Expression Of Downstream Targets Research Articles

Hexokinase 2 nonmetabolic function-mediated phosphorylation of IκBα enhances pancreatic ductal adenocarcinoma progression.

Aberrant signaling in tumor cells induces nonmetabolic functions of some metabolic enzymes in many cellular activities. As a key glycolytic enzyme, the nonmetabolic function of hexokinase 2 (HK2) plays a role in tumor immune evasion. However, whether HK2, dependent of its nonmetabolic activity, plays a role in human pancreatic ductal adenocarcinoma (PDAC) tumorigenesis remains unclear. Here, we demonstrated that HK2 acts as a protein kinase and phosphorylates IκBα at T291 in PDAC cells, activating NF-κB, which enters the nucleus and promotes the expression of downstream targets under hypoxia. HK2 nonmetabolic activity-promoted activation of NF-κB promotes the proliferation, migration, and invasion of PDAC cells. These findings provide new insights into the multifaceted roles of HK2 in tumor development and underscore the potential of targeting HK2 protein kinase activity for PDAC treatment.

The Ins and Outs of Homeodomain-Leucine Zipper/Hormone Networks in the Regulation of Plant Development

The generation of complex plant architectures depends on the interactions among different molecular regulatory networks that control the growth of cells within tissues, ultimately shaping the final morphological features of each structure. The regulatory networks underlying tissue growth and overall plant shapes are composed of intricate webs of transcriptional regulators which synergize or compete to regulate the expression of downstream targets. Transcriptional regulation is intimately linked to phytohormone networks as transcription factors (TFs) might act as effectors or regulators of hormone signaling pathways, further enhancing the capacity and flexibility of molecular networks in shaping plant architectures. Here, we focus on homeodomain-leucine zipper (HD-ZIP) proteins, a class of plant-specific transcriptional regulators, and review their molecular connections with hormonal networks in different developmental contexts. We discuss how HD-ZIP proteins emerge as key regulators of hormone action in plants and further highlight the fundamental role that HD-ZIP/hormone networks play in the control of the body plan and plant growth.

Triptolide, a Cancer Cell Proliferation Inhibitor, Causes Zebrafish Muscle Defects by Regulating Notch and STAT3 Signaling Pathways.

Triptolide is a natural compound in herbal remedies with anti-inflammatory and anti-proliferative properties. We studied its effects on critical signaling processes within the cell, including Notch1 and STAT3 signaling. Our research showed that triptolide reduces cancer cell proliferation by decreasing the expression of downstream targets of these signals. The levels of each signal-related protein and mRNA were analyzed using Western blot and qPCR methods. Interestingly, inhibiting one signal with a single inhibitor alone did not significantly reduce cancer cell proliferation. Instead, MTT assays showed that the simultaneous inhibition of Notch1 and STAT3 signaling reduced cell proliferation. The effect of triptolide was similar to a combination treatment with inhibitors for both signals. When we conducted a study on the impact of triptolide on zebrafish larvae, we found that it inhibited muscle development and interfered with muscle cell proliferation, as evidenced by differences in the staining of myosin heavy chain and F-actin proteins in confocal fluorescence microscopy. Additionally, we noticed that inhibiting a single type of signaling did not lead to any significant muscle defects. This implies that triptolide obstructs multiple signals simultaneously, including Notch1 and STAT3, during muscle development. Chemotherapy is commonly used to treat cancer, but it may cause muscle loss due to drug-related adverse reactions or other complex mechanisms. Our study suggests that anticancer agents like triptolide, inhibiting essential signaling pathways including Notch1 and STAT3 signaling, may cause muscle atrophy through anti-proliferative activity.

Regulation of Notch signaling by non-muscle myosin II Zipper in Drosophila

The Notch pathway is an evolutionarily conserved signaling system that is intricately regulated at multiple levels and it influences different aspects of development. In an effort to identify novel components involved in Notch signaling and its regulation, we carried out protein interaction screens which identified non-muscle myosin II Zipper (Zip) as an interacting partner of Notch. Physical interaction between Notch and Zip was further validated by co-immunoprecipitation studies. Immunocytochemical analyses revealed that Notch and Zip co-localize within same cytoplasmic compartment. Different alleles of zip also showed strong genetic interactions with Notch pathway components. Downregulation of Zip resulted in wing phenotypes that were reminiscent of Notch loss-of-function phenotypes and a perturbed expression of Notch downstream targets, Cut and Deadpan. Further, synergistic interaction between Notch and Zip resulted in highly ectopic expression of these Notch targets. Activated Notch-induced tumorous phenotype of larval tissues was enhanced by over-expression of Zip. Notch-Zip synergy resulted in the activation of JNK pathway that consequently lead to MMP activation and proliferation. Taken together, our results suggest that Zip may play an important role in regulation of Notch signaling.

NCI 159456 PERK Inhibitor as a Targeted Therapy for Lung Cancer: An In Vitro Study.

