Integration Of Signaling Research Articles

Abstract 3490: Unveiling sex differences in lung adenocarcinoma through multi-omics integrative protein signaling networks

Abstract Sex differences in lung adenocarcinoma (LUAD) are evident in incidence rates, prognostic outcomes, and therapy responses, yet the underlying molecular mechanisms driving these disparities remain underexplored. In this study, we conducted a comprehensive proteogenomic analysis encompassing 38 females and 73 males with LUAD from the Clinical Proteomic Tumor Analysis Consortium (CPTAC) dataset. Employing Transcription Inference using Gene Expression and Regulatory data (TIGER), we inferred sex-differentially activated transcription factors (TFs) from The Cancer Genome Atlas (TCGA) LUAD gene expression data and identified sex-differentially activated kinases using CPTAC protein phosphorylation data. We further constructed a comprehensive kinase-TF signaling network by integrating these sex-differentially activated kinases with TFs, identifying all paths shorter than 3 in the protein interaction networks to highlight druggable pathways. Our analyses revealed that many proteins exhibit not only sex-biased abundance but also sex-biased phosphorylation and acetylation. Furthermore, these sex-biased proteins were associated with critical biological pathways including cell proliferation, immune response, and metabolism. Using kinase-TF signaling networks, we found substantial sex bias in the activities of clinically actionable TFs and kinases, including the glucocorticoid receptor (NR3C1), AR, AURKA, CDK6, and MAPK14. Leveraging the PRISM cancer cell line screening database, we identified several small-molecule drugs, such as glucocorticoid receptor agonists and aurora kinase inhibitors, potentially exhibiting sex-specific efficacy as LUAD therapeutics. Our findings showed that the activity of some clinically relevant TFs and kinases differ by sex in LUAD, underscoring the need to consider sex as a biological variable and the utility of multi-omics integrative protein signaling networks in advancing our understanding of cancer biology and the development of sex-aware therapeutics. Citation Format: Chen Chen, Enakshi Saha, Dawn L. DeMeo, John Quackenbush, Camila M. Lopes-Ramos. Unveiling sex differences in lung adenocarcinoma through multi-omics integrative protein signaling networks [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 3490.

Integrated single-cell sequencing, spatial transcriptome sequencing and bulk RNA sequencing highlights the molecular characteristics of parthanatos in gastric cancer.

Parthanatos is a novel programmatic form of cell death based on DNA damage and PARP-1 dependency. Nevertheless, its specific role in the context of gastric cancer (GC) remains uncertain. In this study, we integrated multi-omics algorithms to investigate the molecular characteristics of parthanatos in GC. A series of bioinformatics algorithms were utilized to explore clinical heterogeneity of GC and further predict the clinical outcomes. Firstly, we conducted a comprehensive analysis of the omics features of parthanatos in various human tumors, including genomic mutations, transcriptome expression, and prognostic relevance. We successfully identified 7 cell types within the GC microenvironment: myeloid cell, epithelial cell, T cell, stromal cell, proliferative cell, B cell, and NK cell. When compared to adjacent non-tumor tissues, single-cell sequencing results from GC tissues revealed elevated scores for the parthanatos pathway across multiple cell types. Spatial transcriptomics, for the first time, unveiled the spatial distribution characteristics of parthanatos signaling. GC patients with different parthanatos signals often exhibited distinct immune microenvironment and metabolic reprogramming features, leading to different clinical outcomes. The integration of parthanatos signaling and clinical indicators enabled the creation of novel survival curves that accurately assess patients' survival times and statuses. In this study, the molecular characteristics of parthanatos' unicellular and spatial transcriptomics in GC were revealed for the first time. Our model based on parthanatos signals can be used to distinguish individual heterogeneity and predict clinical outcomes in patients with GC.

Fluorescence Biosensing Based on Bifurcated DNA Scaffold-Aggregated Ag Nanocluster via Responsive Conformation Switch of Quasi-Molecular Beacon.

