Overexpression Of c-Jun Research Articles

Abstract 1548: Potent activity of CAR T cells targeting the oncofetal protein GPC2 engineered to recognize low antigen density in neuroblastoma

Abstract Background: CAR T-cells targeting solid tumor antigens have not yet demonstrated similar success as seen for CD19, BCMA and CD22 in B-cell malignancies. Truly tumor-specific antigens are rare, yet pediatric solid tumors manifest stalled fetal developmental programs and often overexpress oncofetal antigens, normally restricted to prenatal tissues. Antigen density is a major factor determining CAR potency and whether such nonmutant antigens can be effectively and safely targeted remains unknown. Here, we engineered CAR T-cells targeting GPC2 to recognize clinically relevant antigen thresholds and assess safety. Methods: Expression of GPC2 was assessed during organ development (Cardoso-Moreira et al. 2019), by IHC in prenatal, infant and pediatric brain and in ssRNAseq datasets of fetal (La Manno et. al 2016) and adult brain (Allen Brain Atlas). To measure the molecules/cell of GPC2 and other immunotherapy-relevant targets on bone-marrow infiltrating neuroblastoma (NB) cells of high-risk patients, we developed a Flow Cytometric quantification assay and engineered GPC2-CAR T-cells to recognize relevant antigen thresholds. To assess binding specificity towards GPC2, a membrane proteome array encompassing >5,300 human proteins was utilized. Taking advantage of the murine cross-reactivity of our lead GPC2-CAR, toxicity was assessed in a relevant xenograft model. Results: During organ development, we found GPC2 expression restricted to the developing brain and a gradual decrease until silenced after birth. IHC of prenatal brain revealed an inverse correlation between GPC2 and gestational age (r = -0.775, p = 0.008) and very low staining in infant and pediatric brain (H-scores <100). Analysis of fetal midbrain ssRNAseq data spatializes GPC2 to the neuronal progenitor compartment, a correlation absent in adult brain. We measured a mean of ~4782 GPC2 molecules/cell on patient's metastatic NB cells, a significantly lower number than on NB cell lines (p=0.0496). We found KYMRIAH-based CAR T-cell constructs unable to control tumor due to subthreshold antigen density. Sequential modifications to the CAR architecture, such as CD28-derived hinge-transmembrane and signaling domains domains resulted in a potent lead GPC2-CAR capable of impressive tumor regression in three xenograft models of NB (metastatic, orthotopic and patient-derived xenograft). Potency of our lead GPC2-CAR could be further enhanced by overexpression of c-Jun, mediating long-term complete responses. We found no clinically relevant off-target binding to other membrane proteins and no clinical or histological evidence of toxicity. Conclusion: Our results demonstrate safety and efficacy of a lead GPC2-CAR and illuminate oncofetal antigens as a promising class of targets for CAR T cell therapy of solid tumors. This work establishes a strong basis for testing GPC2 CAR T-cells in early phase clinical trials. Citation Format: Sabine Heitzeneder, Kristopher R. Bosse, Zhongyu Zhu, Dontcho Jelev, Shaurya Dhingra, Robbie Majzner, Elena Sotillo-Pineiro, Samantha Buongervino, Peng Xu, Jing Huang, Alberto Delaidelli, Martin Hasselblatt, Kevin Parker, Hima Anbunathan, Anya Alag, Jennifer Hwang, Min Huang, Dorota D. Klysz, Johanna L. Theruvath, Jose G. Vilches-Moure, Ansuman T. Satpathy, Poul H. Sorensen, Dimiter S. Dimitrov, John M. Maris, Crystal L. Mackall. Potent activity of CAR T cells targeting the oncofetal protein GPC2 engineered to recognize low antigen density in neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1548.

Induction of the Proinflammatory Chemokine Interleukin-8 Is Regulated by Integrated Stress Response and AP-1 Family Proteins Activated during Coronavirus Infection.

