Receptor Ligands Research Articles

Abstract 493: Unlocking solid tumors to nano-immunotherapy

Abstract Despite the remarkable efficiency of cancer immunotherapies, only a low percentage of patients achieve long-lasting clinical responses. Non-tumor cells within the tumor microenvironment (TME), including tumor vasculature and immune stromal cells, dictate therapeutic efficacy. The presence of germinal centers (GC) within the TME of cancer patients, including infiltrating T follicular helper (Tfh) cells and B cells, has been considered predictive of response to immunotherapies. We are developing nano-immunotherapies to induce immunological memory to control tumor relapse without any follow-up treatment.Nanomaterials co-incorporated tumor-associated antigens, clinically relevant toll-like receptor ligands, and regulators of tumor progression, namely the PD-L1/PD-1 expression and the secretion of TGF-β. Nanoparticle (NP) physicochemical properties were fully addressed. The immunotherapeutic potential of this nano-immunotherapy was addressed in melanoma, colorectal cancer (CRC), pancreatic cancer, and triple-negative breast cancer (TNBC) 3D models. These were created using patient-derived tumor cells, cancer-associated fibroblasts, and endothelial cells, to which paired patients' peripheral blood mononuclear cells were added to assess T-cell infiltration and tumor sprouting to identify the best nano-immunotherapy dosage/schedule. Our in vivo studies evaluated tumor volume, and animals’ survival, and characterized the tumor-infiltrating immune cells within TME, including the differentiation of Tfh cells and primary GC by flow cytometry.Our nano-immunotherapy remodeled the TME of B16F10 melanoma, MC38 CRC, 4T1 and E0771 TNBC, and KPC PDAC. Different TME subpopulations were identified as major blockers of anti-tumor immunity, and this knowledge guided the selection of combination approaches using modulators of those tumor immune evasion-related pathways (e.g., PD-L1, FAK). Our combination nano-immunotherapies tailored to address the tumor immune suppression profile found in vaccinated animals, significantly delayed tumor development, and increased disease-free survival rates. We found that the adjuvants delivered by our NP led to a ~4-fold increase in antibody production, being far more effective in generating GC responses. Thus, the effectiveness of combinational immunotherapy is at least partially related to the modulation of Tfh cells driving the formation of GC responses.Our nano-immunotherapy is a promising clinically translatable approach to defeating solid tumors. Acknowledgments: This work was supported by PTDC/BTM-SAL/4350/2021 and LCF/PR/HR22/52420016. Citation Format: Barbara Carreira, Rita C. Acúrcio, Ana I. Matos, Liane M. Moura, Ana C. Santos, Raquel Gouveia, Jéssica Cordeiro, Carina Peres, Daniela Vaskovich-Koubi, Ron Kleiner, Sabina Pozzi, Ronit Satchi-Fainaro, Helena F. Florindo. Unlocking solid tumors to nano-immunotherapy [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 493.

Abstract 3208: A nano sized system for the treatment of gastrointestinal cancers

Abstract Despite the recent progress in cancer immunotherapies, most patients do not benefit from the currently available immune checkpoint blockers, and many develop severe immune-related adverse events. Therefore, it is urgent to develop alternative, effective immunotherapeutic approaches. An example of non-responsive cancers are gastrointestinal (GI) cancers, which account for 26% of the global cancer incidence and 35% of all cancer-related deaths. In many GI cancers, the failure of immunotherapies to yield clinical response is partially attributed to the inefficient antigen presentation by DC, which results in insufficient T cell stimulation despite their expression of tumor-associated antigens (TAA). To overcome these hurdles, we designed novel polymeric-based nanoparticles (NP) to deliver a combination of GI cancers antigens of CEACAM5 protein and toll-like receptor ligands to DC. We chose to encapsulate CEACAM5 antigens because it is overexpressed in GI cancers, such as gastric cancer, colorectal cancer, and pancreatic cancer. With this approach, we aim to modulate the tumor milieu-immune cell interactions to an anti-cancer phenotype in order to control tumor growth and improve overall survival. Our NP present an average diameter below 200 nm, narrow polydispersity index, slightly negative surface charge, spherical morphology, and high antigens and immune potentiators loading. Cy5-labeled NPs were found to be extensively internalized by DC, which triggered their activation in vivo, shown by the increasing expression of DC-related co-stimulatory molecules such as CD80, CD86, and CD40. Furthermore, we assessed the immunotherapeutic effect of our NP on mouse models and on a human 3D multi-cellular spheroid ex-vivo model, composing the patient’s tumor cells and peripheral blood mononuclear cells (PBMC). We found that the NP successfully induced a potent immune-mediated anti-tumor response against CEACAM5-positive GI tumors, sensitizing the tumor microenvironment to other therapies and decreasing tumor volume, ultimately leading to prolonged survival. Taken together, the developed NP induced a strong antigen-specific immune response and overall sensitized CEACAM5-expressing GI cancers to the clinically relevant treatments in vivo, that so far have shown limited clinical outcomes in addition to a specific T cell anti-tumor activity that was shown on our patient-derived ex vivo model. Citation Format: Daniella Vaskovich, Rita Acúrcio, Ron Kleiner, Barbara Carreira, Ana Matos, Helena Florindo, Ronit Satchi-Fainaro. A nano sized system for the treatment of gastrointestinal 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 3208.

