Plants utilize receptor-like proteins and receptor-like kinases (RLPs/RLKs) to perceive and respond to a wide variety of invading pathogens and insect herbivores. While the strategies employed by microbial pathogens to suppress plant immunity have been well characterized, it remains unclear how herbivorous insects counteract receptor-mediated defenses. Here, we show that salivary effectors evolve independently in whiteflies and planthoppers to dampen RLP4-mediated plant immunity. RLP4, as a leucine-rich repeat RLP (LRR-RLP), confers plant resistance against herbivorous insects by forming the RLP4/SOBIR1 complexes. In the whitefly Bemisia tabaci, BtRDP, the Aleyrodidae-specific salivary sheath protein, interacts with RLP4 from multiple plant species and promotes its ubiquitin-dependent degradation. Overexpression of NtRLP4 in transgenic plants exerts a detrimental effect on B. tabaci by exploiting the crosstalk between the salicylic acid and jasmonic acid pathways. Conversely, overexpression of BtRDP or silencing of NtRLP4 effectively alleviates such negative effects. In planthopper Nilaparvata lugens, the Delphacidae-restricted salivary protein NlSP104 also targets and promotes the degradation of OsRLP4 from rice plants. These findings reveal convergent evolution of salivary proteins in insects and underscore the complex interactions between plants and herbivorous insects.

Sleep plays a critical role in animal physiology, primarily governed by the brain, and its disruption is prevalent in various brain disorders. Mettl5 is associated with intellectual disability (ID), which often includes sleep disturbances. However, the mechanism underlying these sleep disruptions in ID remains poorly understood. In this study, we investigated the sleep phenotypes resulting from Drosophila Mettl5 mutations. Rescue experiments revealed that Mettl5 functions predominantly within neurons and glia marked by Mettl5-Gal4 to regulate sleep. Previous work established that Mettl5 forms a complex with Trmt112 to influence rRNA methylation. Notably, a mutation in Trmt112 recapitulated these sleep disturbances, implicating translational regulation by the Mettl5/Trmt112 complex. Subsequent RNA-seq and Ribo-seq analyses of Mettl51bp mutants uncovered downstream effects, including altered expression of proteasome components and clock genes. Rescue experiments confirmed that the net increase in PERIOD protein underlies the sleep phenotype. This study illuminates the interplay between ribosome function, clock genes, and the proteasome in sleep regulation, highlighting the integrated roles of protein synthesis and degradation. These findings could potentially provide an example for in vivo study of rRNA methylation function, expand our understanding of protein homeostasis in sleep, and offer insights into the sleep phenotypes associated with ID.

It is now recognized that mitochondria play a crucial role in tumorigenesis; however, it has become clear that tumor metabolism varies significantly between cancer types. The failure of recent clinical trials aimed at directly targeting tumor respiration through oxidative phosphorylation inhibitors underscores the critical need for further studies providing an in-depth evaluation of mitochondrial bioenergetics. Accordingly, we comprehensively assessed the bulk tumor and mitochondrial metabolic phenotype in murine HER2-driven mammary cancer tumors and benign mammary tissue. Transcriptomic and proteomic profiling revealed a broad downregulation of mitochondrial genes/proteins in tumors, including OXPHOS subunits comprising Complexes I-IV. Despite reductions in tumor mitochondrial proteins, mitochondrial respiration was several-fold higher compared to benign mammary tissue, which persisted regardless of normalization method (wet weight, total protein content, and when corrected for mitochondrial content). This upregulated respiratory capacity could not be explained by OXPHOS uncoupling, suggesting HER2 signaling regulates intrinsic mitochondrial bioenergetics. In further support, lapatinib, an EGFR/HER2 tyrosine kinase inhibitor, attenuated mitochondrial respiration in NF639 murine mammary tumor epithelial cells. Together, this data highlights that the typical correlation between mitochondrial content and respiratory capacity may not apply to all tumor types and implicates HER2-linked activation of mitochondrial respiration supporting tumorigenesis in this model.