Non-small cell lung cancer (NSCLC) represents the most common histological type of lung cancer, characterized by a five-year survival rate of 15% and poor prognosis. Accumulating evidence indicates a prominent role of endoplasmic reticulum (ER) stress and the protein kinase RNA-like ER kinase (PERK)-dependent pathway of the unfolded protein response (UPR) in the pathogenesis of NSCLC. Increased expression of downstream targets of PERK was observed in various subtypes of NSCLC, and it was associated with a more aggressive phenotype, high risk of recurrence, and poor prognosis. Therefore, the present study aimed to investigate the biological effect of the selective PERK inhibitor NCI 159456 on A549 NSCLC cells and Human Pulmonary Fibroblasts (HPF) in vitro. Treatment of both normal and ER-stressed A549 cells with NCI 159456 resulted in a significant increase in the mRNA expression level of pro-apoptotic genes like activating transcription factor 4 (ATF4), DNA damage inducible transcript 3 (DDIT3), and BCL2 Associated X, Apoptosis Regulator (BAX) as well as a decreased level of the anti-apoptotic gene B-cell lymphoma 2 (Bcl-2). Cytotoxicity and genotoxicity analyses revealed that NCI 159456 significantly decreased viability and increased DNA damage in A549 cells under normal and ER stress conditions. Caspase-3 and reactive oxygen species (ROS) detection assays demonstrated that NCI 159456 significantly induced apoptosis and increased the ROS level in normal and ER-stressed A549 cells. Importantly, treatment with the inhibitor did not affect substantially normal HPF cells at any used concentration. The results indicate that PERK inhibitors could potentially be applied as a targeted therapy for NSCLC.

Abstract 6516: Single-cell protein activity inference reveals lymphocytic immune infiltration in diffuse intrinsic pontine glioma induced by focused ultrasound

Abstract Diffuse intrinsic pontine glioma (DIPG) is a highly aggressive pediatric brain tumor, located in the brainstem, and traditionally thought to be immune-excluded with no significant infiltration by cytotoxic T-cells, and therefore not amenable to immunotherapies. Focused ultrasound technology (FUS) for drug delivery is being investigated for DIPG in both preclinical and clinical trial settings. In this study, we sought to study the effects of FUS and radiation therapy (RT) on immune cell infiltration in DIPG tumors to assess their ability to alter the immune microenvironment and potentially enhance combination immunotherapies. Specifically, we generated single-cell RNAseq profiles for 27,780 cells from syngeneic orthotopic DIPG tumors (KPAP cell line containing H3K27M mutation, ATRX and P53 loss, and PDGFRA overexpression) after 14 days of tumor growth across the following treatment arms: vehicle control, FUS, and RT (n=2 each group). FUS was applied by 4-point sonication around the tumor with a pressure of 0.6 Mpa at days 8 and 11 post tumor implantation (PTI). RT was administered in fractions of 3 Gy daily from days 8 to 11 PTI (12Gy total). We leveraged a set of algorithms for network-based inference of regulatory protein activity to infer a set of context-matched gene regulatory networks from which protein activity can be inferred by expression of downstream targets. By this approach, we identified distinct clusters of immune cells segregating by cell type as inferred by singleR and activity of known lineage markers. Strikingly, we found that both RT and FUS significantly depleted a TGFb-expressing population of immune-suppressive macrophages as compared to vehicle controls. Importantly, FUS only additionally increased T-cell infiltration into the tumor micro-environment over three-fold as compared to vehicle controls, which was not observed in response to RT. Further protein validation is underway. This has significant implications for therapeutic combinations of FUS-mediated drug delivery with immune checkpoint inhibitors in DIPG, such that in addition to drug delivery, FUS-mediated immune modulatory effects may provide more proinflammatory response to enhance immunotherapy. Citation Format: Erin White, Hong-Jian Wei, Ester Calvo Fernández, Hanna Minns, Jovana Pavisic, Robyn Gartrell, Cheng-Chia Wu, Andrea Califano, Aleksandar Obradovic. Single-cell protein activity inference reveals lymphocytic immune infiltration in diffuse intrinsic pontine glioma induced by focused ultrasound [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6516.

Abstract 6814: E2F1-induced autocrine IL-6 inflammatory loop mediates cancer-immune cell crosstalk that regulates metastatic properties in melanoma