Infection induces the production of proinflammatory cytokines and chemokines such as interleukin-8 (IL-8) and IL-6. Although they facilitate local antiviral immunity, their excessive release leads to life-threatening cytokine release syndrome, exemplified by the severe cases of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. In this study, we investigated the roles of the integrated stress response (ISR) and activator protein-1 (AP-1) family proteins in regulating coronavirus-induced IL-8 and IL-6 upregulation. The mRNA expression of IL-8 and IL-6 was significantly induced in cells infected with infectious bronchitis virus (IBV), a gammacoronavirus, and porcine epidemic diarrhea virus, an alphacoronavirus. Overexpression of a constitutively active phosphomimetic mutant of eukaryotic translation initiation factor 2α (eIF2α), chemical inhibition of its dephosphorylation, or overexpression of its upstream double-stranded RNA-dependent protein kinase (PKR) significantly enhanced IL-8 mRNA expression in IBV-infected cells. Overexpression of the AP-1 protein cJUN or its upstream kinase also increased the IBV-induced IL-8 mRNA expression, which was synergistically enhanced by overexpression of cFOS. Taken together, this study demonstrated the important regulatory roles of ISR and AP-1 proteins in IL-8 production during coronavirus infection, highlighting the complex interactions between cellular stress pathways and the innate immune response.

The role of c-Jun in enhancing TCR-T function in treatment of Hepatocellular Carcinoma

Abstract Hepatocellular carcinoma (HCC) is responsible for most of primary liver cancer cases and is the sixth most common cancer in the world with a general 5 year survival rate of 18% due to lack of effective therapy. Adoptive T-cell therapy (ACT) with T-cell receptor–engineered T-cell (TCR-T) against HCC-associated antigen a-fetoprotein (AFP), can redirect human T cells to recognize and kill HCC tumor cells. However, the clinical efficacy of TCR-T therapy is dependent on the proper expansion, in vivo persistence of T cells that is markedly reduced due to exhaustion-associated dysfunction, and activation induced cell death. To improve persistence of TCR-T cells without modifying TCR affinity, we engineered TCR-T cells to overexpress c-Jun, an AP-1 transcription factor associated with T cell development and activation. In this study, we showed that TCR-T cells overexpressing c-Jun have enhanced expansion capacity in culture, improved cytokine production against HepG2 tumor cells without altering their cytotoxic potential. Interestingly TCR-T cells overexpressing c-Jun showed resistance to activation induced cell death with decreased expression of inhibitory receptors after stimulation with HepG2 tumor cells. Thus, c-Jun overexpression could be a potential therapeutic agent to enhance the anti-tumor efficacy of TCR-T cells against HCC.

Upregulation of Extracellular Vesicles-Encapsulated miR-132 Released From Mesenchymal Stem Cells Attenuates Ischemic Neuronal Injury by Inhibiting Smad2/c-jun Pathway via Acvr2b Suppression.

Ischemic cerebrovascular disease is a significant and common public health issue worldwide. The emerging roles of mesenchymal stem cells (MSCs)-derived extracellular vesicles (EVs) in ischemic neuronal injury continue to be investigated. The current study aimed to investigate the role of EV-derived miR-132 from MSCs in ischemic neuronal injury. EVs were initially isolated from bone MSCs (BMSCs) and subsequently evaluated. A middle cerebral artery occlusion (MCAO) mouse model was constructed with the neurological function evaluated through a series of neurological scores, a pole test, and a foot fault test. Histopathological changes, neuron viability, and apoptosis, as well as cerebral infarction, were detected by hematoxylin and eosin (HE) staining and 2,3,5-triphenyltetrazolium hydrochloride (TTC) staining. The targeting relationship between microRNA (miR)-132 and Activin receptor type IIB (Acvr2b) was further confirmed based on dual-luciferase reporter gene assay results. Loss- and gain-of-function assays were conducted to elucidate the role of miR-132, EV-derived miR-132, Acvr2b, and Smad2 in oxygen-glucose deprivation (OGD)-treated neurons, and in mice models. Neuronal cell viability and apoptosis were evaluated via Cell Counting kit-8 (CCK-8) and flow cytometry. Our results indicated that Acvr2b was highly expressed, while miR-132 was poorly expressed in the MCAO mice and OGD-treated neurons. Acvr2b silencing or upregulation of miR-132 led to an elevation in neuronal activity, decreased neuronal apoptosis, reduced expression of Bax, and cleaved-caspase 3, as well as increased Bcl-2 expression. Acvr2b expression was targeted and inhibited by miR-132. EV-derived Acvr2b promoted activation of phosphorylated-Smad2 (p-Smad2)/c-jun signaling pathway, ultimately inducing neuronal injury. Our study provides evidence demonstrating that the overexpression of c-jun inhibits the protective role of MSCs-derived EV-miR-132 in neuronal injury. Upregulation of EV-derived miR-132 released from MSCs attenuates ischemic neuronal injury by inhibiting Smad2/c-jun pathways via the suppression of Acvr2b.