Continuous flash suppression (CFS), in which a dynamic masker presented to one eye suppresses awareness of a stimulus in the other eye, is widely used to study visual subconsciousness. Although some studies report preserved high-level processing under CFS, these effects have been increasingly questioned and may partly reflect residual low-level feature processing. A key unresolved issue is how strongly neuronal responses in V1, where inputs from the two eyes first converge, are affected by CFS, and how much the remaining signals can support downstream processing. Here, we used two-photon calcium imaging to record large populations of V1 neurons in awake, fixating macaques while presenting grating stimuli under CFS. CFS strongly suppressed V1 orientation responses in an ocular-dominance-dependent manner, nearly abolishing responses in neurons preferring the masker eye or both eyes, and significantly reducing responses in neurons preferring the grating eye. Modeling analyses further indicated that V1 population activity under CFS may still support coarse orientation classification but not accurate stimulus reconstruction. These results suggest that CFS substantially degrades orientation information in V1. The residual signals may support limited low-level processing but are likely insufficient for downstream higher-level visual and cognitive tasks.

Cyclic nucleotide-binding domain (CNBD) ion channels are vital for cellular signaling and excitability, with activation regulated by cyclic adenosine- or guanosine-monophosphate (cAMP, cGMP) binding. However, the allosteric mechanisms underlying this activation, particularly the energetics that describe conformational changes within individual domains and between domains, remain unclear. The prokaryotic CNBD channel SthK has been a useful model for better understanding these allosteric mechanisms. Previously, we applied time-resolved transition metal ion Förster resonance energy transfer (tmFRET) to investigate the conformational dynamics and energetics in the CNBD of a soluble C-terminal fragment of the SthK protein, SthKCterm (Eggan et al., 2024). Here, we used a similar strategy to measure the conformational energetics of the CNBD in the full-length channel, SthKFull, and compared them to measurements from SthKCterm. We incorporated the noncanonical amino acid Acd as a FRET donor and a metal bound to a chelator conjugated to a cysteine as an acceptor. We used time-correlated single-photon counting (TCSPC) to measure time-resolved FRET and fit the TCSPC data to obtain donor-acceptor distance distributions in the absence and presence of cAMP. The distance distributions allowed us to quantify the energetics of coupling between the C-terminal domains and the transmembrane domains by comparing the donor-acceptor distance distributions for SthKCterm and SthKFull. Our data indicate that the presence of the SthK transmembrane domains makes the activating conformational change in the CNBD more favorable. These findings highlight the power of time-resolved tmFRET to uncover the structural and energetic landscapes of allosteric proteins and of the ligand-mediated mechanism in CNBD channels specifically.

The rapid emergence of mineralized structures in diverse animal groups during the late Ediacaran and early Cambrian periods likely resulted from modifications of pre-adapted biomineralization genes inherited from a common ancestor. As the oldest extant phylum with mineralized structures, sponges are key to understanding animal biomineralization. Yet, the biomineralization process in sponges, particularly in forming spicules, is not well understood. To address this, we conducted transcriptomic, genomic, and proteomic analyses on the calcareous sponge Sycon ciliatum, supplemented by in situ hybridization. We identified 829 genes overexpressed in regions of increased calcite spicule formation, including 17 calcarins-proteins analogous to corals' galaxins localized in the spicule matrix and expressed in sclerocytes. Their expression varied temporally and spatially, specific to certain spicule types, indicating that fine-tuned gene regulation is crucial for biomineralization control. Similar subtle expression changes are also relevant in stony coral biomineralization. Tandem gene arrangements and expression changes suggest that gene duplication and neofunctionalization have significantly shaped S. ciliatum's biomineralization, similar to that in corals. These findings suggest a parallel evolution of carbonate biomineralization in the calcitic S. ciliatum and aragonitic corals, exemplifying the evolution of mechanisms crucial for animals to act as ecosystem engineers and form reef structures.

