Jeffrey N. Myers, George A. Calin, Patrick Hwu, Moran Amit. Adrenergic regulation of the anti-tumor immune response in p53-deficient tumors [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Innovating through Basic, Clinical, and Translational Research; 2023 Jul 7-8; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2023;29(18_Suppl):Abstract nr PO-076.

Stem cell competition driven by the Axin2-p53 axis controls brain size during murine development.

Cell competition acts as a quality-control mechanism that eliminates cells less fit than their neighbors to optimize organ development. Whether and how competitive interactions occur between neural progenitor cells (NPCs) in the developing brain remains unknown. Here, we show that endogenous cell competition occurs and intrinsically correlates with the Axin2 expression level during normal brain development. Induction of genetic mosaicism predisposes Axin2-deficient NPCs to behave as "losers" in mice and undergo apoptotic elimination, but homogeneous ablation of Axin2 does not promote cell death. Mechanistically, Axin2 suppresses the p53 signaling pathway at the post-transcriptional level to maintain cell fitness, and Axin2-deficient cell elimination requires p53-dependent signaling. Furthermore, mosaic Trp53 deletion confers a "winner" status to p53-deficient cells that outcompete their neighbors. Conditional loss of both Axin2 and Trp53 increases cortical area and thickness, suggesting that the Axin2-p53 axis may coordinate to survey cell fitness, regulate natural cell competition, and optimize brain size during neurodevelopment.

Hippo dictates signaling for cellular homeostasis and immune defense in Crassostrea hongkongensis hemocytes.

The Hippo signaling pathway is an evolutionarily conserved signaling cascade that plays a crucial role in regulating cell proliferation, differentiation, and apoptosis. It has been shown to be a key regulator of cell fate and cellular homeostasis in various immune processes. Despite its well-established functions in vertebrate immunity, its roles in marine invertebrate immunity remain poorly understood. Therefore, our present work provides fresh mechanistic insights into how the Hippo pathway orchestrates hemocytic functions in Crassostrea hongkongensis, with implications for studies on its major forms and modifications in animal evolution. The complete set of Hippo pathway genes, including SAV1, MOB1, LATS, YAP/TAZ, TEAD, and MST, were identified from the C. hongkongensis genome. Quantitative PCR assays were conducted to examine the mRNA expression levels of these genes in different tissues and the levels of these genes in hemocytes before and after bacterial challenges. The study also examined the crosstalk between the Hippo pathway and other immune pathways, such as the AP-1 and p53-dependent p21 signaling cascades. RNA interference was used to knock down MST and TEAD, and MST is a core orchestrator of non-canonical Hippo signaling, to investigate its impact on phagocytosis and bacterial clearance in hemocytes. The results demonstrated that members of the Hippo pathway were highly expressed in hemocytes, with their expression levels significantly increasing following bacterial challenges. Crosstalk between the Hippo pathway and other immune pathways triggered hemocytic apoptosis, which functioned similarly to the canonical Mst-Lats-Yap signaling pathway in Drosophila and mammals. Knocking down MST resulted in increased phagocytosis and boosted the efficiency of bacterial clearance in hemocytes, presumably due to mobilized antioxidant transcription by Nrf for maintaining immune homeostasis. This study provides novel insights into the regulatory mechanisms underlying the Hippo pathway in immune responses of C. hongkongensis hemocytes. The study highlights the importance of the Hippo pathway in maintaining immune homeostasis and orchestrating hemocytic functions in oysters. Moreover, this study demonstrates the divergence of the Hippo pathway's roles in marine invertebrate immunity from mammalian observations, indicating the need for further comparative studies across species. These findings have significant implications for future research aimed at elucidating the evolutionary trajectory and functional diversity of the Hippo signaling pathway in animal evolution.