Abstract 3162: Antagonizing beta-2 adrenergic receptor’s function triggers death in glioma cells and down-regulates survivin

Abstract Introduction: Epidemiological studies showed that beta-adrenergic antagonists caused a low incidence of several cancer types. Glioblastoma highly expresses beta-2-adrenergic receptors. GBM accounts for 50% of central nervous system cancers. After standard-of-care therapeutic intervention, there is a survival time of 15 months. Our goal is to develop a translational therapy that will trigger the death of GBM cells while preserving healthy cells Methodology: Salmeterol xinafoate is a biased agonist that activates the G-protein pathway. Biased agonists activate signaling through the G-protein or the beta-arrestin pathway of G-protein coupled receptors. Dose-response experiments were performed using beta1 and beta2-adrenergic receptor antagonists and biased beta2-adrenergic receptor agonists to determine loss of glioma cell viability in GBM5, GBM12, and LN229 glioma cell lines. Western immunoblotting gauged survivin expression levels in all three lines. Epoxomicin was used to inhibit the proteasome complex. Annexin V and propidium iodide were employed to measure apoptotic or necrotic cell death. Results: Disrupting beta2-adrenergic receptor signaling by salmeterol xinafoate led to cell death of glioma cells but spared healthy neuron-glia cells using similar doses. Salmeterol xinafoate revealed greater efficacy for killing glioma cells than beta1 and beta2-adrenergic receptor antagonists. To determine the mechanism of glioma cell death, LN229 cells were treated with escalating doses of Salmeterol xinafoate, and at 24 hours, annexin V staining showed early apoptosis, and Annexin V/propidium iodide co-staining also showed late apoptosis. Propidium iodide revealed minor necrosis in LN229 glioma cells. Western immunoblotting showed salmeterol xinafoate downregulated survivin in a dose-dependent manner. However, the epoxomicin's inhibition of the proteasome complex prevented dose-escalated downregulation of survivin for all three glioma cell lines. Conclusion: The bitopic structure of salmeterol enables this drug to bind to the beta2-adrenergic receptors ligand binding site and an exosite site by its aryloxyalkyl tail that may contribute to the increased efficacy. Salmeterol xinafoate triggers glioma cell death by apoptosis at least in one glioma cell line. Salmeterol Xinafoate down-regulates survivin through protease degradation. Promising results show that the FDA-approved drug salmeterol xinafoate enables a favorable transition to the clinic. Repurposing this drug for GBMs may be combined with the current standard-of-care chemotherapies to enhance more significant loss of tumor volume and increase patient survival. Citation Format: Orli Algranatti, Ji Yae Lee, James M. Angelastro. Antagonizing beta-2 adrenergic receptor’s function triggers death in glioma cells and down-regulates survivin [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 3162.

Abstract 6013: VIO-01, a pan-DDR DNA decoy mediating DNA repair abrogation and unleashing the anti-tumor immune response

Abstract Background: Targeting specific DNA Damage Response (DDR) proteins has been worldwide studied and developed, with the example of PARP inhibitors being the only approved treatments in this field. Here, we describe the antineoplastic and immunomodulatory effects of VIO-01, a first-in-class DNA decoy-cholesterol conjugate, that operates as a pan repair proteins decoy, resulting in constitutive exhaustion of the DNA damage response. Methods: VIO-01-induced DDR proteins trapping and cell cytotoxicity were examined in both homologous recombination proficient (HRP) and deficient (HRD) cancer cells. DNA repair efficacy was monitored by analyzing repair protein recruitment to damage sites. RNAseq analysis in HRP/HRD ovarian cancer cells was employed to uncover the molecular mechanisms underlying VIO-01 effects. Effects on the innate and adaptive immune responses were assessed by following T-cell mediated anti-tumor cytotoxicity. VIO-01 antitumor efficacy and biodistribution were also assessed. Additionally, the interest of cholesterol as a vector for VIO-01 was analyzed compared to other ligands like the folate receptor ligand. Results: VIO-01 bounds to several DDR proteins, such as PARP1, KU70/80, MRN complex and MSH2/MSH3 with high affinity, resulting in an abrogation of single- and double-strand break repair. In line with this, and through global transcriptome analysis, VIO-01 treatment resulted in notable downregulation of Base Excision Repair, NHEJ and Nucleotide Excision Repair pathways in both HRP/HRD cell lines. Additionally, VIO-01 elicited the activation of the immune system and inflammatory responses in ovarian cancer cells. Conjointly, VIO-01 treatment induced a significant differentiation of monocytes into non-classical monocytes, leading to a shaping of the tumor microenvironment via CD8+ T cell recruitment in early stage of activation. The activity of VIO-01 was specific to tumor cells, while sparing healthy and immune cells, at odds with PARP inhibitors. In line with in vitro results, VIO-01 mediated antitumor efficacy in vivo coupled to tumor-targeting T-cell responses. These interesting effects were driven by a favorable ADME/PK profile, showing a long-lasting VIO-01 residence time VIO-01 into tumors (at least 3 days post treatment), coupled to a clear hijacking from the liver and a rapid blood clearance, ensuring a minimal toxicity. Moreover, regulatory toxicology studies demonstrated a favorable safety profile of VIO-01 in non-human primate with the major findings being a transient increase in complement factors induced by the binding of VIO-01 to factor H, which was not observed in human serum, predicting favorable safety profile in humans. Conclusions: Our results provide a preclinical rationale for using VIO-01 to trigger DNA damage exhaustion and an antitumor immune response, paving the way for rapid clinical application in patients bearing HRD or HRP tumors. Citation Format: Vlada Zakharova, Chloe Doizelet, Nicolas Babault, Marie-Christine Lienafa, Fetta Mazed, Megane Debiais, Pierre Vilela, Emilie Perroud, Wael Jdey. VIO-01, a pan-DDR DNA decoy mediating DNA repair abrogation and unleashing the anti-tumor immune response [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 6013.