Abstract Melanoma is a cancer that is metastatic, drug-refractory and fatal if managed late or inadequately. The transcription factor E2F1 is a key player in melanoma pathology, promoting metastatic traits like EMT, neoangiogenesis, chemoresistance and evasion of immune surveillance. Particularly, high levels of E2F1 trigger tumor progression by developing protein-protein interactions (PPI) with coregulators that enhance its potential to activate a prometastatic gene regulatory network (GRN). We aimed to investigate the extent to which E2F1-expressing melanoma cells influence the immune response. Methods: We combined transcriptomic approaches with bioinformatics and structural modeling to search for target genes involved in E2F1-activated immunomodulatory and prometastatic GRNs in aggressive melanoma. Immunoprecipitation and ChIP were performed to verify both PPI and protein-DNA interactions. Functional analyses, including qRT-PCR, immunoblotting and luciferase assays, were performed to verify expression and regulation of downstream targets. Additionally, we established a melanoma-immune cell coculture system with patient-derived CD4+ and CD8+ T cells. Cytokine arrays and ELISA were used for measurement of cytokine secretion to analyze the E2F1-induced tumor-immuno-crosstalk. Results: We identified an E2F1-dependent GRN with IL-6 as a central factor mediating EMT and immune regulation. Mechanistically, E2F1-induced activation and secretion of IL-6 leads to an autocrine inflammatory forward-feedback loop, which in turn further promotes invasiveness and EMT marker expression in melanoma cells. Interestingly, IL6 activated STAT3 associates with E2F1 in a structure-dependent manner, forming a complex that enhances IL-6 expression by binding to a composite E2F1:STAT3-responsive promoter element. Moreover, we observed that the E2F1-IL6 axis dispenses immunomodulatory effects on surrounding CD4+ and CD8+ T cells, further stimulating IL-6 release, but also leading to secretion of IL-10 into the TME. Although IL-10 has an immunosuppressive function, it also showed anti-metastatic properties in melanoma cells. Thus, in contrast to IL-6, IL-10 decreased cell motility and reduced the expression of Vim, Slug, and Snai1, while simultaneous knockdown of E2F1 increased these effects. The findings are supported by clinical data sets showing that melanoma patients who have high levels of E2F1 and IL-6 and low IL-10 exhibit poor survival. Conclusion: Our results unveil a novel gene regulatory program by which E2F1-associated IL-6 release induces the formation of an E2F1:STAT3 complex, resulting in a forward-feedback loop that promotes cancer invasion and regulates T cells response through intercellular cytokine signaling in the tumor microenvironment. Citation Format: Prabir Dhar, Krishna P. Singh, Shailendra K. Gupta, Dhanya Peringot, Alf Spitschak, Brigitte M. Pützer. E2F1-induced autocrine IL-6 inflammatory loop mediates cancer-immune cell crosstalk that regulates metastatic properties in melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6814.

Abstract 5965: First-in-class chemical induced proximity system achieves dose-dependent control of TP53 activation in gastric cancer

Abstract Background: Gastric cancer (GC) remains a common and deadly disease, and inactivating TP53 mutations occur in up to 68% of GC tumors. p53 is a master regulator of multiple cellular pathways, such as the cell cycle, apoptosis, and senescence. Previously, we demonstrated the ability to activate endogenous target gene expression using a novel chemical induced proximity system comprised of deactivated Cas9 (dCas9) and “chemical epigenetic modifiers” or “CEMs” (PMID: 31712774). In this study, we applied our dCas9-CEM system for the first time in oncology to activate TP53 gene expression and selectively kill targeted cells in a preclinical model of GC. Methods: AGS cells were derived from a gastric adenocarcinoma, and harbor wild-type TP53. They were transduced with our dCas9 system and either sgRNAs targeting TP53 or a non-targeting (NT) sgRNA. After 48 h CEM87 incubation (1-25 nM), expression of TP53 and downstream targets (CDKN1A and BBC3) were evaluated by RT-qPCR. After 48 h CEM87 incubation (0.1-25 nM), colony-forming potential was assessed by crystal violet staining after 10 days. After 24 h incubation (5-25 nM), cells were fixed and stained with propidium iodide (PI), and DNA content was assessed by flow cytometry to determine cell cycle fractions. After 48 h incubation (50-200 nM), cells were fixed and stained with Annexin V and PI, and apoptosis was assessed by flow cytometry. Last, cells were treated with nine ascending concentrations (10 pM-100 μM) for 48 h, and cell viability was measured using CellTiter-Glo™. IC50 values were calculated using four-parameter non-linear regression. Results: AGS-TP53 cells show significantly increased expression of TP53 and downstream target genes CDKN1A and BBC3 versus AGS-NT cells after 10 nM CEM87 (105%, 99%, and 43% increase, P=0.004, 0.002, and 0.03, respectively; n=3). Colony formation was significantly reduced in AGS-TP53 versus AGS-NT cells after 10 nM CEM87 (60.2% reduction, P=0.003; n=3). Treatment with 10 nM CEM87 also led to a G2/M stall after significantly reducing cells in both G1 (47.4% vs 38.9%, P<0.0001; n=3) and S-phase (25.1% vs 20.3%%, P<0.0001; n=3) in AGS-TP53 versus AGS-NT cells. Treatment with 200 nM CEM87 significantly increased early apoptosis in AGS-TP53 versus AGS-NT cells (28.7% vs 6.6%, respectively, P<0.0001; n=3). Last, CEM87 was 50 times more potent in AGS-TP53 cells than AGS-NT cells (IC50: 3 vs 146 nM; n=8). Conclusions: These data highlight how our innovative system upregulates TP53 expression and impacts downstream phenotypic effects of TP53 activation in a preclinical model of GC. Citation Format: Ryan M. Kemper, Travis J. Nelson, Nathaniel A. Hathaway, Daniel J. Crona. First-in-class chemical induced proximity system achieves dose-dependent control of TP53 activation in gastric cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5965.