Abstract 1289: The role of mutant p53-mediated mechanisms in modulating the tumor microenvironment and promoting lung metastasis

Abstract INTRODUCTION/METHODS: The multistep process of the tumor invasion-metastasis cascade is a major barrier to effective therapy. Mutations of the tumor suppressor p53 are frequently detected in esophageal squamous cell carcinoma (ESCC) cases, which in turn correlate with high metastatic rates. To understand the mutant p53-mediated mechanisms in promoting ESCC metastasis, we conducted RNA-Seq, ChIP-Seq and cytokine array on isogenic primary and metastatic tumor cells harvested from our mouse model of esophageal cancer, L2-Cre; LSL-Trp53R172H; Rosa26LSL-YFP, in which we have identified macrophage colony stimulating factor 1 (CSF1) to be a direct gene target of p53-R172H. We have targeted CSF1/CSF1R signaling to assess its pro-tumorigenic roles. In addition to studying tumor intrinsic mechanisms, we have established multiplex immunofluorescence and flow cytometry approaches to characterize the changes in tumor microenvironment (TME). Overall, this study aims to investigate the role and mediators of CSF1 signaling through its cognate receptor CSF1R by which missense p53 mutations can promote lung metastasis. RESULTS/DISCUSSION: We demonstrate that metastatic ESCC has increased Csf1 expression compared to primary tumors and this is dependent upon p53 mutation status, which is reinforced by TCGA data and patient-derived tissue microarrays (TMAs). The TMA analysis also suggested that CSF1 expression is higher in tumors that are poorly differentiated. Furthermore, through the proximity ligation assay (PLA), we have identified that bromodomain and extra-terminal (BET) protein BRD4, which binds to acetylated histones to regulate transcription interacts with p53-R172H at higher levels compared to wild-type p53 to induce Csf1 expression. We show that the BRD4/CSF1/CSF1R signaling axis fosters tumor invasion, subcutaneous tumor growth and metastatic burden in lungs in a mutant p53 background. In accordance, targeting CSF1/CSF1R signaling reduces phosphorylation of STAT3 in the metastatic tumors, which suggests a mechanism for increased invasiveness and metastasis. In parallel, upon inhibiting this axis, we have identified decreased infiltration of F4/80+CD163+ and F4/80+CD206+ M2-polarized macrophages, as well as CD31+ endothelial cells, and increased number of CD3+CD8+ cytotoxic T-cells at the metastatic tumor sites, indicating that the CSF1/CSF1R pathway plays a critical role in shaping the pro-metastatic and immunosuppressive TME. Finally, analysis of squamous cell carcinoma (SCC) datasets reveals that specific p53 mutations are associated with differential survival rates and CSF1 expression. CONCLUSION: We have demonstrated novel roles and mechanisms of mutant p53-dependent CSF1-CSF1R signaling pathway in fostering ESCC tumor invasion and lung metastasis that may be applicable to other SCCs. We believe this can open up new avenues for therapeutic applications. Citation Format: Gizem Efe, Katherine M. Cunningham, Qiaosi Tang, Kensuke Sugiura, Karen Dunbar, Kensuke Suzuki, Lois Resnick-Silverman, Alison M. Taylor, James J. Manfredi, Carol L. Prives, Anil K. Rustgi. The role of mutant p53-mediated mechanisms in modulating the tumor microenvironment and promoting lung metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1289.

The long noncoding RNA Meg3 mediates TLR4-induced inflammation in experimental obstructive nephropathy.

Kidney inflammation contributes to the progression of chronic kidney disease (CKD). Modulation of Toll-like receptor 4 (TLR4) signaling is a potential therapeutic strategy for this pathology, but the regulatory mechanisms of TLR4 signaling in kidney tubular inflammation remains unclear. Here, we demonstrated that tubule-specific deletion of TLR4 in mice conferred protection against obstruction-induced kidney injury, with reduction in inflammatory cytokine production, macrophage infiltration and kidney fibrosis. Transcriptome analysis revealed a marked down-regulation of long noncoding RNA (lncRNA) Meg3 in the obstructed kidney from tubule-specific TLR4 knockout mice compared with wild-type control. Meg3 was also induced by lipopolysaccharide in tubular epithelial cells via a p53-dependent signaling pathway. Silencing of Meg3 suppressed LPS-induced cytokine production of CCL-2 and CXCL-2 and the activation of p38 MAPK pathway in vitro and ameliorated kidney fibrosis in mice with obstructive nephropathy. Together, these findings identify a proinflammatory role of lncRNA Meg3 in CKD and suggest a novel regulatory pathway in TLR4-driven inflammatory responses in tubular epithelial cells.

Organophosphorus Flame Retardant TCPP Induces Cellular Senescence in Normal Human Skin Keratinocytes: Implication for Skin Aging.