Abstract 5228: Engineering CD123-targeting vδ1-enriched γδT cells with enhanced preclinical efficacy and safety profile

Abstract Acute myeloid leukemia (AML) is an aggressive hematological malignancy that, despite advances in treatment, has an overall 5-year survival rate of less than 30%. There is an urgent unmet need for new and effective therapies for AML. Chimeric antigen receptor (CAR)-modified cell therapies are emerging as promising options. However, the selection of suitable antigens is challenging, due to the heterogeneous expression of potential antigen targets in AML. Most antigens, such as CD123, are expressed on both leukemic cells and normal hematopoietic stem and progenitor cells. In addition, conventional autologous or allogeneic T cell therapies present many limitations, such as cytokine release syndrome (CRS) and graft-vs-host disease (GvHD). We therefore evaluated CD123-targeting CAR designs in vδ1-enriched γδT cells, an innate immune cell platform that is inherently non-MHC restricted, has low risks of CRS and GvHD, and is capable of discriminating between malignant and healthy cells in preclinical models. We engineered vδ1-enriched γδT cells from peripheral blood of healthy individuals to express various CD123-targeting CAR constructs. The CD123-targeting CAR-engineered vδ1-enriched γδT cells expressed high levels of natural cytotoxicity receptors, suggesting capability to target innate receptor ligands on AML cell surface. CD123-targeting CAR-expressing vδ1-enriched γδT cells demonstrated enhanced cytotoxicity in vitro against multiple AML cell lines, in both short-term co-culture and repeated antigen stimulation assays. CD123-targeting CAR-expressing vδ1-enriched γδT cells also showed enhanced efficacy and survival in vivo against mouse models of human AML. Interestingly, attenuation of CAR activity via novel designs preserved the inherent selectivity of vδ1-enriched γδT cells against AML cells relative to normal hematopoietic stem or progenitor cells, which also express CD123. Together, our results demonstrated through rational CAR designing and engineering in vδ1-enriched γδT cells, an allogeneic cell therapy targeting CD123 can be generated with selective cytotoxicity against AML cells while sparing normal hematopoietic stem cells. Such product designs have the potential to be a transformative therapeutic option for a broad population of AML patients. Citation Format: Mei Rosa Ng, Sarah L. Hayward, Di Zhang, Gary Gu, Bri Paulhus, Donjeta Gjuka, Collin Crowley, Sophie McLaughlin, Anya Lublinsky, Angel Mathew, Evans Berreondo Giron, Doanh Mai, Madisen Lee, Megha Wal, Rebecca Alade, Andre Simoes, Robin Lytle, James Gavin, Shira Cramer, Zhong-hua Yan, Taylor Hickman, Rashmi Choudhary, Cori Abikoff, Yana Wang, Istvan Kovacs, Nandhu M. Sobhana, Lan Cao. Engineering CD123-targeting vδ1-enriched γδT cells with enhanced preclinical efficacy and safety profile [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 5228.

Abstract 7463: Chromosomal instability elicits anti-tumor immunity in pancreatic cancer