Nerve Growth Factor (NGF) modulates in vitro induced myofibroblasts by highlighting a differential protein signature

We previously described the profibrogenic effect of NGF on conjunctival Fibroblasts (FBs) and its ability to trigger apoptosis in TGFβ1-induced myofibroblasts (myoFBs). Herein, cell apoptosis/signalling, cytokines’ signature in conditioned media and inflammatory as well as angiogenic pathway were investigated. Experimental myoFBs were exposed to NGF (0.1–100 ng/mL), at defined time-point for confocal and biomolecular analysis. Cells were analysed for apoptotic and cell signalling activation in cell extracts and for some inflammatory and proinflammatory/angiogenic factors’ activations. NGF triggered cJun overexpression and phospho-p65-NFkB nuclear translocation. A decreased Bcl2:Bax ratio and a significant expression of smad7 were confirmed in early AnnexinV-positive myoFBs. A specific protein signature characterised the conditioned media: a dose dependent decrease occurred for IL8, IL6 while a selective increase was observed for VEGF and cyr61 (protein/mRNA). TIMP1 levels were unaffected. Herein, NGF modulation of smad7, the specific IL8 and IL6 as well as VEGF and cyr61 modulation deserve more attention as opening to alternative approaches to counteract fibrosis.

An in vivo study of the effect of c-Jun on intervertebral disc degeneration in rats

ABSTRACT Intervertebral disc degeneration (IDD) has been well-recognized as one of the causes of vast lower back pain. The objective of the current study intends to elucidate the influence and regulatory molecular mechanisms of c-Jun on IDD. This study established an IDD model of Sprague-Dawley (SD) rats by needle puncture. An LV5-c-Jun lentiviral vector was constructed and injected into rats’ intervertebral disc (IVD) tissue to increase the c-Jun expression following the establishment of modeling. The pathological changes of IVD tissue structure and collagen fibers were visualized following the processes of hematoxylin-eosin (HE) staining method and transmission electron microscopy. Real-time PCR, western blot, immunohistochemistry, and ELISA assays were performed to detect the expression levels of TGF-β, TIMP-3, COL2A1, and inflammatory cytokines. The collagen fibers were arranged in parallel and the surface was smooth after c-Jun overexpression, whereas the collagen fibers in the control group were disorderly arranged with a rough surface. The findings indicated that c-Jun was responsible for upregulating expression levels of TGF-β, TIMP-3, and COL2A1 in the mRNA and proteins, but simultaneously downregulating expression levels of inflammatory factors IL-1β, IL-17, IL-6, and TNF-α. c-Jun overexpression produced a positive effect on IDD, inhibited inflammatory response in vivo, and might delay the degeneration of IVD. Thus, c-Jun may act as a novel potential agent in treating IDD.

Loss of Fbxw7 triggers mammary tumorigenesis associated with E2F/c-Myc activation and Trp53 mutation

Fbw7 is a tumor suppressor that regulates the degradation of oncogenic substrates such as c-Jun, c-Myc, Notch1 intracellular domain (ICD), and cyclin E by functioning as the substrate recognition protein in the Skp1-Cullin-F-box (SCF) ubiquitin ligase complex. Consequently, low expression or loss of FBXW7 in breast cancer has been hypothesized to result in the accumulation of oncogenic transcription factors that are master regulators of proliferation, apoptosis, and ultimately transformation. Despite this, the direct effect of Fbw7 loss on mammary gland morphology and tumorigenesis has not been examined. Here, we demonstrate that conditional deletion of Fbxw7 in murine mammary tissue initiates breast tumor development and also results in lactation and involution defects. Further, while Fbxw7 loss results in the overexpression of Notch1-ICD, c-Jun, cyclin E, and c-Myc, the downstream transcription factor pathways associated with c-Myc and cyclin E are the most dysregulated, including at the single-cell level. These pathways are dysregulated early after Fbxw7 loss, and their sustained loss results in tumorigenesis and reinforced c-Myc and cyclin E-E2F pathway disruption. We also find that loss of Fbxw7 is linked to the acquisition of Trp53 mutations, similar to the mutational spectrum observed in patients. Our results demonstrate that the loss of Fbxw7 promotes the acquisition of Trp53 mutations and that the two cooperate in breast tumor development. Targeting c-Myc, E2F, or p53 may therefore be a beneficial treatment strategy for FBXW7-altered breast cancer patients.