Tris (1-chloro-2-propyl) phosphate (TCPP) is one of the most frequently detected organophosphorus flames in the environment. Continuous daily exposure to TCPP may harm human skin. However, little is known about the adverse effects of TCPP on human skin. In this study, we first evaluated the detrimental effects and tried to uncover the underlying mechanisms of TCPP on human skin keratinocytes (HaCaT) after 24 h exposure. We found that TCPP caused a concentration-dependent decrease in HaCaT cell viability after exposure to 1.56-400 μg/mL for 24 h, with an IC50 of 275 μg/mL. TCPP also promoted the generation of intracellular reactive oxygen species (ROS) and triggered DNA damage, evidenced by an increase of phosphorylated histone H2A.X (γH2A.X) in the nucleus. Furthermore, the cell cycle was arrested at the G1 phase at 100 μg/mL by upregulation of the mRNA expression of p53 and p21 and downregulation of cyclin D1 and CDK4 expression. Additionally, both the senescence-associated-β-galactosidase activity and related proinflammatory cytokine IL-1β and IL-6 were elevated, indicating that TCPP exposure caused cellular senescence may be through the p53-dependent DNA damage signal pathway in HaCaT cells. Taken together, our data suggest that flame-retardant exposure may be a key precipitating factor for human skin aging.

A Low-Phenylalanine-Containing Whey Protein Hydrolysate Stimulates Osteogenic Activity through the Activation of p38/Runx2 Signaling in Osteoblast Cells.

A phenylalanine (Phe)-restricted diet is indispensable for individuals suffering from phenylketonuria (PKU). Our previous study reported a low-Phe-containing whey protein hydrolysate (LPH) prepared from a selected whey protein hydrolysate (TA2H). This study aimed to investigate the osteogenic activity of LPH and TA2H in MC3T3-E1 preosteoblast cells and explore the underlying mechanism. Results showed that the treatment of TA2H and LPH (at the final concentrations of 100–1000 μg/mL) had a stimulatory effect on the proliferation, differentiation, and mineralization of MC3T3-E1 cells. The LPH of 1000 μg/mL significantly increased cell proliferation (2.15- ± 0.11-fold) and alkaline phosphatase activity (1.22- ± 0.07-fold), promoted the protein and mRNA levels of runt-related transcription factor 2 (Runx2, 2.50- ± 0.14-fold and 2.97- ± 0.23-fold, respectively), enhanced the expression of differentiation biomarkers (type-I collagen, osteocalcin, and osteopontin), increased calcium deposition (1.56- ± 0.08-fold), and upregulated the ratio of osteoprotegerin/receptor activator of nuclear factor-κB ligand. The exploration of signaling pathways indicated that the activated p38-dependent Runx2 signaling contributed to the LPH-induced osteogenesis. These results provided evidence, for the first time, that a prepared low-Phe whey protein hydrolysate positively modulated the activity of osteoblasts through the p38/Runx2 pathway, thereby providing a new osteoinductive protein substitute to make functional PKU food.

Abstract 2421: Elucidation of CSF1 autocrine signaling mediated by mutant p53 in metastatic esophageal carcinoma