Abstract Chromosomal instability (CIN) is a hallmark of solid cancers and an established driver of disease progression, therapy resistance, metastasis and on the whole poor patient outcome. Pancreatic cancer is particularly devastating, defined by dismal survival rates with many patients presenting with metastatic disease upon diagnosis, and 5-year survival only reaching ~10%. Despite recent efforts to improve prognosis for pancreatic cancer patients with immunotherapies alone and in combination with other therapeutics progress remains limited. Despite CIN’s established role as a driver of disease progression, we have made the paradoxical observation that chromosomally unstable, murine pancreatic cancer cells elicit an anti-tumor immune response. CIN was induced experimentally in a chromosomally stable, metastatic model of pancreatic cancer. Modeling metastatic dissemination by injection of cells into the tail vein of mice led to robust tumor formation in the lungs where CIN-low cells formed many small lesions while CIN-high cells formed fewer but larger tumors. We investigated if the tumors were infiltrated differentially by immune cells by immunofluorescence and found that CIN-high tumors were more readily infiltrated by effector T-cells. This observation motivated us to test immunotherapy response in CIN-low and CIN-high cells. Subcutaneously implanted tumor cells were treated with an immune checkpoint inhibitor and isotype control antibody which revealed that CIN-high cells responded to immunotherapy while CIN-low cells did not. To put forward potential mechanisms explaining CIN-driven anti-tumor immunity, we performed molecular analyses of CIN-low and CIN-high tumors. First, we found that inflammatory pathways were significantly up-regulated in CIN-high tumors compared with CIN-low tumors. These findings were validated by cytokine measurements and RNA in situ hybridization. To dissect inflammatory pathway activation mechanistically, we engineered CIN-high cells using CRISPR/Cas9 that were deficient for key inflammatory cytokines and transcription factors. These experiments suppressed cell-intrinsic and ligand-induced inflammation. Yet, we failed to determine singular cytokine expression as mediator of anti-tumor immunity. To rule out the contribution of ligand-induced inflammation in the tumor microenvironment, we probed immune checkpoint blockade in combination with antibodies blocking inflammatory ligand receptors in CIN-high cells. We found that immune checkpoint blockade was cancer cell-intrinsic and independent of blocking antibodies. In summary, our data implicates CIN as a determinant of therapeutic and genetic anti-tumor immunity in a preclinical model of pancreatic cancer. Citation Format: Daniel Bronder, Roshan K. Sriram, Mercedes Duran, Melody Di Bona, Sarina McElduff, Samuel F. Bakhoum. Chromosomal instability elicits anti-tumor immunity 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 7463.

Stereoselective Synthesis of the Gal-α-(1→3)-Gal-β-(1→3)-GlcNAc Trisaccharide: a new Ligand for DCAR and Mincle C-Type Lectin Receptors.

In this work, we have discovered that the Gal-α-(1→3)-Gal-β-(1→3)-GlcNAc trisaccharide, a fragment of the B antigen Type-1, is a new ligand of two C-type lectin receptors (CLRs) i. e. DCAR and Mincle which are key players in different types of autoimmune diseases. Accordingly, we report here on a straightforward methodology to access pure Gal-α-(1→3)-Gal-β-(1→3)-GlcNAc trisaccharide. A spacer with a terminal primary amine group was included at the reducing end of the GlcNAc residue thus ensuring the further functionalization of the trisaccharide Gal-α-(1→3)-Gal-β-(1→3)-GlcNAc.

Virus-Induced Acute Respiratory Distress Syndrome Causes Cardiomyopathy Through Eliciting Inflammatory Responses in the Heart.

Viral infections can cause acute respiratory distress syndrome (ARDS), systemic inflammation, and secondary cardiovascular complications. Lung macrophage subsets change during ARDS, but the role of heart macrophages in cardiac injury during viral ARDS remains unknown. Here we investigate how immune signals typical for viral ARDS affect cardiac macrophage subsets, cardiovascular health, and systemic inflammation. We assessed cardiac macrophage subsets using immunofluorescence histology of autopsy specimens from 21 patients with COVID-19 with SARS-CoV-2-associated ARDS and 33 patients who died from other causes. In mice, we compared cardiac immune cell dynamics after SARS-CoV-2 infection with ARDS induced by intratracheal instillation of Toll-like receptor ligands and an ACE2 (angiotensin-converting enzyme 2) inhibitor. In humans, SARS-CoV-2 increased total cardiac macrophage counts and led to a higher proportion of CCR2+ (C-C chemokine receptor type 2 positive) macrophages. In mice, SARS-CoV-2 and virus-free lung injury triggered profound remodeling of cardiac resident macrophages, recapitulating the clinical expansion of CCR2+ macrophages. Treating mice exposed to virus-like ARDS with a tumor necrosis factor α-neutralizing antibody reduced cardiac monocytes and inflammatory MHCIIlo CCR2+ macrophages while also preserving cardiac function. Virus-like ARDS elevated mortality in mice with pre-existing heart failure. Our data suggest that viral ARDS promotes cardiac inflammation by expanding the CCR2+ macrophage subset, and the associated cardiac phenotypes in mice can be elicited by activating the host immune system even without viral presence in the heart.

BMP9 is a key player in endothelial identity and its loss is sufficient to induce arteriovenous malformations.

BMP9 is a high affinity ligand of ALK1 and endoglin receptors that are mutated in the rare genetic vascular disorder hereditary hemorrhagic telangiectasia (HHT). We have previously shown that loss of Bmp9 in the 129/Ola genetic background leads to spontaneous liver fibrosis via capillarization of liver sinusoidal endothelial cells (LSEC) and kidney lesions. We aimed to decipher the molecular mechanisms downstream of BMP9 to better characterize its role in vascular homeostasis in different organs. For this, we performed an RNA-seq analysis on LSEC from adult WT and Bmp9-KO mice and identified over 2000 differentially expressed genes. Gene ontology analysis showed that Bmp9 deletion led to a decrease in BMP and Notch signalling, but also LSEC capillary identity while increasing their cell cycle. The gene ontology term 'glomerulus development' was also negatively enriched in Bmp9-KO mice vs. WT supporting a role for BMP9 in kidney vascularization. Through different imaging approaches (electron microscopy, immunostainings), we found that loss of Bmp9 led to vascular enlargement of the glomeruli capillaries associated with alteration of podocytes. Importantly, we also showed for the first time that the loss of Bmp9 led to spontaneous arteriovenous malformations (AVMs) in the liver, gastrointestinal tract, and uterus. Altogether, these results demonstrate that BMP9 plays an important role in vascular quiescence both locally in the liver by regulating endothelial capillary differentiation markers and cell cycle but also at distance in many organs via its presence in the circulation. It also reveals that loss of Bmp9 is sufficient to induce spontaneous AVMs, supporting a key role for BMP9 in the pathogenesis of HHT.