Wnt proteins are critical signaling molecules in developmental processes across animals. Despite intense study, their evolutionary roots have remained enigmatic. Using sensitive sequence analysis and structure modeling, we establish that the Wnts are part of a vast assemblage of domains, the Lipocone superfamily, defined here for the first time. It includes previously studied enzymatic domains like the phosphatidylserine synthases (PTDSS1/2) and the TelC toxin domain from Streptococcus intermedius, the enigmatic VanZ proteins, the animal Serum Amyloid A (SAA), and a further host of uncharacterized proteins in a total of 30 families. Although the metazoan Wnts are catalytically inactive, we present evidence for a conserved active site across this superfamily, versions of which are consistently predicted to operate on head groups of either phospholipids or polyisoprenoid lipids, catalyzing transesterification and phosphate-containing head group cleavage reactions. We argue that this superfamily originated as membrane proteins, with one branch (including Wnt and SAA) evolving into diffusible versions. By comprehensively analyzing contextual information networks derived from comparative genomics, we establish that they act in varied functional contexts, including regulation of membrane lipid composition, extracellular polysaccharide biosynthesis, and biogenesis of bacterial outer-membrane components, like lipopolysaccharides. On multiple occasions, members of this superfamily, including the bacterial progenitors of Wnt and SAA, have been recruited as effectors in biological conflicts spanning inter-organismal interactions and anti-viral immunity in both prokaryotes and eukaryotes. These findings establish a unifying theme in lipid biochemistry, explain the origins of Wnt signaling, and provide new leads regarding immunity across the tree of life.

Sickness-induced sleep is a behavior conserved across species that promotes recovery from illness, yet the underlying mechanisms are poorly understood. Here, we show that interleukin-6-like cytokine signaling from the Drosophila gut to brain glial cells regulates sleep. Under healthy conditions, this pathway promotes wakefulness. However, elevated gut cytokine signaling in response to oxidative stress - triggered by immune and inflammatory responses in the intestine - induces sleep. The cytokines Unpaired 2 and -3 are upregulated by oxidative stress in enteroendocrine cells and activate JAK-STAT signaling in glial cells, including those of the blood-brain barrier (BBB). This activity maintains elevated sleep during oxidative-stress-induced intestinal disturbances, suggesting that the JAK-STAT pathway in glia inhibits wake-promoting signaling to facilitate sleep-dependent restoration under these conditions. We find that the enteric peptide Allatostatin A (AstA) enhances wakefulness, and during intestinal oxidative stress, gut-derived Unpaired 2/3 inhibits AstA receptor expression in BBB glia, thereby sustaining an elevated sleep state during gut inflammation or illness. Taken together, our work identifies a gut-to-glial communication pathway that couples sleep with intestinal homeostasis and disease, enhancing sleep during intestinal sickness, and contributing to our understanding of how sleep disturbances arise from gastrointestinal disturbances.

β-Adrenoceptors (β-ARs) regulate cardiac function during sympathetic nerve stimulation. β-ARs are present in both the cardiac T-tubule (TTM) and outer surface membrane (OSM), but how their location impacts their function is unknown. Here, we developed a technology based on size exclusion to explore the function of β-ARs located in the OSM. We synthesized a PEG-Iso molecule by covalently linking isoprenaline (Iso) to a 5000 Da PolyEthylene-Glycol (PEG) chain to increase the size of the β-AR agonist and prevent it from accessing the T-tubule network. The affinity of PEG-Iso and Iso on β1- and β2-ARs was measured using radioligand binding. Molecular dynamics simulation was used to assess PEG-Iso conformation and visualize the accessibility of the Iso moiety to water. Using confocal microscopy, we show that PEGylation constrains molecules outside the T-tubule network of adult rat ventricular myocytes (ARVMs) due to the presence of the extracellular glycocalyx. β-AR activation in OSM with PEG-Iso produced a lower stimulation of [cAMP]i than Iso but a larger stimulation of cytosolic PKA at equivalent levels of [cAMP]I and similar effects on excitation-contraction coupling parameters. However, PEG-Iso produced a much lower stimulation of nuclear cAMP and PKA than Iso. Thus, OSM β-ARs in ARVMs control mainly cytosolic cAMP/PKA pathway and contractility, while TTM β-ARs control mainly nuclear cAMP, PKA, and consequent nuclear protein phosphorylation. Size exclusion strategy using ligand PEGylation provides a unique approach to evaluate the respective contribution of T-tubule vs. OSM proteins in cardiac cells.