Abstract Introduction: Esophageal cancer is a highly aggressive cancer and is the 6th leading cause of cancer mortality worldwide, with esophageal squamous cell carcinoma (ESCC) being the most prevalent subtype. Mutations in the tumor suppressor TP53 (human homolog of mouse Trp53), which encodes p53, are detected frequently in ESCC that in turn correlate with poor survival and high metastatic rates. We have conducted RNA-Seq and cytokine array on primary and metastatic tumor cells harvested from our mouse models with Trp53R172H mutation. We have identified colony stimulating factor 1 (CSF1) to be upregulated. Our data suggest the existence of CSF1-CSF1R autocrine signaling, and its functional relationship to the promotion of metastasis in ESCC, recognizing that CSF1 is known to be critical in the polarization of macrophages to M2-like tumor associated macrophages. Our goal is to investigate the role and mediators of CSF1/CSF1R signaling by which missense TP53 mutations can promote invasion and lung metastasis in ESCC. Methods: We used L2-Cre; LSL-Trp53R172H; Rosa26LSL-YFP mice and 2D/3D cell culture systems to model ESCC. To assess the functional role of CSF1, we established a CRISPR approach to knockout Csf1in our cells introducing ribonucleoprotein (RNP) Cas9/gRNA complex via nucleofection and overexpressed Csf1. We also utilized a small molecule inhibitor of CSF1R, GW2580. We conducted TCGA analysis on the association of distinct TP53 mutations with ESCC prognosis and Csf1 expression, as well as Csf1 expression in metastatic tumors. Based on this, we modeled ESCC by introducing various DNA contact and conformational p53 mutations into esophageal cells with wildtype p53 via base editing. Results: TCGA data and our models demonstrate that metastatic ESCCs have increased Csf1 expression compared to primary tumors and it is dependent upon p53 mutation status. In addition, analysis of ESCC patients’ datasets reveal that specific p53 mutations are associated with markedly differential overall survival rates and Csf1 expression. We show that Csf1 function is related to tumor cell invasion, primary tumor growth and metastatic tumor burden in a mutant p53 background. We have identified both bromodomain and extra-terminal motif (BET) protein BRD4 and transcription factor nuclear factor kappa B (NF-κB) as proteins that interact with Trp53R172H at higher levels compared to wildtype p53 to induce Csf1 expression. In addition, inhibition of CSF1R with small molecule GW2580 downregulated JAK-STAT3 pathway mediating epithelial-mesenchymal transition (EMT) mechanisms that would contribute to increased invasiveness and metastasis. Conclusion: We have demonstrated novel roles and mechanisms of mutant p53-dependent CSF1-CSF1R autocrine signaling and its mediators in promoting lung metastasis from ESCC that may be applicable to other squamous cell cancers. We believe this mechanism can be targeted therapeutically. Citation Format: Gizem Efe, Qiaosi Tang, Ricardo Cruz-Acuna, Kensuke Suzuki, Kensuke Sugiura, Joel Gabre, Uma Sachdeva, Wei Gu, Carol L. Prives, Anil K. Rustgi. Elucidation of CSF1 autocrine signaling mediated by mutant p53 in metastatic esophageal carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2421.

Antisense Oligonucleotide-Based Therapy on miR-181a-5p Alleviates Cartilage Degradation of Temporomandibular Joint Osteoarthritis via Promoting SIRT1.

Temporomandibular joint osteoarthritis (TMJOA) condylar cartilage degeneration and abnormal subchondral bone pathological remodeling induce pain and joint dysfunction, and cartilage degeneration is considered irreversible. Very few therapeutic approaches are administrated in practice. Nucleotides have demonstrated considerable potential as a next-generation medication, and they have been applied in several models of osteoarthritis. There is a need to establish an effective protocol for TMJOA gene therapy. In the current study unilateral anterior crossbite (UAC) surgery was used to simulate mechanical stress-induced TMJOA in mice. Degeneration of condylar cartilage and destruction of subchondral bone were observed in damaged joints, and miR-181a-5p was elevated in chondrocytes. Intra-articular injection of miR-181a-5p antisense oligonucleotide (ASO) could reduce the cartilage damage and alleviate UAC-induced TMJOA progression, but it did not restore injured subchondral bone. Mechanically, miR-181a-5p evidently targeted the 3’ untranslated region of Sirt1 directly, resulting in inhibition of silent information regulator 1 expression and promoting apoptosis by elevating p53-dependent signaling, indicating that miR181a-5p ASO promoted chondrocyte survival. The present study suggests that ASO-based gene therapy may be an effective TMJOA treatment.

Novel Targeting of DNA Methyltransferase Activity Inhibits Ewing Sarcoma Cell Proliferation and Enhances Tumor Cell Sensitivity to DNA Damaging Drugs by Activating the DNA Damage Response.

DNA methylation is an important component of the epigenetic machinery that regulates the malignancy of Ewing sarcoma (EWS), the second most common primary bone tumor in children and adolescents. Coordination of DNA methylation and DNA replication is critical for maintaining epigenetic programming and the DNMT1 enzyme has been demonstrated to have an important role in both maintaining the epigenome and controlling cell cycle. Here, we showed that the novel nonnucleoside DNMT inhibitor (DNMTi) MC3343 induces a specific depletion of DNMT1 and affects EWS tumor proliferation through a mechanism that is independent on DNA methylation. Depletion of DNMT1 causes perturbation of the cell cycle, with an accumulation of cells in the G1 phase, and DNA damage, as revealed by the induction of γH2AX foci. These effects elicited activation of p53-dependent signaling and apoptosis in p53wt cells, while in p53 mutated cells, persistent micronuclei and increased DNA instability was observed. Treatment with MC3343 potentiates the efficacy of DNA damaging agents such as doxorubicin and PARP-inhibitors (PARPi). This effect correlates with increased DNA damage and synergistic tumor cytotoxicity, supporting the use of the DNMTi MC3343 as an adjuvant agent in treating EWS.