Abstract 1602: Leukemia inhibitory factor (LIF)-factor XIIIA mediated macrophage-stromal crosstalk in pancreatic cancer

Abstract Background: Pancreatic ductal adenocarcinoma (PDAC) is a lethal malignancy marked by a highly immunosuppressive tumor microenvironment (TME). Macrophages are known to promote immunosuppression and exhibit a profibrotic transcriptional profile with potential involvement in extracellular matrix (ECM) remodeling. Previously, we identified hyperactivation of cancer cell intrinsic cyclic AMP response element binding protein 1 (CREB) regulates leukemia inhibitory factor (LIF) to mediate macrophage infiltration and polarization within the TME of PDAC. Subsequently, we observed that disrupting LIF-LIF receptor (LIFR) signaling therapeutically not only diminishes the infiltration of tumor-associated macrophages (TAMs) but also results in a decrease in ECM deposition. This study delves into the potential regulatory pathway through which LIF induces remodeling of the ECM within the PDAC TME. Methods: We targeted LIF-induced macrophage-stromal crosstalk using the LIFR antagonist (EC359) in an orthotopically tumor implanted LSL-KrasG12D/+; Trp53 R172H/+; Pdx1Cre/+ (KPC) mice model of PDAC. Sirius red staining in tumor sections was performed to assess ECM (collagen) deposition. Moreover, we performed RNA transcriptomics-based analysis of recombinant (rLIF) induced bone marrow derived macrophages harvested from syngeneic C57BL/6 mice. The Cancer Genome Atlas (TCGA) pancreatic cancer (PAAD) data set along with single-cell RNA sequencing (scRNA seq) were employed to assess the expression of major downstream targets of LIF. To validate the RNA-seq results, we conducted immunohistochemical analysis (IHC) on mouse PDAC tissue sections and quantitative polymerase (qPCR) analysis on RAW 264 macrophage cell line treated with rLIF or KPC CREBWT PDAC tumor cells conditioned media. Results: We observed a drastic decrease in both TAM infiltration and, of note, ECM deposition in orthotopically implanted KPC pancreas tumor mice tissue treated with EC359 as compared to vehicle. Mechanistically, our RNA-seq analysis unveiled a significant upregulation of Factor XIIIA (FXIIIa), a major downstream target of LIF. Notably, TCGA data revealed that FXIIIa mRNA expression is upregulated across PDAC tumors marked by poor survival outcomes. Moreover, scRNA seq and IHC established that TAMs highly express FXIIIa in the PDAC TME. In exploring the effect of LIF-LIFR signaling in the ECM, mouse RAW 264.7 macrophage lines incubated in high LIF conditions confirmed transcriptional upregulation of ECM-associated proteins such as FXIIIa as well as fibronectin. Conclusion: Our investigation provides valuable insights into the possible mechanism by which LIF triggers ECM remodeling, thereby contributing to the desmoplastic stroma. Significantly, our findings propose that LIF-regulated FXIIIa signaling in macrophages serves as a plausible mediator of the LIF-induced ECM remodeling in PDAC. Citation Format: Nishah Jaferi, Siddharth Mehra, Varun Krishnamoorthy, Sudhakar Jinka, Anna Bianchi, Vanessa T. Garrido, Luis A. Nivelo, Ban Yuguang, Nagaraj S. Nagathihalli. Leukemia inhibitory factor (LIF)-factor XIIIA mediated macrophage-stromal crosstalk in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1602.

Abstract 3018: Transcriptional dysregulation in germline TP53-driven breast cancers