Horizontal gene transfer (HGT) plays an important evolutionary role in prokaryotes, but it is less frequent in mammals. We previously reported that cell-free chromatin particles (cfChPs) - chromosomal fragments released from the billions of dying cells that circulate in human blood - are horizontally transferred to healthy cells with biological effects. However, the underlying mechanism and function of these effects remained unclear. We treated NIH3T3 mouse fibroblasts cells with cfChPs isolated from human serum and serially passaged the cells. The intracellular activities of cfChPs were analysed using chromatin fibre fluorography, cytogenetic analysis, immunofluorescence, and fluorescent in situ hybridisation. We discovered that the internalised cfChPs were almost exclusively comprised of non-coding DNA, and the disparate DNA sequences contained within them had randomly combined to form complex concatemers, some of which were multi-mega base pairs in size. The concatemers autonomously performed many functions attributable to the nuclear genome such as DNA, RNA and protein synthesis. They harboured human LINE-1 and Alu elements, with the potential to rearrange themselves within the mouse genome. Our results suggest that a cell simultaneously harbours two autonomous genome forms: one that is inherited (hereditary genome) and numerous others that are acquired (satellite genomes). The satellite genomes may have evolutionary functions given their ability to serve as vehicles for transposable elements and to generate a plethora of novel proteins. Our results also suggest that 'within-self' HGT may occur in mammals on a massive scale via the medium of cfChP concatemers that have undergone extensive and complex modifications resulting in their behaviour as 'foreign' genetic elements.

Lysogens, bacteria with one or more viruses (prophages) integrated into their genomes, are abundant in the gut of animals. Prophages often influence bacterial traits; however, the influence of prophages on the gut microbiota-host immune axis in animals remains poorly understood. Here, we investigate the influence of the prophage SfPat on Shewanella fidelis 3313, a persistent member of the gut microbiome of the model marine tunicate, Ciona robusta. Establishment of a SfPat deletion mutant (ΔSfPat) reveals the influence of this prophage on bacterial physiology in vitro and during colonization of the Ciona gut. In vitro, deletion of SfPat reduces S. fidelis 3313 motility and swimming while increasing biofilm formation. To understand the in vivo impact of these prophage-induced changes in bacterial traits, we exposed metamorphic stage 4 Ciona juveniles to wildtype (WT) and ΔSfPat strains. During colonization, ΔSfPat localizes to overlapping and distinct areas of the gut compared to the WT strain. We examined the differential expression of various regulators of cyclic-di-GMP, a secondary signaling molecule that mediates biofilm formation and motility. The pdeB gene, which encodes a bacterial phosphodiesterase known to influence biofilm formation and motility by degrading cyclic-di-GMP, is upregulated in the WT strain but not in ΔSfPat when examined in vivo. Expression of the Ciona gut immune effector, VCBP-C, is enhanced during colonization by ΔSfPat compared to the WT strain; however, VCBP-C binding to the WT strain does not promote the excision of SfPat in an SOS-dependent pathway. Instead, VCBP-C binding significantly reduces the expression of a phage major capsid protein. Our findings suggest that SfPat influences host perception of this important colonizing commensal and highlights the significance of investigating tripartite dynamics between prophages, bacteria, and their animal hosts to better understand the gut microbiota-host immune axis.