The c-Jun signaling pathway has a protective effect on nucleus pulposus cells in patients with intervertebral disc degeneration.

Among a range of diverse clinical symptoms, intervertebral disc degeneration (IDD) contributes mostly to the onset of lower back pain. The present study aimed to investigate the effects of c-Jun on nucleus pulposus (NP) cells of IDD and its regulation on molecular mechanisms. Intervertebral disc (IVD) tissues were collected from patients suffering from IDD disease, and NP cells were subsequently isolated and cultured. By overexpressing c-Jun in NP cells, expression levels of mRNAs and proteins of IDD-related genes and inflammatory cytokines were subjected to reverse transcription-quantitative PCR, western blot and ELISA assays. Additional transforming growth factor-β (TGF-β) antibodies were administrated to suppress the function of TGF-β. Cell proliferation and apoptosis were determined via Cell Counting Kit-8 and TUNEL assays, respectively. The results demonstrated that the overexpression of c-Jun robustly upregulated both mRNA and protein expression of TGF-β, TIMP metallopeptidase inhibitor 3, aggrecan and collagen type II alpha 1 chain and simultaneously downregulated the expression of the inflammatory cytokines TNF-α, interleukin (IL)-1β, IL-6 and IL-17. Furthermore, following c-Jun overexpression, survival rates of NP cells were increased while apoptosis rates were decreased. However, the addition of a TGF-β antibody significantly promoted apoptosis and restricted cell survival, which differed from the results of the c-Jun overexpression group. The present study hypothesized therefore that c-Jun may positively regulate TGF-β expression within NP cells of IDD, which could promote the proliferation of IDD-NP cells and accelerate cell viability via reducing apoptosis and the inflammatory response.

MiR-139 protects against oxygen-glucose deprivation/reoxygenation (OGD/R)-induced nerve injury through targeting c-Jun to inhibit NLRP3 inflammasome activation

BackgroundCerebral ischemia/reperfusion (I/R) injury after ischemic stroke is usually accompanied with the activation of inflammasome which seriously impairs neurological function. MiR-139 has been reported to be associated with inflammatory regulation in multiple diseases. However, its effect and mechanism on inflammation regulation after cerebral I/R injury are still poorly understood. MethodsAn in vitro model of cerebral I/R injury was constructed with oxygen-glucose deprivation/reoxygenation (OGD/R) treatment. TargetScan bioinformatics analysis and dual luciferase reporter assay were utilized to confirm the targeted relationship between miR-139 and c-Jun. Cell pyroptosis was verified by flow cytometry and Caspase-1 Detection Kit. qRT-PCR assay was performed to detect the expression levels of miR-139, c-Jun, NLRP3 and ASC. Western blotting was applied to measure the protein levels of c-Jun and pyroptosis-related markers NLRP3, ASC, caspase-1, GSDMDNterm. The ELISA assay was applied to measure the release of IL-1β, IL-18 and LDH. ResultsMiR-139 was significantly downregulated whereas c-Jun was obviously upregulated after OGD/R treatment. TargetScan analysis predicted that c-Jun was a potential target of miR-139, which was verified by the dual-luciferase reporter assay. Also, overexpression of miR-139 repressed c-Jun expression. Furthermore, miR-139 inhibited OGD/R-induced cell pyroptosis and the upregulation of NLRP3, caspase-1, ASC, GSDMDNterm, and the release of IL-1β, IL-18 and LDH, while miR-139 inhibition exerted the opposite effects. However, overexpression of c-Jun aggravated OGD/R-induced nerve injury and partly abolished the neuroprotective effect of miR-139. ConclusionUpregulation of miR-139 exerted neuroprotection against OGD/R-induced nerve injury by negatively regulating c-Jun/NLRP3 inflammasome signaling. This study offered insights for providing potential therapeutic targets for treating cerebral I/R injury.