Fundamental Mechanisms in Membrane Receptology: Old Paradigms, New Concepts and Perspectives

Receptology, the science of receptors, is a multidimensional field of research which can be dissected into biosynthesis, membrane sorting, ligand binding and signal transduction. Plasma membrane receptors connect the cells with their environment and transmit signals that are translated into biological information. The historical paradigm of ligand–receptor interactions is the lock-and-key model. This model presupposes that both partners have a precise 3D shape that perfectly fits together to form the ligand–receptor complex. However, this simple model suffers from severe limitations due to several levels of simplifications: (i) water molecules and membrane lipids are not considered; (ii) not all ligands have a stable 3D structure; (iii) the ligand-binding pocket of the receptor is often flexible and conformationally rearranged after the initial binding step (induced fit mechanism) and/or subjected to conformational selection by the ligand; (iv) there are signal transduction mechanisms which can be either purely mechanical (conformational change of the receptor induced after binding of the ligand), lipid-assisted (e.g., by raft lipids such as cholesterol or gangliosides), or in some instances of quantic nature (detection of odorant molecules). The aim of the present review is to challenge the old paradigms and present new concepts of membrane receptology that consider the impact of critical parameters such as water molecules, membrane lipids, electrostatic surface potential and quantum mechanisms.

The monocyte-derived cytokine response in whole blood from preterm newborns against sepsis-related bacteria is similar to term newborns and adults.

Sepsis is characterized by a dysregulated innate immune response. It is a leading cause of morbidity and mortality in newborns, in particular for newborns that are born premature. Although previous literature indicate that the pro-inflammatory response may be impaired in preterm newborns, serum levels of monocyte-derived cytokines, such as TNF-α and IL-6, vary highly between newborns and can reach adult-like concentrations during sepsis. These contradictory observations and the severe consequences of neonatal sepsis in preterm newborns highlight the need for a better understanding of the pro-inflammatory cytokine response of preterm newborns to improve sepsis-related outcomes. Using an in vitro model with multiple read outs at the transcriptional and protein level, we consistently showed that the monocyte-derived cytokine response induced by sepsis-related bacteria is comparable between preterm newborns, term newborns and adults. We substantiated these findings by employing recombinant Toll-like receptor (TLR) ligands and showed that the activation of specific immune pathways, including the expression of TLRs, is also similar between preterm newborns, term newborns and adults. Importantly, we showed that at birth the production of TNF-α and IL-6 is highly variable between individuals and independent of gestational age. These findings indicate that preterm newborns are equally capable of mounting a pro-inflammatory response against a broad range of bacterial pathogens that is comparable to term newborns and adults. Our results provide a better understanding of the pro-inflammatory response by preterm newborns and could guide the development of interventions that specifically modulate the pro-inflammatory response during sepsis in preterm newborns.

Validation of CDC45 as a novel biomarker for diagnosis and prognosis of gastric cancer.

Cell division cycle protein 45 (CDC45) has been demonstrated to play vital roles in the progression of various malignancies. However, the clinical significance of CDC45 in gastric cancer (GC) remains unreported. In this study, we employed the TCGA database and the TCGA & GTEx dataset to compare the mRNA expression levels of CDC45 between gastric cancer tissues and adjacent or normal tissues (p < 0.05 was considered statistically significant), which was further validated in multiple datasets including GSE13911, GSE29272, GSE118916, GSE66229, as well as RT-qPCR. Furthermore, we harnessed the Human Protein Atlas (HPA) to evaluate the protein expression of CDC45, which was subsequently verified through immunohistochemistry (IHC). To ascertain the diagnostic utility of CDC45, receiver operating characteristic (ROC) curves and the area under the ROC curve (AUC) were calculated in TCGA database, and further validated it in TCGA & GTEx and GSE66229 datasets. The Kaplan-Meier method was used to reveal the prognostic importance of CDC45 in The Cancer Genome Atlas (TCGA) database and authenticated through the GSE66229, GSE84433, and GSE84437 datasets. Through cBioPortal, we identified co-expressed genes of CDC45, and pursued enrichment analysis. Additionally, we availed gene set enrichment analysis (GSEA) to annotate the biological functions of CDC45. Differential expression analysis revealed that CDC45 was significantly upregulated at both the mRNA and protein levels in GC (all p < 0.05). Remarkably, CDC45 emerged as a promising prognostic indicator and a novel diagnostic biomarker for GC. In a comprehensive the drug susceptibility analysis, we found that patients with high expression of CDC45 had high sensitivity to various chemotherapeutic agents, among which 5-fluorouracil, docetaxel, cisplatin, and elesclomol were most evident. Furthermore, our findings suggested a plausible association between CDC45 and immune cell infiltration. Enrichment analysis revealed that CDC45 and its associated genes may play crucial roles in muscle biofunction, whereas GSEA demonstrated significant enrichment of gene sets pertaining to G protein-coupled receptor ligand binding and G alpha (i) signaling events. Our study elucidates that upregulation of CDC45 is intricately associated with immune cell infiltration and holds promising potential as a favorable prognostic marker and a novel diagnostic biomarker for GC.