Abstract Background: Pathogenic germline variants (PGVs) in TP53 is associated with multicancer risk syndrome known as Li-Fraumeni Syndrome (LFS) and females have 80-90% risk of breast cancer at very early age compared to sporadic breast cancers. The mechanism of p53 tumor suppression relies on its function as a transcriptional activator; however, there is little data on the specific transcriptional defects in human breast tumors driven by germline mutant p53. Methods: To investigate the specific transcriptional defects due to TP53 PGVs in human breast, we performed bulk RNA-seq on invasive ductal carcinoma (IDC), ductal carcinoma in situ (DCIS) and surrounding and contralateral normal breast tissue in LFSBC compared to non-LFS sporadic BC. We also compared our results to breast tumors from The Cancer Genome Atlas (TCGA) with and without acquired TP53 mutations. Results: Our RNA-seq analysis revealed significant changes in the expression of key pathways in LFSBC compared to non-LFS breast cancer. Notably, cancer hallmark pathways such as apoptosis and reactive oxygen species were downregulated, while androgen response and cholesterol homeostasis pathways were upregulated in LFSBC compared to non-LFSBC. Expression of downstream targets of p53 such as BAX, tumor suppressors such as CDKN1A (p21) and BTG2, were significantly down in LFSBC suggesting impaired transcriptional activity of mutated TP53 in LFS patients. We also observed downregulation of cell cycle regulators in LFS breast tumors compared to their surrounding and contralateral tissues. Although, there were no significant transcriptional differences as the tumor progressed from LFS DCIS to IDC, indicating that the damage caused by the mutated p53 had already occurred at the early stage. Additionally, we also investigated the immune cell composition in LFS-BC and non-LFSBC. LFSBC exhibited a higher relative proportion of CD4+ T cells, CD20+ B cells, NK cells, macrophages and monocytes compared to all sporadic non-LFSBC subtypes (TCGA) and age matched non-LFSBC. This providing novel insights into the immune landscape associated with LFS-driven breast cancer. Conclusion: Our findings suggest that breast tissues with an inherited TP53 mutation undergo a defective transcriptional state, shifting away from apoptosis, and experiencing dysregulation of cell cycle at an early stage as DCIS, potentially contributing to the early onset of LFS breast cancer. The preliminary analysis of immune repertoire in LFSBC opens avenues for deeper investigations into spatial transcriptomic profiles of germline TP53-mutated breast tumors also the immune cells. Overall, this study provides valuable insights into the TP53 mutation driven breast cancers in humans. Citation Format: Nabamita Boruah, Renyta Moses, Ryan Hausler, Heena Desai, Anh Le, Gregory Kelly, Ashvathi Raghavakaimal, Mohana Narasimhamurthy, Anupma Nayak, Arnold J. Levine, Kara N. Maxwell. Transcriptional dysregulation in germline TP53-driven breast cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3018.

Differential Expression of MicroRNA MiR-145 and MiR-155 Downstream Targets in Oral Cancers Exhibiting Limited Chemotherapy Resistance.

New evidence has suggested that non-coding microRNAs play a significant role in mediating and modulating chemotherapy resistance, particularly among oral cancers. One recent study found that the upregulation of miR-145 and the downregulation of miR-155 strongly correlated with a limited chemotherapy resistance to Cisplatin, 5-Fluorouracil, and Paclitaxel, although the mechanism(s) responsible for these observations remain unidentified. Using commercially available cell lines of oral squamous cell carcinoma, RNA was isolated, converted into cDNA, and subsequently screened for the expression of downstream targets of miR-145 and miR-155 using qPCR. These results demonstrated the upregulation of miR-21, miR-125, miR-133, miR-365, miR-720, and miR-1246, as well as the downregulation of miR-140, miR-152, miR-218, miR-221, and miR-224. This screening also confirmed the differential expression and regulation of mir-145 and miR-155 among the cell lines with limited chemotherapy resistance (SCC15). In addition, several downstream targets of these specific microRNAs were upregulated by all oral cancer cell lines, such as MBTD1 and FSCN1, or downregulated in all cell lines, such as CLCN3, FLI-1, MRTFB, DAB, SRGAP1, and ABHD17C. However, three miR-145 downstream targets were identified in the least chemotherapy-resistant cells, exhibiting the differential upregulation of KCNA4 and SRGAP2, as well as the downregulation of FAM135A, with this expression pattern not detected in any of the other oral cancer cell lines. These data strongly support that the differential regulation of these three downstream targets may be related to the chemosensitivity of this oral cancer cell line. The potential involvement of these targets must be further investigated to determine how and whether mechanisms of these cellular pathways may be involved in the observed lack of chemotherapy resistance. These data may be important to design targets or treatments to reduce chemotherapy resistance and improve patient treatment outcomes.

Dimethyloxalylglycine pretreatment of living donor alleviates both donor and graft liver ischemia-reperfusion injury in rats.

Background: Under the circumstance of the increasing waiting list for liver transplantation, living donor liver transplantation (LDLT) can alleviate the shortage of liver donors to some extent. However, how to reduce both donor and graft ischemia-reperfusion injury (IRI) is still an unsolved problem in LDLT. Hypoxia-induced transcription factor 1 (HIF1) activation is considered an important mechanism of cellular adaptation to hypoxia, and early activation of HIF1 may be a new way to alleviate liver IRI. Therefore, we aimed to investigate the impact of the HIF1 stabilizer dimethyloxalylglycine (DMOG) on IRI and the survival rate of donors and recipients of rat LDLT. Methods: Seventy percent partial liver resection and 30% partial liver transplantation were used to simulate donor and recipient of clinical LDLT. Rats were treated with DMOG (40mg/kg) or with an equivalent amount of saline. The expression of HIF1 and downstream targets was analyzed after 2h of reperfusion. Liver function and histopathology, apoptosis and oxidative stress levels were detected 6h after reperfusion. At the same time, the 7-day survival rate of rats was calculated. Results: DMOG pretreatment significantly reduced IR-induced injury in the donor and recipient, which was manifested by reducing liver function damage and promoting tissue recovery. Meanwhile, compared with the untreated group, the oxidative stress level and the cell apoptosis rate were decreased in the group pretreated with DMOG. In addition, the transcription and expression of HIF1 target genes in the DMOG group were significantly enhanced. Remarkably, DMOG also increased the survival rate of the recipient. Conclusion: This study provides the first evidence that DMOG pretreatment of donors significantly alleviates liver IRI in both donors and recipients and increases the survival rate of recipients in LDLT. Therefore, DMOG may be a promising strategy for improving LDLT in the future.