To address the limitations of typical hairpin-structural molecular beacons, exploring the ability of a quasi-molecular beacon (qMB) to create label-free fluorescence biosensors is intriguing and remains a challenge. Herein, we propose the first example of modular qMB with the feature of a stimulation-responsive conformation switch to develop an aggregated Ag nanocluster (aAgNC) in a bifurcated DNA scaffold for fluorescently sensing a specific initiator (I*). This qMB was well designed to program four functional modules: I*-recognizable element adopting metastable stem-loop bihairpin structure and two DNA splits (exposed C3GT4 and locked C4AC4T) of aAgNC template that is separated by a tunable hairpin spacer for the customized combination of selective recognition and signaling readout. When presenting I* in an assay route, the specific hybridization induces the directional disassembly of the bihairpin unit, on which the qMB is configurationally switched to liberate the locked split. Thus, the bifurcated parent template pair of C3GT4/C4AC4T is proximal, affording in situ nucleation and clustering of emissive aAgNC. By collecting the fluorescence signal, the quantitative detection of I* is achieved. Benefiting from the ingenious programming of qMB, the recognizing and signaling integration actuates the construction of a facile and convenient fluorescent biosensor featuring rapid reaction kinetics, a wide linear range, high sensitivity, and specificity. This would provide a new paradigm to exploit versatile qMB-based biosensing platforms via stimulation-responsive conformation switches for developing various DNA-scaffolded Ag clusters.

Ethylene Modulates Rice Root Plasticity under Abiotic Stresses

Plants live in constantly changing environments that are often unfavorable or stressful. Root development strongly affects plant growth and productivity, and the developmental plasticity of roots helps plants to survive under abiotic stress conditions. This review summarizes the progress being made in understanding the regulation of the phtyohormone ethylene in rice root development in response to abiotic stresses, highlighting the complexity associated with the integration of ethylene synthesis and signaling in root development under adverse environments. Understanding the molecular mechanisms of ethylene in regulating root architecture and response to environmental signals can contribute to the genetic improvement of crop root systems, enhancing their adaptation to stressful environmental conditions.

Epigenetic integration of signaling from the regenerative environment.

Skeletal muscle has an extraordinary capacity to regenerate itself after injury due to the presence of tissue-resident muscle stem cells. While these muscle stem cells are the primary contributor to the regenerated myofibers, the process occurs in a regenerative microenvironment where multiple different cell types act in a coordinated manner to clear the damaged myofibers and restore tissue homeostasis. In this regenerative environment, immune cells play a well-characterized role in initiating repair by establishing an inflammatory state that permits the removal of dead cells and necrotic muscle tissue at the injury site. More recently, it has come to be appreciated that the immune cells also play a crucial role in communicating with the stem cells within the regenerative environment to help coordinate the timing of repair events through the secretion of cytokines, chemokines, and growth factors. Evidence also suggests that stem cells can help modulate the extent of the inflammatory response by signaling to the immune cells, demonstrating a cross-talk between the different cells in the regenerative environment. Here, we review the current knowledge on the innate immune response to sterile muscle injury and provide insight into the epigenetic mechanisms used by the cells in the regenerative niche to integrate the cellular cross-talk required for efficient muscle repair.

Arabidopsis BBX14 is involved in high light acclimation and seedling development.

The development of photosynthetically competent seedlings requires both light and retrograde biogenic signaling pathways. The transcription factor GLK1 functions at the interface between these pathways and receives input from the biogenic signal integrator GUN1. BBX14 was previously identified, together with GLK1, in a core module that mediates the response to high light (HL) levels and biogenic signals, which was studied by using inhibitors of chloroplast development. Our chromatin immunoprecipitation-Seq experiments revealed that BBX14 is a direct target of GLK1, and RNA-Seq analysis suggests that BBX14 may function as a regulator of the circadian clock. In addition, BBX14 plays a role in chlorophyll biosynthesis during early onset of light. Knockout of BBX14 results in a long hypocotyl phenotype dependent on a retrograde signal. Furthermore, the expression of BBX14 and BBX15 during biogenic signaling requires GUN1. Investigation of the role of BBX14 and BBX15 in GUN-type biogenic (gun) signaling showed that the overexpression of BBX14 or BBX15 caused de-repression of CA1 mRNA levels, when seedlings were grown on norflurazon. Notably, transcripts of the LHCB1.2 marker are not de-repressed. Furthermore, BBX14 is required to acclimate plants to HL stress. We propose that BBX14 is an integrator of biogenic signals and that BBX14 is a nuclear target of retrograde signals downstream of the GUN1/GLK1 module. However, we do not classify BBX14 or BBX15 overexpressors as gun mutants based on a critical evaluation of our results and those reported in the literature. Finally, we discuss a classification system necessary for the declaration of new gun mutants.