Parkinson's disease (PD) is characterized by the death of substantia nigra pars compacta (SNc) dopamine (DA) neurons, but the pathophysiological mechanisms that precede and drive their death remain unknown. The activity of DA neurons is likely altered in PD, but we understand little about if or how chronic changes in activity may contribute to degeneration. To address this question, we developed a chemogenetic (DREADD) mouse model to chronically increase DA neuron activity and confirmed this increase using ex vivo electrophysiology. Chronic hyperactivation of DA neurons resulted in prolonged increases in locomotor activity during the light cycle and decreases during the dark cycle, consistent with chronic changes in DA release and circadian disturbances. We also observed early, preferential degeneration of SNc projections, recapitulating the PD hallmarks of selective vulnerability of SNc axons and the comparative resilience of ventral tegmental area axons. This was followed by the eventual loss of midbrain DA neurons. Continuous DREADD activation resulted in a sustained increase in baseline calcium levels, supporting a role for increased calcium in the neurodegeneration process. Finally, spatial transcriptomics from DREADD mice examining midbrain DA neurons and striatal targets, and cross-validation with human patient samples, provided insights into potential mechanisms of hyperactivity-induced toxicity and PD. Our results thus reveal the preferential vulnerability of SNc DA neurons to increased neural activity and support a potential role for increased neural activity in driving degeneration in PD.

This study investigated 17α-estradiol's effects on aged hypothalamic physiological activity via long-term administration. Single-nucleus transcriptomic sequencing (snRNA-seq) was performed on pooled hypothalami from each group: aged male Norway brown rats treated with 17α-estradiol (O.T), aged controls (O), and young controls (Y). Supervised clustering of neurons (based on neuropeptides/receptors) evaluated subtype responses to aging and 17α-estradiol. Aging-induced elevation of neuronal cellular metabolism, stress, and reduced synapse formation-related pathways were significantly attenuated by 17α-estradiol. Neuron population analysis showed that subtypes regulating food intake, reproduction, blood pressure, stress response, and electrolyte balance were sensitive to 17α-estradiol. 17α-estradiol increased serum oxytocin (Oxt) and hypothalamic-pituitary-gonadal (HPG) axis activity (elevated plasma Gnrh, total testosterone; reduced estradiol). Gnrh1 upregulation mediated its effects on energy homeostasis, neural synapse, and stress response. Notably, Crh neurons in O.T showed prominent stress phenotypes, distinct from Agrp/Ghrl neurons. Thus, HPG axis and energy metabolism may be key 17α-estradiol targets in male hypothalamus. Additionally, our results demonstrate that supervised clustering (based on neuropeptides/receptors) effectively assesses the responses of hypothalamic neuron subtypes to aging and 17α-estradiol treatment.

Functional diversification of homologous neuronal microcircuits is a widespread feature observed across brain regions, as well as across species, while its molecular and developmental mechanisms remain largely unknown. We address this question in Drosophila larvae by focusing on segmentally homologous Wave command-like neurons, which diversify their wiring and function in a segment-specific manner. Anterior Wave (a-Wave) neurons extend axons anteriorly and connect to circuits inducing backward locomotion, whereas posterior Wave (p-Wave) neurons extend axons posteriorly and trigger forward locomotion. Here, we show that Frizzled receptors DFz2 and DFz4, together with the DWnt4 ligand, regulate the segment-specific Wave axon projection. DFz2 knockdown (KD) not only reroutes Wave axons to posterior neuromeres but also biases its motor command to induce forward instead of backward locomotion as tactile response. Thus, segment-specific axon guidance diversifies the function of homologous command neurons in behavioral regulation. Since control of anterior-posterior (A-P) axon guidance by Wnt/Fz signaling is evolutionarily conserved, our results reveal a potentially universal molecular principle for formation and diversification of the command system in the nerve cord. Furthermore, this work indicates that sensorimotor transduction can be rerouted by manipulating a single gene in a single class of neurons, potentially facilitating the evolutionary flexibility in action selection.