KIAA1429 regulates cell proliferation by targeting c-Jun messenger RNA directly in gastric cancer.

N6-methyladenosine (m6A) modification regulatory proteins are involved in the development of many types of cancer. KIAA1429 serves as a scaffold in bridging the catalytic core components of the m6A methyltransferase complex. The role of KIAA1429 in gastric cancer and its related mechanism has not been reported upon. The expression of KIAA1429 was detected in human gastric cancer tissues and cell lines by quantitative real-time polymerase chain reaction and western blot. The effects of KIAA1429 on gastric cancer proliferation were evaluated by cell counting kit assays, colony formation assays, flow cytometry assay, and in vivo experiments withnude mice.And messenger RNA (mRNA) high-throughput sequencing, RNA immunoprecipitation assay (RIP), luciferase assay, and a rescue experiment were used to identify the relationship between KIAA1429 and its specific targeted gene, c-Jun. We found that KIAA1429 was upregulated in gastric cancer tissues, and expressed lower in adjacent tissues. The upregulated KIAA1429 promoted proliferation and downregulated KIAA1429 was proved to inhibit proliferation of gastric cancer in vitro and in vivo. Then, we identified the potential KIAA1429 regulating gene as c-Jun by mRNAs high-throughput sequencing and RIP assay. By luciferase assay, we verified that KIAA1429 regulated the expression of c-Jun inan m6A-independent manner. Finally, the overexpression of c-Jun rescued the inhibition of proliferation caused by KIAA1429 knockdown in gastric cancer cells. KIAA1429 could act as an oncogene in gastric cancer by stabilizing c-Jun mRNA inan m6A-independent manner. Thishighlights thefunctional role for KIAA1429 as a potential prognostic biomarker and therapeutic target in gastric cancer.

Inside the USCAP Journals

Inside the USCAP Journals

Inside the USCAP Journals

Inside the USCAP Journals

C-Jun Overexpression Accelerates Wound Healing in Diabetic Rats by Human Umbilical Cord-Derived Mesenchymal Stem Cells.

Objective Mesenchymal stem cells (MSCs) are considered a promising therapy for wound healing. Here, we explored the role of c-Jun in diabetic wound healing using human umbilical cord-derived MSCs (hUC-MSCs). Methods Freshly isolated hUC-MSCs were subjected to extensive in vitro subcultivation. The cell proliferative and migratory capacities were assessed by the Cell Counting Kit-8 and scratch assays, respectively. c-Jun expression was evaluated by RT-PCR and western blot analysis. The function of c-Jun was investigated with lentivirus transduction-based gene silencing and overexpression. Diabetes mellitus was induced in SD rats on a high-glucose/fat diet by streptozocin administration. Wounds were created on the dorsal skin. The effects of c-Jun silencing and overexpression on wound closure by hUC-MSCs were examined. Reepithelialization and angiogenesis were assessed by histological and immunohistochemical analysis, respectively. Platelet-derived growth factor A (PDGFA), hepatocyte growth factor (HGF), and vascular endothelial growth factor (VEGF) levels were determined by western blot analysis. Results hUC-MSCs showed gradually decreased cell proliferation, migration, and c-Jun expression during subcultivation. c-Jun silencing inhibited cell proliferation and migration, while c-Jun overexpression enhanced proliferation but not migration. Compared with untransduced hUC-MSCs, local subcutaneous injection of c-Jun-overexpressing hUC-MSCs accelerated wound closure, enhanced angiogenesis and reepithelialization at the wound bed, and increased PDGFA and HGF levels in wound tissues. Conclusion c-Jun overexpression promoted hUC-MSC proliferation and migration in vitro and accelerated diabetic wound closure, reepithelization, and angiogenesis by hUC-MSCs in vivo. These beneficial effects of c-Jun overexpression in diabetic wound healing by hUC-MSCs were at least partially mediated by increased PDGFA and HGF levels in wound tissues.

Prevention of CAR-T-cell dysfunction.

The overexpression of the transcription factor c-Jun improves chimeric-antigen-receptor T-cell functionality and enhances the killing of low-antigen-expressing liquid and solid cancers in mice.