Zerumbone (ZER), a Potential Anticancer for Breast Mediates Cancer Cell Death Through Targeting β-catenin Signaling Pathway in Tumor Regression in Sprague Dawley Rat Mammary Gland Tumors

Background & Objective: Canonical WNT/Wingless pathway regulates expression of target genes by modulating intracellular β-catenin leading to cancer growth and survival. Here, we studied inhibition of β-catenin using zerumbone (ZER) or β-catenin-siRNA in Sprague-Dawley rat cancer model. Methodology: Rat mammary gland tumor model was induced with the wild-type LA7 breast cancer cell line or β-catenin-knockdown-LA7 cells. LA7-rats were treated with ZER or β-catenin-siRNA as a positive control and the levels of β-catenin expression and its target gene were assessed using real-time PCR, immunohistochemistry and TUNEL assay. Results: We found good correlation between β-catenin inhibition and induction of apoptosis when using ZER or β-catenin-siRNA. In this aspect, ZER and β-catenin-siRNA inhibited cellular proliferation as reflected by reduced growth of breast cancer and apoptosis induction in rat mammary gland tumor. Further, our studies demonstrate that treatment with ZER and β-catenin-siRNA affected β-catenin-dependent gene expression. β-catenin downregulation by ZER or knockdown by β-catenin-siRNA improves the morphological and histological feature of the breast tissues as confirmed by subtle changes of the genes involved in cell-apoptotic pathways. Conclusion: This indicates that ZER targets similarly to β-catenin-siRNA and possibly be a useful anti-breast cancer by affecting expression of down-stream targets that are key components in cancer development. ZER can be a promising anticancer candidate for treatment of breast.

AHSA1 Regulates Hepatocellular Carcinoma Progression via the TGF-β/Akt-Cyclin D1/CDK6 Pathway.

Activator of heat shock protein 90 (HSP90) ATPase Activity 1 (AHSA1) regulates proliferation, apoptosis, migration, and invasion of osteosarcoma and hepatocellular carcinoma (HCC). However, the novel mechanism of AHSA1 in the tumor biology of hepatocellular carcinoma (HCC) remains unclear. We analyzed AHSA1 expression in 85 pairs of clinical samples of HCC and the Cancer Genome Atlas database. The role of AHSA1 in HCC was proved by cell proliferation, colony formation, migration, cell cycle analysis in vitro, xenograft models and tumor metastasis assay in vivo, and bioinformatics. High AHSA1 expression was demonstrated in HCC and associated with invasive depth, clinical stage, and poor overall survival of patients. UnivariateCox analysis confirmed that AHSA1 was an independent prognostic factor for patients with HCC. Meanwhile, AHSA1 upregulation promoted cell proliferation, colony formation, and cell migration in vitro and tumor cell proliferation and metastasis of HCC cells in vivo. AHSA1 upregulation increased the cell cycle transition from G1 to S phase by increasing the expression of cyclinD1, cyclinD3, and cyclin-dependent kinase 6(CD). Transforming growth factor beta 1 (TGF-β1)-induced protein kinase B (Akt) signaling regulated the expression of downstream targets, including cyclinD1. AHSA1 expression was closely correlated with the expression of TGF-β, Akt, cyclinD1, cyclinD3, and CDK6 using the Gene Expression Profiling Interactive Analysis database. AHSA1 upregulation participated in HCC progression by regulating TGF-β/Akt-cyclinD1/CDK6 signaling. AHSA1 might serve as a biomarker for predicting the clinical outcome of patients with HCC. It is vital in tumor metastasis and disease progression of HCC and may facilitate the development of clinical intervention strategies against HCC.