The lysosomal damage response is important for the maintenance of cellular homeostasis in human cells. Although the mechanisms underlying the repair and autophagic elimination of damaged lysosomes have been elucidated, the early signal transduction pathways and genes induced in response to lysosomal damage remain elusive. We performed transcriptome and proteome analyses and found that the TAB-TAK1-IKK-NF-κB axis is activated by K63-linked ubiquitin chains that accumulate on damaged lysosomes. This activates the expression of various transcription factors and cytokines that promote anti-apoptosis and intercellular signaling. The findings highlight the crucial role of ubiquitin-regulated signal transduction and gene expression in cell survival and cell-cell communication in response to lysosomal damage. The results suggest that the ubiquitin system is not only involved in the removal of damaged lysosomes by lysophagy, but also functions in the activation of cellular signaling for cell survival.

Representation of external and internal states in the brain plays a critical role in enabling suitable behavior. Recent studies suggest that state representation and state value can be simultaneously learned through Temporal-Difference-Reinforcement-Learning (TDRL) and Backpropagation-Through-Time (BPTT) in recurrent neural networks (RNNs) and their readout. However, neural implementation of such learning remains unclear as BPTT requires offline update using transported downstream weights, which is suggested to be biologically implausible. We demonstrate that simple online training of RNNs using TD reward prediction error and random feedback, without additional memory or eligibility trace, can still learn the structure of tasks with cue-reward delay and timing variability. This is because TD learning itself is a solution for temporal credit assignment, and feedback alignment, a mechanism originally proposed for supervised learning, enables gradient approximation without weight transport. Furthermore, we show that biologically constraining downstream weights and random feedback to be non-negative not only preserves learning but may even enhance it because the non-negative constraint ensures loose alignment-allowing the downstream and feedback weights to roughly align from the beginning. These results provide insights into the neural mechanisms underlying the learning of state representation and value, highlighting the potential of random feedback and biological constraints.

Diabetes affects >10% of adults worldwide and is caused by impaired production or response to insulin, resulting in chronic hyperglycemia. Pancreatic islet β-cells are the sole source of endogenous insulin, and our understanding of β-cell dysfunction and death in type 2 diabetes (T2D) is incomplete. Single-cell RNA-seq data supports heterogeneity as an important factor in β-cell function and survival. However, it is difficult to identify which β-cell phenotypes are critical for T2D etiology and progression. Our goal was to prioritize specific disease-related β-cell subpopulations to better understand T2D pathogenesis and identify relevant genes for targeted therapeutics. To address this, we applied a deep transfer learning tool, DEGAS, which maps disease associations onto single-cell RNA-seq data from bulk expression data. Independent runs of DEGAS using T2D or obesity status identified distinct β-cell subpopulations. A singular cluster of T2D-associated β-cells was identified; however, β-cells with high obese-DEGAS scores contained two subpopulations derived largely from either non-diabetic (ND) or T2D donors. The obesity-associated ND cells were enriched for translation and unfolded protein response genes compared to T2D cells. We selected CDKN1C and DLK1 for validation by immunostaining in human pancreas sections from healthy and T2D donors. Both CDKN1C and DLK1 were heterogeneously expressed among β-cells. CDKN1C was increased in β-cells from T2D donors, in agreement with the DEGAS predictions, while DLK1 appeared depleted from T2D islets of some donors. In conclusion, DEGAS has the potential to advance our holistic understanding of the β-cell transcriptomic phenotypes, including features that distinguish β-cells in obese ND or lean T2D states. Future work will expand this approach to additional human islet omics datasets to reveal the complex multicellular interactions driving T2D.