Loss of LRP1 promotes acquisition of contractile-myofibroblast phenotype and release of active TGF-β1 from ECM stores

In healing tissue, fibroblasts differentiate to α-smooth muscle actin (SMA)-expressing contractile-myofibroblasts, which pull the wound edges together ensuring proper tissue repair. Uncontrolled expansion of the myofibroblast population may, however, lead to excessive tissue scarring and finally to organ dysfunction. Here, we demonstrate that the loss of low-density lipoprotein receptor-related protein (LRP) 1 overactivates the JNK1/2-c-Jun-Fra-2 signaling pathway leading to the induction of α-SMA and periostin expression in human lung fibroblasts (hLF). These changes are accompanied by increased contractility of the cells and the integrin- and protease-dependent release of active transforming growth factor (TGF)-β1 from the extracellular matrix (ECM) stores. Liberation of active TGF-β1 from the ECM further enhances α-SMA and periostin expression thus accelerating the phenotypic switch of hLF. Global gene expression profiling of LRP1-depleted hLF revealed that the loss of LRP1 affects cytoskeleton reorganization, cell-ECM contacts, and ECM production. In line with these findings, fibrotic changes in the skin and lung of Fra-2 transgenic mice were associated with LRP1 depletion and c-Jun overexpression. Altogether, our results suggest that dysregulation of LRP1 expression in fibroblasts in healing tissue may lead to the unrestrained expansion of contractile myofibroblasts and thereby to fibrosis development. Further studies identifying molecules, which regulate LRP1 expression, may provide new therapeutic options for largely untreatable human fibrotic diseases.

C-Jun overexpression in CAR T cells induces exhaustion resistance.

SUMMARYCAR T cells mediate antitumor effects in a small subset of cancer patients1–3, but dysfunction due to T cell exhaustion is an important barrier to progress4–6. To investigate the biology of exhaustion in human T cells expressing CAR receptors, we used a model system employing a tonically signaling CAR, which induces hallmark features of exhaustion6. Exhaustion was associated with a profound defect in IL-2 production alongside increased chromatin accessibility of AP-1 transcription factor motifs and overexpression of bZIP and IRF transcription factors that have been implicated in mediating the dysfunctional program present in exhausted cells7–10. Here we demonstrate that engineering CAR T cells to overexpress c-Jun, a canonical AP-1 factor, enhanced expansion potential, increased functional capacity, diminished terminal differentiation and improved antitumor potency in five different in vivo tumor models. We conclude that a functional deficiency in c-Jun mediates dysfunction in exhausted human T cells and that engineering CAR T cells to overexpress c-Jun renders them exhaustion-resistant, thereby addressing a major barrier to progress for this emerging class of therapeutics.

LINC00116 enhances cervical cancer tumorigenesis through miR-106a/c-Jun pathway.

Some studies imply that LINC00116 is involved in cervical cancer progression; however, the molecular mechanism by which LINC00116 modulating tumorigenesis of cervical cancer remains not clear. Reverse transcription-quantitative PCR (RT-qPCR)and the Western blot approaches were employed to probe genes expression levels. To examine the tumorigenic abilities of cervical cancer cells, MTT assay, Transwell assay, and wound-healing assay were used to investigate proliferation, invasion, and migration of HeLa or C-33A cells. LINC00116 knockdown attenuates cell proliferation, invasion, and migration of cervical cancer cells. miR-106a directly binds LINC00116 and regulate each other. Moreover, miR-106a inhibitor remarkably enhanced tumorigenesis of shLINC00116 HeLa cells. Through bioinformatic and dual-luciferase reporter assay, the results showed that miR-106a mimic directly targeted and downregulated the c-Jun. c-Jun overexpression could greatly rescue miR-106a mimic-modulated cervical cancer tumorigenesis. LINC00116 knockdown and miR-106a mimic-modulated programmed cell death ligand 1 (PD-L1) expression, which could be reverted by c-Jun introduction. LINC00116, PD-L1, and JUN were both upregulated in cervical cancer tumors compared to normal tissues. Lower expression levels of LINC00116 and JUN, as well as higher level of miR-106a were closely associated with higher overall survival of cervical cancer patients. Here, we report a novel role for LINC00116 in tumorigenesis of cervical cancer by regulating miR-106a/c-Jun axis. Our findings provide a foundation for understanding cervical cancer and facilitate the development of therapeutical approaches by targeting LINC00116.

Interaction Of c-Jun And HOTAIR- Increased Expression Of p21 Converge In Polyphyllin I-Inhibited Growth Of Human Lung Cancer Cells.