Simple ammonium salt and sigma-1 receptor ligand dipentylammonium provides neuroprotective effects in cell culture and Caenorhabditis elegans models of Alzheimer's disease

The sigma-1 receptor (σ-1R), a chaperone protein located at the mitochondria-associated membrane (MAM) of the endoplasmic reticulum, can interact with and modify the signaling pathways of various proteins, thereby modulating many disease pathologies, including Alzheimer's disease (AD). The σ-1R ligand dipentylammonium (DPA) was analyzed for its anti-AD properties using PC12 cells (in vitro) and Caenorhabditis elegans (in vivo) models along with molecular docking (in silico) analysis. DPA at 1 and 10 µM concentrations was able to significantly potentiate NGF-induced neurite growth length by 137.7 ± 12.0 and 187.8 ± 16.4, respectively, when compared to the control 76.9 ± 7.4. DPA also regulated neurite damage caused by Aβ(25−35) treatment in differentiated PC12 cells by improving cell viability and neurite length. In C. elegans, DPA could significantly extend the median and maximum lifespan of Aβ transgenic strain CL2006 without impacting wild-type nematodes. Additionally, it could significantly reduce the paralysis phenotype of another Aβ transgenic strain, CL4176, thereby improving the overall health in AD pathogenesis. This effect depended on σ-1R, as DPA could not modulate the lifespan of σ-1R mutant TM3443. This was further confirmed using agonist PRE084 and antagonist BD1047, wherein the agonist alone could extend the lifespan of CL2006, while the antagonist suppressed the effect of DPA in CL2006. Interestingly, neither had an TM3443. Further, molecular docking analysis showed that DPA had a similar binding affinity as that of PRE084, BD1047 and pentazocine against the σ-1R receptor in humans and C. elegans, which collectively suggests the anti-AD properties of DPA.

The Effect of Retinoic Acid on Arsenite-Transformed Malignant UROtsa Bladder Cancer Cells: In Vitro Model of Basal Muscle-Invasive Bladder Cancer.

Bladder cancer (BC) is the eighth most common cause of cancer death in the United States of America. BC is classified into non-muscle-invasive bladder cancer (NMIBC) and muscle-invasive bladder cancer (MIBC). Genetically, MIBCs are categorized into the more aggressive basal subtype or less aggressive luminal subtype. All-trans retinoic acid (tretinoin), the ligand for the RAR-RXR retinoic acid receptor, is clinically used as a differentiation therapy in hematological malignancies. This study aims to determine the effects of retinoic acid on arsenite-transformed malignant urothelial cells (UROtsa As), serving as a model for basal muscle-invasive bladder cancer. We treated three independent isolates of arsenite-transformed malignant human urothelial UROtsa cells (UROtsa As) with tretinoin for 48 h. Cell viability, proliferation, and apoptosis were analyzed using crystal violet staining and flow cytometry. mRNA and protein level analyses were performed using RT-qPCR and the Simple Western™ platform, respectively. Tretinoin was found to reduce cell proliferation and urosphere formation, as well as decrease the expression of basal markers (KRT1, KRT5, KRT6, EGFR) and increase the expression of luminal differentiation markers (GATA3, FOXA1). Mechanistically, the antiproliferative effect of tretinoin was attributed to the downregulation of c-myc. Our results suggest that targeting the retinoic acid pathway can diminish the aggressive behavior of basal muscle-invasive urothelial cancer and may enhance patient survival.

Androgens and Notch signaling cooperate in seminiferous epithelium to regulate genes related to germ cell development and apoptosis

It was reported previously that in adult males disruption of both androgen and Notch signaling impairs spermatid development and germ cell survival in rodent seminiferous epithelium. To explain the molecular mechanisms of these effects, we focused on the interaction between Notch signaling and androgen receptor (AR) in Sertoli cells and investigate its role in the control of proteins involved in apical ectoplasmic specializations, actin remodeling during spermiogenesis, and induction of germ cell apoptosis. First, it was revealed that in rat testicular explants ex vivo both testosterone and Notch signaling modulate AR expression and cooperate in the regulation of spermiogenesis-related genes (Nectin2, Afdn, Arp2, Eps8) and apoptosis-related genes (Fasl, Fas, Bax, Bcl2). Further, altered expression of these genes was found following exposure of Sertoli cells (TM4 cell line) and germ cells (GC-2 cell line) to ligands for Notch receptors (Delta-like1, Delta-like4, and Jagged1) and/or Notch pathway inhibition. Finally, direct interactions of Notch effector, Hairy/enhancer-of-split related with YRPW motif protein 1, and the promoter of Ar gene or AR protein were revealed in TM4 Sertoli cells. In conclusion, Notch pathway activity in Sertoli and germ cells regulates genes related to germ cell development and apoptosis acting both directly and indirectly by influencing androgen signaling in Sertoli cells.