Structural and enzymatic evidence for the methylation of the ACK1 tyrosine kinase by the histone lysine methyltransferase SETD2

SETD2 (SET-domain containing protein 2) is a histone methyltransferase (HMT) of the SET family responsible for the trimethylation of K36 of histone H3, thus producing the epigenetic mark H3K36me3. Recent studies have shown that certain SET family HMTs, such as SMYD2, SMYD3 or SETDB1 can also methylate protein kinases and therefore be involved in signaling pathways. Here we provide structural and enzymatic evidence showing that SETD2 methylates the protein tyrosine kinase ACK1 in vitro. ACK1 is recognized as a major integrator of signaling from various receptor tyrosine kinases. Using ACK1 peptides and recombinant proteins, we show that SETD2 methylates the K514 residue of ACK1 generating K514 mono, di or tri-methylation. Interestingly, K514 is found in a “H3K36-like” motif of ACK1 which is known to be post-translationally modified and to be involved in protein-protein interaction. The crystal structure of SETD2 catalytic domain in complex with an ACK1 peptide further provides the structural basis for the methylation of ACK1 K514 by SETD2. Our work therefore strongly suggests that ACK1 could be a novel non-histone substrate of SETD2 and further supports that SET HMTs, such as SETD2, could be involved in both epigenetic regulations and cell signaling.

Genome-wide identification of QTLs associated with iron deficiency tolerance in soybean reveals GATA12 as the integrator of iron signaling in roots

Genome-wide identification of QTLs associated with iron deficiency tolerance in soybean reveals GATA12 as the integrator of iron signaling in roots

Detecting and dissecting signaling crosstalk via the multilayer network integration of signaling and regulatory interactions.

The versatility of cellular response arises from the communication, or crosstalk, of signaling pathways in a complex network of signaling and transcriptional regulatory interactions. Understanding the various mechanisms underlying crosstalk on a global scale requires untargeted computational approaches. We present a network-based statistical approach, MuXTalk, that uses high-dimensional edges called multilinks to model the unique ways in which signaling and regulatory interactions can interface. We demonstrate that the signaling-regulatory interface is located primarily in the intermediary region between signaling pathways where crosstalk occurs, and that multilinks can differentiate between distinct signaling-transcriptional mechanisms. Using statistically over-represented multilinks as proxies of crosstalk, we infer crosstalk among 60 signaling pathways, expanding currently available crosstalk databases by more than five-fold. MuXTalk surpasses existing methods in terms of model performance metrics, identifies additions to manual curation efforts, and pinpoints potential mediators of crosstalk. Moreover, it accommodates the inherent context-dependence of crosstalk, allowing future applications to cell type- and disease-specific crosstalk.

The heat response regulators HSFA1s promote Arabidopsis thermomorphogenesis via stabilizing PIF4 during the day.

During summer, plants often experience increased light inputs and high temperatures, two major environmental factors with contrasting effects on thermomorphological traits. The integration of light and temperature signaling to control thermomorphogenesis in plants is critical for their acclimation in such conditions, but the underlying mechanisms remain largely unclear. We found that heat shock transcription factor 1d (HSFA1d) and its homologs are necessary for plant thermomorphogenesis during the day. In response to warm daytime temperature, HSFA1s markedly accumulate and move into the nucleus where they interact with phytochrome-interacting factor 4 (PIF4) and stabilize PIF4 by interfering with phytochrome B-PIF4 interaction. Moreover, we found that the HSFA1d nuclear localization under warm daytime temperature is mediated by constitutive photomorphogenic 1-repressed GSK3-like kinase BIN2. These results support a regulatory mechanism for thermomorphogenesis in the daytime mediated by the HSFA1s-PIF4 module and uncover HSFA1s as critical regulators integrating light and temperature signaling for a better acclimation of plants to the summer high temperature.

The Tetraspanin Tspan8 Associates with Endothelin Converting Enzyme ECE1 and Regulates Its Activity.