BackgroundLung cancer is a leading cause of cancer-related death worldwide. Previously we demonstrated that polyphyllin I (PPI), a bioactive component extracted from Paris polyphylla, inhibited the growth of non-small cell lung cancer (NSCLC) cells through the SAPK/JNK-mediated suppressing p65, DNMT1 and EZH2 expressions. However, the molecular mechanism underlying anti-lung cancer effect by PPI still remain elusive.PurposeIn this current study, we further explored the molecular mechanism underlying the anti-lung cancer effect of PPI.MethodsMTT, Cell-LightTM EdU DNA cell proliferation and colony formation assays were used to measure cell growth. Western blot were used to examine protein levels of c-Jun and p21. The expression level of long non-codingth RNA HOX transcript antisense RNA (HOTAIR) was measured by qRT-PCR. The p21 promoter activity was measured by Dual-Luciferase Reporter Assay System. The transient transfection experiments were used to silence and overexpression of c-Jun, p21 and HOTAIR. Tumor xenograft and bioluminescent imaging experiments were carried out to confirm the in vitro findings.Results We showed that PPI suppressed growth of NSCLC cells. Mechanistically, we observed that PPI reduced expression of HOTAIR, while increased transcription factor c-Jun protein levels. Additionally, PPI also induced protein expression and promoter activity of p21, a cyclin-dependent kinase inhibitor. While exogenously expressed HOTAIR showed no effect on c-Jun levels, silencing of c-Jun significantly reversed the PPI-inhibited HOTAIR expression. Moreover, excessive expressed c-Jun further enhanced PPI-inhibited HOTAIR expression and PPI-induced p21 protein levels. Intriguingly, overexpression of HOTAIR and silencing of c-Jun overcame the PPI-induced p21 protein and promoter activity. Finally, silencing of p21 neutralized the PPI-inhibited cell proliferation. Similar results were also found in one xenograft mouse model.Conclusion Our results demonstrate that PPI inhibits growth of NSCLC cells through regulation of HOTAIR and c-Jun expressions, which lead to induction of p21 gene. The interactions among HOTAIR, c-Jun and p21 regulatory axis converge in the overall anti-lung cancer effect of PPI. This study unveils an additional new mechanism for the anti-lung cancer role of PPI.

Neuromedin B mediates IL-6 and COX-2 expression through NF-κB/P65 and AP-1/C-JUN activation in human primary myometrial cells.

Neuromedin B (NMB) and its receptor regulate labor onset by mediating inflammatory factors; however the underlying mechanisms remain poorly understood. The present study is aimed to investigate the mechanisms of NMB-induced cyclo-oxygenase 2 (COX-2) expression and interleukin (IL)-6 generation in human primary myometrial cells. The results indicated that NMB could increase phosphorylation of nuclear factor κB (NF-κB) transcription factor p65 (p65) and Jun proto-oncogene, activator protein 1 (AP-1) transcription factor subunit (c-Jun), and in turn, markedly up-regulated the expression levels of COX-2 and IL-6. This up-regulation was significantly attenuated by knockdown of p65 or c-Jun, and enhanced by overexpression of p65 or c-Jun. Furthermore, we identified a potential interaction between p65 and c-Jun following NMB stimulation. In addition, a significant positive correlation was observed between the amount of phosphorylated p65 and the levels of COX-2 and IL-6, and between the amount of phosphorylated c-Jun and COX-2 and IL-6 levels. These data suggested that NMB-induced COX-2 and IL-6 expression were mediated via p65 and c-Jun activation.

Abstract PL01-05: Next-generation CAR T cells designed to overcome tumor resistance