TRAF3 regulation of proximal TLR signaling in B cells.

Toll-like receptors are pattern recognition receptors that bridge the innate and adaptive immune responses and are critical for host defense. Most studies of Toll-like receptors have focused upon their roles in myeloid cells. B lymphocytes express most Toll-like receptors and are responsive to Toll-like receptor ligands, yet Toll-like receptor-mediated signaling in B cells is relatively understudied. This is an important knowledge gap, as Toll-like receptor functions can be cell type specific. In striking contrast to myeloid cells, TRAF3 inhibits TLR-mediated functions in B cells. TRAF3-deficient B cells display enhanced IRF3 and NFκB activation, cytokine production, immunoglobulin isotype switching, and antibody production in response to Toll-like receptors 3, 4, 7, and 9. Here, we address the question of how TRAF3 impacts initial B-cell Toll-like receptor signals to regulate downstream activation. We found that TRAF3 in B cells associated with proximal Toll-like receptor 4 and 7 signaling proteins, including MyD88, TRAF6, and the tyrosine kinase Syk. In the absence of TRAF3, TRAF6 showed a greater association with several Toll-like receptor signaling proteins, suggesting that TRAF3 may inhibit TRAF6 access to Toll-like receptor signaling complexes and thus early Toll-like receptor signaling. In addition, our results highlight a key role for Syk in Toll-like receptor signaling in B cells. In the absence of TRAF3, Syk activation was enhanced in response to ligands for Toll-like receptors 4 and 7, and Syk inhibition reduced downstream Toll-like receptor-mediated NFκB activation and proinflammatory cytokine production. This study reveals multiple mechanisms by which TRAF3 serves as a key negative regulator of early Toll-like receptor signaling events in B cells.

Construction of an immune-related gene signature for overall survival prediction and immune infiltration in gastric cancer.

Treatment options for patients with gastric cancer (GC) continue to improve, but the overall prognosis is poor. The use of PD-1 inhibitors has also brought benefits to patients with advanced GC and has gradually become the new standard treatment option at present, and there is an urgent need to identify valuable biomarkers to classify patients with different characteristics into subgroups. To determined the effects of differentially expressed immune-related genes (DEIRGs) on the development, prognosis, tumor microenvironment (TME), and treatment response among GC patients with the expectation of providing new biomarkers for personalized treatment of GC populations. Gene expression data and clinical pathologic information were downloaded from The Cancer Genome Atlas (TCGA), and immune-related genes (IRGs) were searched from ImmPort. DEIRGs were extracted from the intersection of the differentially-expressed genes (DEGs) and IRGs lists. The enrichment pathways of key genes were obtained by analyzing the Kyoto Encyclopedia of Genes and Genomes (KEGGs) and Gene Ontology (GO) databases. To identify genes associated with prognosis, a tumor risk score model based on DEIRGs was constructed using Least Absolute Shrinkage and Selection Operator and multivariate Cox regression. The tumor risk score was divided into high- and low-risk groups. The entire cohort was randomly divided into a 2:1 training cohort and a test cohort for internal validation to assess the feasibility of the risk model. The infiltration of immune cells was obtained using 'CIBERSORT,' and the infiltration of immune subgroups in high- and low-risk groups was analyzed. The GC immune score data were obtained and the difference in immune scores between the two groups was analyzed. We collected 412 GC and 36 adjacent tissue samples, and identified 3627 DEGs and 1311 IRGs. A total of 482 DEIRGs were obtained. GO analysis showed that DEIRGs were mainly distributed in immunoglobulin complexes, receptor ligand activity, and signaling receptor activators. KEGG pathway analysis showed that the top three DEIRGs enrichment types were cytokine-cytokine receptors, neuroactive ligand receptor interactions, and viral protein interactions. We ultimately obtained an immune-related signature based on 10 genes, including 9 risk genes (LCN1, LEAP2, TMSB15A mRNA, DEFB126, PI15, IGHD3-16, IGLV3-22, CGB5, and GLP2R) and 1 protective gene (LGR6). Kaplan-Meier survival analysis, receiver operating characteristic curve analysis, and risk curves confirmed that the risk model had good predictive ability. Multivariate COX analysis showed that age, stage, and risk score were independent prognostic factors for patients with GC. Meanwhile, patients in the low-risk group had higher tumor mutation burden and immunophenotype, which can be used to predict the immune checkpoint inhibitor response. Both cytotoxic T lymphocyte antigen4+ and programmed death 1+ patients with lower risk scores were more sensitive to immunotherapy. In this study a new prognostic model consisting of 10 DEIRGs was constructed based on the TME. By providing risk factor analysis and prognostic information, our risk model can provide new directions for immunotherapy in GC patients.

CYP7A1 Gene Induction via SHP-Dependent or Independent Mechanisms can Increase the Risk of Drug-Induced Liver Injury Independently or in Synergy with BSEP Inhibition.