The Hippo pathway controls organ development, homeostasis, and regeneration primarily by modulating YAP/TEAD-mediated gene expression. Although emerging studies report Hippo-YAP dysfunction after viral infection, it is largely unknown in the context of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Here, we analyzed RNA sequencing data from human-induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) and SARS-CoV-2-infected human lung samples, and observed a decrease in YAP target gene expression. In screening SARS-CoV-2 nonstructural proteins, we found that nonstructural protein 13 (NSP13), a conserved coronavirus helicase, inhibits YAP transcriptional activity independent of the upstream Hippo kinases LATS1/2. Consistently, introducing NSP13 into mouse cardiomyocytes suppresses an active form of YAP (YAP5SA) in vivo. Subsequent investigations on NSP13 mutants revealed that NSP13 helicase activity, including DNA binding and unwinding, is crucial for suppressing YAP transactivation in HEK293T cells. Mechanistically, TEAD4 serves as a platform to recruit NSP13 and YAP. NSP13 likely inactivates the YAP/TEAD4 transcription complex by remodeling chromatin to recruit proteins, such as transcription termination factor 2 (TTF2), to bind the YAP/TEAD/NSP13 complex. These findings reveal a novel YAP/TEAD regulatory mechanism and uncover molecular insights into Hippo-YAP regulation after SARS-CoV-2 infection in humans.

All cells secrete extracellular vesicles (EVs) containing nucleic acid cargo, including microRNAs (miRNAs), that regulate the function of receiving cells. G protein-coupled receptors (GPCRs) affect intracellular function via multiple signaling cascades. However, the mechanisms of GPCR intercellular signaling through EV miRNA activity remain unknown. Human U2 osteosarcoma cells expressing native GPCRs were used to selectively stimulate distinct G protein signaling cascades (Gαi, Gαq, Gα12/13, and β-arrestin) by members of specific receptor subclasses, including the adenosine receptor A1 (ADORA1), the histamine receptor H1 (HRH1), the frizzled class receptor 4 (FZD4), and the atypical chemokine receptor 3 (ACKR3), respectively. We hypothesized that stimulation of specific classes of GPCRs would cause the release of EVs containing miRNAs with receptor-specific up- or downregulated expression, affecting unique pathological downstream signaling cascades. Receptor-specific agonists dose-dependently increased respective signaling cascade intermediates. We found no change in the quantity of EVs (~200 nm diameter), but there were distinct EV miRNA signatures following stimulation of GPCRs. Network analyses of differentially expressed miRNA and their predicted targets validated the linkage between specific receptors and cell function and pathological states. The data can be used to reverse engineer mechanisms involving EV miRNAs for various physiological and pathological processes. GPCRs are major pharmacological targets, so understanding the mechanisms that stimulate or inhibit GPCR-mediated changes in extracellular miRNA signatures could improve long- and short-term therapeutic and unwanted drug effects.

Fibroblast growth factor receptor 2 (FGFR2) is an important member of receptor tyrosine kinase (RTK) family. FGFR2 amplification occurs at a high frequency in gastric cancer (GC) and has been proven to be closely associated with poor prognosis and insensitivity to chemotherapy or immunotherapy. Current FGFR2-targeted therapies have limited efficacy. Hence, how to enhance efficacy and reverse resistance are urgent problems clinically. Src homology region 2-containing protein tyrosine phosphatase 2 (SHP2) serves as the shared downstream mediator of all RTKs and a prominent immunosuppressive molecule. In this study, we identified FGFR2 amplification in 6.2% (10/161) of GC patients in our center. Then we showed that dual blocking SHP2 and FGFR2 enhanced the effects of FGFR2 inhibitor (FGFR2i) in FGFR2-amplified GC both in vitro (human GC cell lines) and in vivo (mouse xenograft tumor models) via suppressing RAS/ERK and PI3K/AKT pathways. We further showed that it overcame FGFR2i resistance by reversing the feedback activation mediated by other RTKs and continuously suppressing FGFR2-initiated downstream pathways. Notably, SHP2 blockade could suppress PD-1 expression and promoted IFN-γ secretion of CD8+ T cells, enhancing the cytotoxic functions of T cells in tumor immune microenvironment. Overall, our findings suggest that dual blocking SHP2 and FGFR2 is a compelling rationale with both targeted treatment and immune regulation for FGFR2-amplified GC.