Abstract Personalized cell therapy using chimeric antigen receptor (CAR) modified T cells has rapidly evolved from a boutique treatment under study at a handful of academic centers to the first global, commercialized cell and gene therapy. Despite significant and ongoing challenges regarding cost and scalability, the impressive and sustained antitumor effects observed in some patients with otherwise incurable B cell malignancies lead to the prediction that applications for this novel class of therapeutics will increase in the coming years. Further optimism derives from the rapid pace of scientific and technological advances occurring in cellular engineering, which are yielding increasingly sophisticated “next generation” CAR T cell therapeutics, endowed with greater tumor specificity and control and designed to overcome tumor resistance. Current concepts regarding mechanisms of tumor resistance to CAR T cells largely implicate tumor evasion or T cell failure, and these are typically mutually exclusive. In B-ALL, tumor evasion most often derives from selection of variants lacking the targeted epitope (CD19neg relapse following CD19-CAR therapy) or expressing reduced levels of the epitope that are below the threshold for CAR recognition (CD22lo relapse following CD22-CAR therapy)[1, 2]. Antigen neg and antigen lo relapse are anticipated to be increasingly problematic as CARs are applied to solid tumors and AML, diseases which display significant target antigen heterogeneity. Several engineering approaches are available to address this problem, including multispecific CARs. We are currently testing a bivalent, CD19/22-CAR in clinical trials in children and adults with B cell malignancies[1], and this agent demonstrates acceptable safety and significant clinical activity. We, and others, have also generated tri- and quad-specific CARs and anticipate increasing importance of such higher order therapeutics in expanding the application of CAR T cells beyond B cell malignancies. The second major mechanism of resistance to CAR T cells is T cell exhaustion, driven by excessive or prolonged CAR signaling in response to high tumor burdens and/or tonic signaling as a result of antigen-independent clustering of the CAR receptor[3]. Functional, phenotypic and transcriptomic profiling of “exhaustion” in CAR T cells reveals it to be essentially identical to exhaustion found in T cells within the tumor microenvironment or those exposed to chronic viral infection. We have therefore utilized a reductionist, in vitro model of a tonic signaling CAR to study the biology of human T cell exhaustion, as a first step toward engineering “exhaustion-resistant” CAR T cells. Retroviral transduction of healthy human T cells with a tonically signaling CAR, rapidly induces phenotypic, functional, transcriptomic and epigenomic hallmarks of T cell exhaustion. Whole genome interrogation using ATAC-Seq reveals that the most differentially accessible chromatin regions in exhausted vs. non-exhausted T cells are those bound by AP1/IRF transcription factors and RNA-Seq confirms overexpression of both the canonical “activating” AP1 transcription factors (TFs), Fos and cJun, as well AP1/IRF TFs, which have been associated with inhibitory transcriptional profiles (JunB, BATF3, ATF3, and IRF4). We hypothesized that an imbalance between classical vs inhibitory TFs might drive dysfunction in exhausted T cells. Consistent with this, overexpression of cJun (cJun OE) in tonically signaling CAR T cells reduced expression of inhibitory receptors, increased IL2 production, and deletion of IRF4 or BAT3 replicated this effect, and cJun OE also enhances long-term proliferative capacity of CAR T cells bearing receptors that do not tonically signal. In vivo, cJun OE CAR T cells demonstrate enhanced efficacy in multiple xenograft tumor models and enhanced persistence. Molecular mapping reveals that the bZIP domain of cJun for exhaustion prevention, whereas phosphorylation sites involved in transactivation and the DNA binding domains is dispensable, which suggests that the major mechanism of action of cJun OE is to compete for binding, and therefore disrupt inhibitory AP1/IRF complexes. cJun OE also enhanced CAR recognition of antigen lo targets, consistent with a model wherein cJun OE amplifies otherwise subthreshold levels of CAR signaling. These data provide new insights into the biology of human T cell exhaustion, highlighting an imbalance between “activating” versus “inhibitory” AP1/IRF complexes in the fundamental pathophysiology of this state, and demonstrate that CAR T cells engineered for “exhaustion resistance” mediate impressively increased potency in preclinical models. 1. Fry, T.J., et al., CD22-targeted CAR T cells induce remission in B-ALL that is naive or resistant to CD19-targeted CAR immunotherapy. Nat Med, 2018. 24(1): p. 20-28. 2. Walker, A.J., et al., Tumor Antigen and Receptor Densities Regulate Efficacy of a Chimeric Antigen Receptor Targeting Anaplastic Lymphoma Kinase. Mol Ther, 2017. 25(9): p. 2189-2201. 3. Long, A.H., et al., 4-1BB costimulation ameliorates T cell exhaustion induced by tonic signaling of chimeric antigen receptors. Nat Med, 2015. 21(6): p. 581-90. Citation Format: Crystal L. Mackall. Next-generation CAR T cells designed to overcome tumor resistance [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 PL01-05.