Tspan8 is a member of the tetraspanins family of cell surface molecules. The ability of tetraspanins to organize membrane microdomains with other membrane molecules and interfere with their function suggests that they could act as surface integrators of external or internal signals. Among the first identified tetraspanins, Tspan8 promotes tumor progression and metastasis, presumably by stimulating angiogenesis and cell motility. In patients, its expression on digestive tract tumors seems to be associated with a bad prognosis. We showed previously that Tspan8 associates with E-cadherin and EGFR and modulates their effects on cell motility. Using Mass spectrometry and western blot, we found a new partner, the endothelin converting enzyme ECE1, and showed that Tspan8 amplifies its activity of conversion of the endothelin-1 precursor bigET1 to endothelin. This was observed by transduction of the colon carcinoma cell line Isreco1, which does not express Tspan8, and on ileum tissue fragments of tspan8ko mice versus wild type mice. Given these results, Tspan8 appears to be a modulator of the endothelin axis, which could possibly be targeted in case of over-activity of endothelins in biological processes of tissues expressing Tspan8.

25-hydroxycholesterol: an integrator of antiviral ability and signaling.

Cholesterol, as an important component in mammalian cells, is efficient for viral entry, replication, and assembly. Oxysterols especially hydroxylated cholesterols are recognized as novel regulators of the innate immune response. The antiviral ability of 25HC (25-Hydroxycholesterol) is uncovered due to its role as a metabolic product of the interferon-stimulated gene CH25H (cholesterol-25-hydroxylase). With the advancement of research, the biological functions of 25HC and its structural functions have been interpreted gradually. Furthermore, the underlying mechanisms of antiviral effect of 25HC are not only limited to interferon regulation. Taken up by the special biosynthetic ways and structure, 25HC contributes to modulate not only the cholesterol metabolism but also autophagy and inflammation by regulating signaling pathways. The outcome of modulation by 25HC seems to be largely dependent on the cell types, viruses and context of cell microenvironments. In this paper, we review the recent proceedings on the regulatory effect of 25HC on interferon-independent signaling pathways related to its antiviral capacity and its putative underlying mechanisms.

Lipopolysaccharide-Induced TRPA1 Upregulation in Trigeminal Neurons is Dependent on TLR4 and Vesicular Exocytosis.

Pain from bacterial infection was believed to be the consequence of inflammation induced by bacterial products. However recent studies have shown that bacterial products can directly activate sensory neurons and induce pain. The mechanisms by which bacteria induce pain are poorly understood, but toll-like receptor (TLR)4 and transient receptor potential A1 (TRPA1) receptors are likely important integrators of pain signaling induced by bacteria. Using male and female mice we show that sensory neuron activation by bacterial lipopolysaccharides (LPS) is mediated by both TRPA1 and TLR4 and involves the mobilization of extracellular and intracellular calcium. We also show that LPS induces neuronal sensitization in a process dependent on TLR4 receptors. Moreover, we show that TLR4 and TRPA1 are both involved in sensory neurons response to LPS stimulation. Activation of TLR4 in a subset of sensory neurons induces TRPA1 upregulation at the cell membrane through vesicular exocytosis, contributing to the initiation of neuronal sensitization and pain. Collectively these data highlight the importance of sensory neurons to pathogen detection, and their activation by bacterial products like LPS as potentially important to early immune and nociceptive responses.SIGNIFICANCE STATEMENT Bacterial infections are often painful and the recent discovery that bacteria can directly stimulate sensory neurons leading to pain sensation and modulation of immune system have highlighted the importance of nervous system in the response to bacterial infection. Here, we showed that lipopolysaccharide, a major bacterial by-product, requires both toll-like receptor (TLR)4 and transient receptor potential A1 (TRPA1) receptors for neuronal activation and acute spontaneous pain, but only TLR4 mediates sensory neurons sensitization. Moreover, we showed for the first time that TLR4 sensitize sensory neurons through a rapid upregulation of TRPA1 via vesicular exocytosis. Our data highlight the importance of sensory neurons to pathogen detection and suggests that TLR4 would be a potential therapeutic target to modulate early stage of bacteria-induced pain and immune response.