Abstract P2098: The Effect Of Cyclic Stretch On Cardiac Fibroblasts

Introduction: Cardiac fibrosis is the accumulation of extracellular matrix proteins in the interstitium and contributes to heart failure. The main source of cardiac fibrosis is cardiac fibroblasts (cFBs), which are activated as a result of pressure overload. Activated fibroblasts, also known as myofibroblasts, express α-smooth muscle actin (α-SMA) and have an increased production of collagen. Here, the objective was to study the behavior of induced pluripotent stem cell (iPSC) derived cFBs in response to pro-fibrotic cytokines and mechanical stress. Methods: Confluent iPSC monolayers were differentiated into cFBs using the protocol in Figure 1A. Afterwards, the differentiated cells were exposed to 10 ng/ml transforming growth factor β (TGFβ) and 10% cyclic stretch at 1 Hz for 3 days using the Flexcell FX-6000 system. Results: iPSC-cFBs had a fibroblast-like morphology. Furthermore, the presence of cardiac ( GATA4, TCF21 ) and fibroblast ( VIM, PDGFRα, DDR2 ) markers at gene and protein levels confirmed the cFB identity. TGF-β stimulation resulted in increased mRNA and protein levels of α-SMA. Additionally, stretching reduced collagen 1 mRNA and protein expression (Fig. 1B/C). Furthermore, stretching was able to protect the cells from TGF-β stimulation, significantly reducing α-SMA and collagen 1 protein and mRNA levels. This was associated with decreased mRNA expression of TGF-β downstream targets ( PAI1). Conclusion: Cyclic mechanical stretch reduces cFB activation and can protect against TGF-β stimulation, resulting in reduced collagen production and transdifferentiation into myofibroblasts.

BSBM-18 SINGLE-CELL PROFILING TUMOR-INFILTRATING IMMUNE CELLS REVEALS CXCL13+ FOLLICULAR HELPER-LIKE CD4+ T CELLS IN HUMAN BRAIN TUMORS

Abstract While therapies enhancing antitumor T cell function have clinical efficacy for patients with brain metastases, patients with high-grade gliomas fail to benefit. How the brain tumor microenvironment differentially shapes T cell function remains unknown. We performed droplet-based single-cell RNA and T cell receptor sequencing on immune cells from tumor and blood of patients with newly diagnosed non-small cell lung cancer brain metastases (n=10) and high-grade glioma (n=12). In total, we examined 220,049 high-quality immune cells, including 159,043 T cells. Tumor-infiltrating T cells in metastases were more abundant (p=0.005), more clonally expanded (p=0.039), and had a less diverse clonal repertoire (p=0.033). While no differences were observed among CD8+ T cells, a population of CXCL13+CD4+ T cells was more abundant in metastases (6.03% vs. 1.82%, p=0.004). This population expressed genes encoding transcriptional factors (BCL6, MAF), co-inhibitory receptors (PDCD1, LAG3, TIGIT), and effector cytokines (IL21, IL4, IFNG) characteristic of follicular helper T cells though notably lacked expression for the canonical chemokine receptor CXCR5. CXCL13+CD4+ T cells were highly expanded, had minimal clonal overlap with other populations, largely confined to the tumor, associated with germinal center B cell and plasma cell signatures, and resembled T cell populations predictive of anti-PD-1 therapy response in extracranial disease. Based on the expression of potential ligand-receptors and downstream signaling targets, CXCL13+CD4+ T cells were predicted to interact with B cells and antigen-presenting cells, as seen in tertiary lymphoid structures. To investigate the relevance of CXCL13+CD4+ T cells in glioblastoma, we analyzed bulk RNA sequencing data from The Cancer Genome Atlas (n=160) and observed a trend towards longer overall survival in patients with high follicular helper-like expression. In summary, we identified a follicular helper-like CD4+ T cell population that is preferentially found in brain metastases and may be a feature of productive antitumor immune responses in human brain tumors.

Gain-of-function mutations in the catalytic domain of DOT1L promote lung cancer malignant phenotypes via the MAPK/ERK signaling pathway.

Lung cancer is a lethal malignancy lacking effective therapies. Emerging evidence suggests that epigenetic enzyme mutations are closely related to the malignant phenotype of lung cancer. Here, we identified a series of gain-of-function mutations in the histone methyltransferase DOT1L. The strongest of them is R231Q, located in the catalytic DOT domain. R231Q can enhance the substrate binding ability of DOT1L. Moreover, R231Q promotes cell growth and drug resistance of lung cancer cells in vitro and in vivo. Mechanistic studies also revealed that the R231Q mutant specifically activates the MAPK/ERK signaling pathway by enriching H3K79me2 on the RAF1 promoter and epigenetically regulating the expression of downstream targets. The combination of a DOT1L inhibitor (SGC0946) and a MAPK/ERK axis inhibitor (binimetinib) can effectively reverse the R231Q-induced phenomena. Our results reveal gain-of-function mutations in an epigenetic enzyme and provide promising insights for the precise treatment of lung cancer patients.

NFAT5 Restricts Bovine Herpesvirus 1 Productive Infection in MDBK Cell Cultures.