Heterogeneous nuclear ribonucleoprotein K promotes the progression of lung cancer by inhibiting the p53-dependent signaling pathway.

BackgroundHeterogeneous nuclear ribonucleoprotein K (hnRNPK) is a nucleic acid‐binding protein. Reportedly, hnRNPK is overexpressed in many human tumors, and such overexpression is associated with poor prognosis, implicating the role of hnRNPK as an oncogene during tumorigenesis. In this study, hnRNPK expression in lung cancer tissues was investigated.MethodsBriefly, hnRNPK was knocked down in lung cancer cell lines, and effects of knockdown on the cell proliferation, migration, and cell cycle were assessed using a cell counting kit‐8 (CCK‐8) assay, colony formation assay, transwell assay and flow cytometry. The effects of hnRNPK knockdown on the p53‐dependent signaling pathway were examined using western blotting. Finally, the effect of hnRNPK knockdown on tumor growth was verified in vivo using a lung cancer xenograft mouse model.ResultshnRNPK knockdown inhibited the cell proliferation, migration and cell cycle. In addition to phenotypic changes, hnRNPK knockdown upregulated expressions of pCHK1, pCHK2, and p53，p21，cyclin D1, thereby mediating the DNA damage response (DDR). The regulatory function of hnRNPK during p53/p21/cyclin D1 signaling in hnRNPK‐knockdown A549 cells was confirmed by suppressed the protein expression of associated signaling pathways, which inhibited DDR.ConclusionhnRNPK plays a crucial role in the progression of lung cancer, ultimately affecting survival rate. Inhibition of progression of lung cancer cells induced by hnRNPK‐knockdown is dependent on activation of p53 by the p53/p21/cyclin D1 pathway.

Phosphoproteomic analysis of thrombin- and p38 MAPK-regulated signaling networks in endothelial cells

Endothelial dysfunction is a hallmark of inflammation and is mediated by inflammatory factors that signal through G protein–coupled receptors including protease-activated receptor-1 (PAR1). PAR1, a receptor for thrombin, signals via the small GTPase RhoA and myosin light chain intermediates to facilitate endothelial barrier permeability. PAR1 also induces endothelial barrier disruption through a p38 mitogen-activated protein kinase–dependent pathway, which does not integrate into the RhoA/MLC pathway; however, the PAR1-p38 signaling pathways that promote endothelial dysfunction remain poorly defined. To identify effectors of this pathway, we performed a global phosphoproteome analysis of thrombin signaling regulated by p38 in human cultured endothelial cells using multiplexed quantitative mass spectrometry. We identified 5491 unique phosphopeptides and 2317 phosphoproteins, four distinct dynamic phosphoproteome profiles of thrombin-p38 signaling, and an enrichment of biological functions associated with endothelial dysfunction, including modulators of endothelial barrier disruption and a subset of kinases predicted to regulate p38-dependent thrombin signaling. Using available antibodies to detect identified phosphosites of key p38-regulated proteins, we discovered that inhibition of p38 activity and siRNA-targeted depletion of the p38α isoform increased basal phosphorylation of extracellular signal–regulated protein kinase 1/2, resulting in amplified thrombin-stimulated extracellular signal–regulated protein kinase 1/2 phosphorylation that was dependent on PAR1. We also discovered a role for p38 in the phosphorylation of α-catenin, a component of adherens junctions, suggesting that this phosphorylation may function as an important regulatory process. Taken together, these studies define a rich array of thrombin- and p38-regulated candidate proteins that may serve important roles in endothelial dysfunction.

Osthole-Mediated Inhibition of Neurotoxicity Induced by Ropivacaine via Amplification of the Cyclic Adenosine Monophosphate Signaling Pathway.