General anesthesia alters CNS and astrocyte expression of activity-dependent and activity-independent genes.

General anesthesia represents a common clinical intervention and yet can result in long-term adverse CNS effects particularly in the elderly or dementia patients. Suppression of cortical activity is a key feature of the anesthetic-induced unconscious state, with activity being a well-described regulator of pathways important for brain health. However, the extent to which the effects of anesthesia go beyond simple suppression of neuronal activity is incompletely understood. We found that general anesthesia lowered cortical expression of genes induced by physiological activity in vivo, and recapitulated additional patterns of gene regulation induced by total blockade of firing activity in vitro, including repression of neuroprotective genes and induction of pro-apoptotic genes. However, the influence of anesthesia extended beyond that which could be accounted for by activity modulation, including the induction of non activity-regulated genes associated with inflammation and cell death. We next focused on astrocytes, important integrators of both neuronal activity and inflammatory signaling. General anesthesia triggered gene expression changes consistent with astrocytes being in a low-activity environment, but additionally caused induction of a reactive profile, with transcriptional changes enriched in those triggered by stroke, neuroinflammation, and Aß/tau pathology. Thus, while the effects of general anesthesia on cortical gene expression are consistent with the strong repression of brain activity, further deleterious effects are apparent including a reactive astrocyte profile.

PpARF6 acts as an integrator of auxin and ethylene signaling to promote fruit ripening in peach.

Although auxin is known to induce ethylene biosynthesis in some Rosaceae fruit crops, the mechanisms underlying the auxin-ethylene interaction during fruit ripening remain largely unknown. Here, the regulatory role of an auxin response factor, PpARF6, in fruit ripening was investigated in peach. Peach fruits showed accelerated ripening after treatment with auxin and PpARF6 was found to be significantly induced. PpARF6 not only could induce ethylene synthesis by directly activating the transcription of ethylene biosynthetic genes, but also competed with EIN3-binding F-box proteins PpEBF1/2 for binding to ethylene-insensitive3-like proteins PpEIL2/3, thereby keeping PpEIL2/3 active. Moreover, PpARF6 showed an interaction with PpEIL2/3 to enhance the PpEIL2/3-activated transcription of ethylene biosynthetic genes. Additionally, ectopic overexpression of PpARF6 in tomato accelerated fruit ripening by promoting the expression of genes involved in ethylene synthesis and fruit texture. In summary, our results revealed a positive regulatory role of PpARF6 in peach fruit ripening via integrating auxin and ethylene signaling.

ISL1 regulates lung branching morphogenesis via Shh signaling pathway

Lung branching morphogenesis relies on a complex coordination of multiple signaling pathways and transcription factors. Here, we found that ablation of the LIM homeodomain transcription factor Islet1 (Isl1) in lung epithelium resulted in defective branching morphogenesis and incomplete formation of five lobes. A reduction in mesenchymal cell proliferation was observed in Isl1ShhCre lungs. There was no difference in apoptosis between the wild-type (ShhCre) and Isl1ShhCre embryos. RNA-Seq and in situ hybridization analysis showed that Shh, Ptch1, Sox9, Irx1, Irx2, Tbx2, and Tbx3 were downregulated in the lungs of Isl1ShhCre embryos. ChIP assay implied the Shh gene served as a direct target of ISL1, since the transcription factor ISL1 could bind to the Shh epithelial enhancer sequence (MACS1). Also, activation of the Hedgehog pathway via ectopic gene expression rescued the defects caused by Isl1 ablation, confirming the genetic integration of Hedgehog signaling. In conclusion, our works suggest that epithelial Isl1 regulates lung branching morphogenesis through administrating the Shh signaling mediated epithelial-mesenchymal communications.

Attending Perceptions on the Use of Preference Signaling in the Ophthalmology Residency Application Process.