Bovine herpesvirus 1 (BoHV-1), an important bovine viral pathogen, causes severe disease in the upper respiratory tract and reproductive system. Tonicity-responsive enhancer-binding protein (TonEBP), also known as nuclear factor of activated T cells 5 (NFAT5), is a pleiotropic stress protein involved in a range of cellular processes. In this study, we showed that the knockdown of NFAT5 by siRNA increased BoHV-1 productive infection and overexpression of NFAT5 via plasmid transfection decreased virus production in bovine kidney (MDBK) cells. Virus productive infection at later stages significantly increased transcription of NFAT5 but not appreciably alter measurable NFAT5 protein levels. Virus infection relocalized NFAT5 protein and decreased the cytosol accumulation. Importantly, we found a subset of NFAT5 resides in mitochondria, and virus infection led to the depletion of mitochondrial NFAT5. In addition to full-length NFAT5, another two isoforms with distinct molecular weights were exclusively detected in the nucleus, where the accumulation was differentially affected following virus infection. In addition, virus infection differentially altered mRNA levels of PGK1, SMIT, and BGT-1, the canonical downstream targets regulated by NFAT5. Taken together, NFAT5 is a potential host factor that restricts BoHV-1 productive infection, and virus infection hijacks NFAT5 signaling transduction by relocalization of NFAT5 molecules in cytoplasm, nucleus, and mitochondria, as well as altered expression of its downstream targets. IMPORTANCE Accumulating studies have revealed that NFAT5 regulates disease development due to infection of numerous viruses, underlying the importance of the host factor in virus pathogenesis. Here, we report that NFAT5 has capacity to restrict BoHV-1 productive infection in vitro. And virus productive infection at later stages may alter NFAT5 signaling pathway as observed by relocalization of NFAT5 protein, reduced accumulation of NFAT5 in cytosol, and differential expression of NFAT5 downstream targets. Importantly, for the first time, we found that a subset of NFAT5 resides in mitochondria, implying that NFAT5 may regulate mitochondrial functions, which will extend our knowledge on NFAT5 biological activities. Moreover, we found two NFAT5 isoforms with distinct molecular weights were exclusively detected in the nucleus, where the accumulation was differentially affected following virus infection, representing a novel regulation mechanism on NFAT5 function in response to BoHV-1infection.

Non-canonical role for the ataxia-telangiectasia-Rad3 pathway in STAT3 activation in human multiple myeloma cells.

The goal of this study was to characterize the relationship between ATR and STAT3 interactions in human multiple myeloma (MM) cells. Various MM cell lines, including IL-6-dependent cells were exposed to ATR inhibitors and effects on STAT3 Tyr705 and Ser727 were monitored by WB analysis and ImageStream analysis. Parallel studies examined induction of cell death, STAT3 DNA binding activity, and expression of STAT3 downstream targets (BCL-XL, MCL-1, c-MYC). Validation was obtained in ATR shRNA knock-down cells, and in cells ectopically expressing BCL-XL, MCL-1, or c-MYC. Analogous studies were performed in primary MM cells and in a MM xenograft model. Multiple pharmacologic ATR inhibitors inhibited STAT3 Tyr705 (but not Ser727) phosphorylation at low uM concentrations and down-regulated BCL-XL, MCL-1, c-MYC in association with cell death induction. Compatible results were observed in ATR shRNA knock-down cells. Cell death induced by ATR inhibitors was significantly attenuated in cells ectopically expressing constitutively active STAT3, BCL-XL, MCL-1, or c-MYC. Concordant results were observed in primary human MM cells and in an in vivo MM xenograft model. Collectively, these findings argue for a non-canonical role for the ATR kinase in STAT3 activation in MM cells, and suggest that STAT3 inactivation contributes to the lethal actions of ATR inhibitors in MM.

O-GlcNAcylation promotes the cytosolic localization of the m6A reader YTHDF1 and colorectal cancer tumorigenesis

O-linked GlcNAc (O-GlcNAc) is an emerging post-translation modification that couples metabolism with cellular signal transduction by crosstalk with phosphorylation and ubiquitination to orchestrate various biological processes. The mechanisms underlying the involvement of O-GlcNAc modifications in N6-methyladenosine (m6A) regulation are not fully characterized. Herein, we show that O-GlcNAc modifies the m6A mRNA reader YTH domain family 1 (YTHDF1) and fine-tunes its nuclear translocation by the exportin protein Crm1. First, we present evidence that YTHDF1 interacts with the sole O-GlcNAc transferase (OGT). Second, we verified Ser196/Ser197/Ser198 as the YTHDF1 O-GlcNAcylation sites, as described in numerous chemoproteomic studies. Then we constructed the O-GlcNAc-deficient YTHDF1-S196A/S197F/S198A (AFA) mutant, which significantly attenuated O-GlcNAc signals. Moreover, we revealed that YTHDF1 is a nucleocytoplasmic protein, whose nuclear export is mediated by Crm1. Furthermore, O-GlcNAcylation increases the cytosolic portion of YTHDF1 by enhancing binding with Crm1, thus upregulating downstream target (e.g. c-Myc) expression. Molecular dynamics simulations suggest that O-GlcNAcylation at S197 promotes the binding between the nuclear export signal motif and Crm1 through increasing hydrogen bonding. Mouse xenograft assays further demonstrate that YTHDF1-AFA mutants decreased the colon cancer mass and size via decreasing c-Myc expression. In sum, we found that YTHDF1 is a nucleocytoplasmic protein, whose cytosolic localization is dependent on O-GlcNAc modification. We propose that the OGT-YTHDF1-c-Myc axis underlies colorectal cancer tumorigenesis.