BackgroundRopivacaine is widely used for clinical anesthesia and postoperative analgesia. However, the neurotoxicity induced by ropivacaine in a concentration- and duration-dependent manner, and it is difficult to prevent neurotoxicity. Osthole inhibits phosphodiesterase-4 activity by binding to its catalytic site to prevent cAMP hydrolysis. The aim of this present study is to explore the precise molecular mechanism of osthole-mediated inhibition of neurotoxicity induced by ropivacaine.Methods:SH-SY5Y cell viability and apoptosis were measured in different concentration and duration. Protein concentration was determined in each signaling pathway. The molecular mechanism of osthole-mediated inhibition of ropivacaine-caused neurotoxicity was evaluated.ResultsThe study demonstrated that osthole inhibits SH-SY5Y cells neurotoxicity in a duration- and concentration-dependent manner. Moreover, ropivacaine significantly increased the expression of caspase-3 by promoting the phosphorylation of p38. Osthole-induced upregulation of cAMP activated cAMP-dependent signaling pathway, sequentially leading to elevated cyclic nucleotide response element-binding protein levels, which inhibits P38-dependent signaling and decreases apoptosis of SH-SY5Y.ConclusionsThis study display the evidence confirmed the molecular mechanism by which osthole amplification of cAMP-dependent signaling pathway, and overexpression of cyclic nucleotide response element-binding protein inhibits P38-dependent signaling and decreases ropivacaine-induced SH-SY5Y apoptosis.

RNA sequencing of glioblastoma tissue slice cultures reveals the effects of treatment at the transcriptional level.

One of the major challenges in cancer research is finding models that closely resemble tumors within patients. Human tissue slice cultures are a promising approach to provide a model of the patient's tumor biology ex vivo. Recently, it was shown that these slices can be successfully analyzed by whole transcriptome sequencing as well as automated histochemistry, increasing their usability as preclinical model. Glioblastoma multiforme (GBM) is a highly malignant brain tumor with poor prognosis and little is known about its genetic background and heterogeneity regarding therapy success. In this study, tissue from the tumors of 25 patients with primary GBM was processed into slice cultures and treated with standard therapy (irradiation and temozolomide). Total RNA sequencing and automated histochemistry were performed to enable analysis of treatment effects at a transcriptional and histological level. Slice cultures from long‐term survivors (overall survival [OS] > 24 months) exhibited more apoptosis than cultures from patients with shorter OS. Proliferation within these slices was slightly increased in contrast to other groups, but not significantly. Among all samples, 58 protein‐coding genes were upregulated and 32 downregulated in treated vs. untreated slice cultures. In general, an upregulation of DNA damage‐related and cell cycle checkpoint genes as well as enrichment of genotoxicity pathways and p53‐dependent signaling was found after treatment. Overall, the current study reproduces knowledge from former studies regarding the feasibility of transcriptomic analyses and automated histology in tissue slice cultures. We further demonstrate that the experimental data merge with the clinical follow‐up of the patients, which improves the applicability of our model system.

The Role of p53-Dependent Signaling Pathways in Survival and Death of Neurons and Glial Cells after Peripheral Nerve Injury

The Role of p53-Dependent Signaling Pathways in Survival and Death of Neurons and Glial Cells after Peripheral Nerve Injury

γ-Glutamyl transpeptidase–activated indole-quinolinium based cyanine as a fluorescence turn-on nucleolus-targeting probe for cancer cell detection and inhibition

A nucleolus as a prominent sub-nuclear, membraneless organelle plays a crucial role in ribosome biogenesis, which is in the major metabolic demand in a proliferating cell, especially in aggressive malignancies. We develop a γ-glutamyltranspeptidase (GGT)-activatable indole-quinolinium (QI) based cyanine consisting of a novel tripeptide fragment (Pro-Gly-Glu), namely QI-PG-Glu as a turn-on red fluorescent probe for the rapid detection of GGT-overexpressed A549 cancer cells in vivo. QI-PG-Glu can be triggered by GGT to rapidly release an activated fluorophore, namely HQI, in two steps including the cleavage of the γ-glutamyl group recognized by GGT and the rapid self-driven cyclization of the Pro-Gly linker. HQI exhibits dramatically red fluorescence upon binding to rRNA for imaging of nucleolus in live A549 cells. HQI also intervenes in rRNA biogenesis by declining the RNA Polymerase I transcription, thus resulting in cell apoptosis via a p53 dependent signaling pathway. Our findings may provide an alternative avenue to develop multifunctional cancer cell-specific nucleolus-targeting fluorescent probes with potential anti-cancer effects.