Objective This article characterizes perspectives of ophthalmologists involved in the residency selection process regarding the potential impact of preference signaling on the ophthalmology residency match. Methods An anonymous online questionnaire generated from SurveyMonkey was approved by the Association of University Professors of Ophthalmology (AUPO) Data Resource Committee for distribution to 391 individuals from the AUPO Departmental Chairs, Program Directors, and Directors of Medical Student Education email listservs in August 2022. Results A total of 96 (24.6%) ophthalmology faculty completed the questionnaire. The majority ( n = 76, 79.2%) agreed or strongly agreed that preference signaling should be implemented in the ophthalmology residency application system. Most respondents agreed or strongly agreed that preference signaling will allow for more holistic reviews of applications ( n = 55, 57.3%), agreed or strongly agreed that it will benefit applicants who do not have connections to home programs or faculty that can reach out to desired programs ( n = 81, 84.4%), and agreed or strongly agreed that it will improve the distribution of interviews to applicants ( n = 76, 79.2%). Participants agreed or strongly agreed that applicants who have signaled interest in their program will receive preference when offering interviews ( n = 59, 61.5%), and those signals will be used as a tiebreaker for similar applications ( n = 75, 78.1%). The majority of participants believed that the ideal number of preference signals' applicants should be given three to four signals ( n = 35, 36.0%) or five to six signals ( n = 29, 30.2%). Conclusion A majority of ophthalmology faculty surveyed support the integration of preference signaling into the ophthalmology residency match.

1588-P: Translatable Modeling of Glucagon Actions in Human iPSC-Derived Hepatocytes Uniquely Predicts Differential Clinical Responses to Glucagon Analog Agonist Molecules

As the development of clinically efficacious glucagon receptor (GCGR) agonists for the treatment of metabolic diseases evolves, the challenge to identify physiologically relevant preclinical models that predict glucagon receptor actions in humans becomes ever more important. While many hepatocyte models are available, most fail to recapitulate fundamental metabolic regulatory responses to key physiologic stimuli including integration of glucagon and insulin actions that govern hepatic carbohydrate or lipid metabolism. We developed and optimized a human induced pluripotent stem cell (iPSC)-derived hepatocyte model to mimic physiologic insulin and glucagon signaling and counter-regulation of metabolic pathways. We leveraged the model to evaluate functional responses of different glucagon receptor agonist molecules to regulate carbohydrate and lipid metabolism. Unlike assays conducted in several other cell line and primary isolated hepatocyte systems, hepatic glucose output assays in the human IPSC hepatocyte model demonstrated substantial potency differences between two novel soluble glucagon analog agonists. These findings predicted differential dose-dependent blood glucose excursions in response to these agonists when administered in a single ascending dose study in healthy human volunteers. Modeling physiologic integration of insulin and glucagon signaling in a clinically translatable human iPSC hepatocyte model can advance understanding of the regulation of nutrient metabolism by glucagon receptor agonists in humans and support the development of novel therapeutic approaches to address metabolic disorders, including type 2 diabetes, obesity, and nonalcoholic fatty liver disease. Disclosure W.Roell: Employee; Eli Lilly and Company, Stock/Shareholder; Eli Lilly and Company. A.Regmi: Employee; Eli Lilly and Company. C.T.Benson: Employee; Eli Lilly and Company, Stock/Shareholder; Eli Lilly and Company. T.Coskun: Employee; Eli Lilly and Company, Stock/Shareholder; Eli Lilly and Company. J.S.Moyers: Employee; Eli Lilly and Company, Stock/Shareholder; Eli Lilly and Company. J.Alsina-fernandez: Employee; Eli Lilly and Company. M.K.Thomas: Employee; Eli Lilly and Company, Stock/Shareholder; Eli Lilly and Company.

Roles of Fibroblast Growth Factors in the Axon Guidance.

Fibroblast growth factors (FGFs) have been widely studied by virtue of their ability to regulate many essential cellular activities, including proliferation, survival, migration, differentiation and metabolism. Recently, these molecules have emerged as the key components in forming the intricate connections within the nervous system. FGF and FGF receptor (FGFR) signaling pathways play important roles in axon guidance as axons navigate toward their synaptic targets. This review offers a current account of axonal navigation functions performed by FGFs, which operate as chemoattractants and/or chemorepellents in different circumstances. Meanwhile, detailed mechanisms behind the axon guidance process are elaborated, which are related to intracellular signaling integration and cytoskeleton dynamics.

Retromer Chaperones: Potential Therapeutics for Treatment of Skin Disease?

Retromer Chaperones: Potential Therapeutics for Treatment of Skin Disease?