Drug-induced liver injury (DILI) is a frequent cause of clinical trial failures during drug development. While inhibiting bile salt export pump (BSEP) is a well-documented DILI mechanism, interference with genes related to bile acid (BA) metabolism and transport can further complicate DILI development. Here, the effects of twenty-eight compounds on genes associated with BA metabolism and transport were evaluated, including those with discontinued development or use, boxed warnings, and clean labels for DILI. The study also included rifampicin and omeprazole, pregnane X receptor and aryl hydrocarbon receptor ligands, and four mitogen-activated protein kinase kinase (MEK1/2) inhibitors. BSEP inhibitors with more severe DILI, notably pazopanib and CP-724714, significantly upregulated the expression of 7 alpha-hydroxylase (CYP7A1), independent of small heterodimer partner (SHP) expression. CYP7A1 expression was marginally induced by omeprazole. In contrast, its expression was suppressed by mometasone (10-fold), vinblastine (18-fold), hexachlorophene (2-fold), bosentan (2.1-fold), and rifampin (2-fold). All four MEK1/2 inhibitors that show clinical DILI were not potent BSEP inhibitors but significantly induced CYP7A1 expression, accompanied by a significant SHP gene suppression. Sulfotransferase 2A1 and BSEP were marginally upregulated, but no other genes were altered by the drugs tested. Protein levels of CYP7A1 were increased with the treatment of CYP7A1 inducers and decreased with obeticholic acid, an farnesoid X receptor ligand. CYP7A1 inducers significantly increased bile acid (BA) production in hepatocytes, indicating the overall regulatory effects of BA metabolism. This study demonstrates that CYP7A1 induction via various mechanisms can pose a risk for DILI, independently or in synergy with BSEP inhibition, and it should be evaluated early in drug discovery. SIGNIFICANCE STATEMENT: Kinase inhibitors, pazopanib and CP-724714, inhibit BSEP and induce CYP7A1 expression independent of small heterodimer partner (SHP) expression, leading to increased bile acid (BA) production and demonstrating clinically elevated drug-induced liver toxicity. MEK1/2 inhibitors that show BSEP-independent drug-induced liver injury (DILI) induced the CYP7A1 gene accompanied by SHP suppression. CYP7A1 induction via SHP-dependent or independent mechanisms can pose a risk for DILI, independently or in synergy with BSEP inhibition. Monitoring BA production in hepatocytes can reliably detect the total effects of BA-related gene regulation for de-risking.

Topical Peroxisome Proliferator-Activated Receptor Agonist Induces Molecular Alterations Enhancing Barrier Function and Water-Holding Capacity of the Human Stratum Corneum In Vivo

The peroxisome proliferator-activated receptor (PPAR) ligands modulate a variety of skin functions but are rarely used in cosmetics. The aim of this double-blind, placebo-controlled, in vivo study was to determine the effect of a topically applied 0.1% PPAR ligand on the composition and physiological parameters of the stratum corneum (SC). By comparing verum and placebo groups post-treatment, we demonstrate (via lipidomic analysis of tape strips) an unstatistically significant trend toward an increase in long-chain triacylglycerols (C50–C56) and medium- and long-chain ceramides (C42–C50) at the superficial SC. By comparing treated and untreated skin using confocal Raman microspectroscopy, we found that the changes in lipid composition in the verum group led to a significant increase in the number of trans conformers and orthorhombic organisation of lipids at the exemplary SC depth. An increase in unfolded states in the secondary and tertiary keratin structures results in an increased ability to bind water. The concentrations of tightly and strongly bound water increase, while weakly bound and unbound water decrease in the entire SC, indicating a transformation of water mobility to a state of increased hydrogen bonding. Thus, the topical PPAR ligands improve the water-holding capacity and the barrier function of the SC.

Are there sex differences in oxytocin and vasopressin V1a receptors ligand binding affinities?

There is substantial evidence for sex differences in the functioning of one of the most common receptor systems; G protein-coupled receptors (GPCRs). There are many points along the GPCR-mediated molecular signaling pathway at which males and females may differ, one of the first of which, chronologically, is in the stability of the interaction between the ligand and the receptor, or its binding affinity. Here we investigate the binding affinities of oxytocin (OT) and vasopressin (AVP) at the oxytocin receptor (OTR) and the vasopressin V1a receptor (V1aR), both of which are present in numerous in brain regions associated with social behavior. In order to investigate sex- and estrous cycle-dependent differences in ligand-receptor binding affinity, male (n = 6) Syrian hamsters (Mesocricetus auratus), females on the day of estrus (E females, n = 6), and females on the second day of diestrus (D2 females n = 6) were chosen for study. Brains from hamsters were mounted on slides and competition and saturation binding assays were conducted. We report a remarkable similarity in the binding affinities of OT and AVP in males and females. Small differences were detected, however, in receptor and ligand specificity in females depending on whether they were in the estrous or diestrous stage of their ovulatory cycle. These data suggest that sex differences in binding affinity are not a likely source of the many sex differences that have been observed in the effects of OT and AVP in hamsters and other species.