Receptor Tyrosine Kinase Signaling Research Articles

Integrative analysis of single-cell transcriptomic and multilayer signaling networks in glioma reveal tumor progression stage

IntroductionTumor microenvironments (TMEs) encompass complex ecosystems of cancer cells, infiltrating immune cells, and diverse cell types. Intercellular and intracellular signals within the TME significantly influence cancer progression and therapeutic outcomes. Although computational tools are available to study TME interactions, explicitly modeling tumor progression across different cancer types remains a challenge.MethodsThis study introduces a comprehensive framework utilizing single-cell RNA sequencing (scRNA-seq) data within a multilayer network model, designed to investigate molecular changes across glioma progression stages. The heterogeneous, multilayered network model replicates the hierarchical structure of biological systems, from genetic building blocks to cellular functions and phenotypic manifestations.ResultsApplying this framework to glioma scRNA-seq data allowed complex network analysis of different cancer stages, revealing significant ligand‒receptor interactions and key ligand‒receptor-transcription factor (TF) axes, along with their associated biological pathways. Differential network analysis between grade III and grade IV glioma highlighted the most critical nodes and edges involved in interaction rewiring. Pathway enrichment analysis identified four essential genes—PDGFA (ligand), PDGFRA (receptor), CREB1 (TF), and PLAT (target gene)—involved in the Receptor Tyrosine Kinases (RTK) signaling pathway, which plays a pivotal role in glioma progression from grade III to grade IV.DiscussionThese genes emerged as significant features for machine learning in predicting glioma progression stages, achieving 87% accuracy and 93% AUC in a 3-year survival prediction through Kaplan-Meier analysis. This framework provides deeper insights into the cellular machinery of glioma, revealing key molecular relationships that may inform prognosis and therapeutic strategies.

CNSC-43. NEUROTRANSMITTER DEPENDENCY UNDERLIES TUMOR GROWTH IN HUMANIZED PEDIATRIC LOW-GRADE GLIOMA MODELS

Abstract Brain tumors arise in close association with neurons, suggesting that these non-neoplastic cells may be critical stromal drivers of brain tumor initiation and growth. Previously, we have shown that murine low-grade optic glioma formation and progression in the setting of Neurofibromatosis type 1 (NF1) is dictated by neurons and neuronal activity. In these studies, these neuronal dependencies reflected neuronal activity-driven enzymatic cleavage of a growth factor (neuroligin-3) from oligodendrocyte precursors cells (tumor initiation) or neuronal production of a paracrine factor to stimulate T cell support of optic glioma growth (tumor progression). Since neurons typically communicate with other neurons through neurotransmitters, we sought to explore the possibility that neurotransmitters operate to modulate low-grade glioma growth using humanized mouse models of pilocytic astrocytoma (PA). Leveraging single cell RNA sequencing of three independent sets of pediatric PAs, we identified neurotransmitter pathway enrichment in the tumor cells. This neurotransmitter pathway enrichment reflected aberrant expression of specific neurotransmitter receptors, which we confirmed in three independently generated PA tissue microarrays and in five distinct primary PA cell lines grown in vitro. Moreover, this aberrant neurotransmitter receptor expression established differential neurotransmitter PA growth dependencies in vitro. Importantly, interruption of neurotransmitter signaling in human PA xenografts attenuated tumor growth and ERK activation in Rag1-/- mice in vivo. Finally, we discovered crosstalk between neurotransmitter receptor and receptor tyrosine kinase signaling that revealed another target for therapeutic inhibition. Taken together, these findings elucidate a previously unknown neurotransmitter PA growth dependency amenable to therapeutic targeting.

CSIG-31. ANNEXIN A7 ALTERNATIVE SPLICING IN GLIOMA STEM CELLS FACILITATES THE DEGRADATION OF EGFR BY PHOSPHORYLATING TYROSINE RESIDUES EXCLUSIVE TO PROTEIN ISOFORM 1

Abstract The majority of GBMs express aberrant receptor tyrosine kinase (RTK) activity, which are canonically endocytosed and degraded. This process is disrupted in GBM to promote receptor recycling and resulting in hyperactive RTK signaling. Exon six of Annexin A7 (ANXA7) can be alternatively spliced to yield two distinct protein isoforms through its inclusion (I1) or exclusion (I2). We investigated ANXA7 isoform differences for epidermal growth factor receptor (EGFR) trafficking fates in PDX glioma stem cells (GSCs). We demonstrated that ANXA7 exon skipping supports the impaired endosomal trafficking of RTKs to favor recycling in GBM. JX14 GSCs were made to overexpress ANXA7-I1 with lentivirus to conduct immunofluorescence experiments measuring EGFR-ANXA7 colocalization (Pearson) with markers of the endocytic pathway for the early endosomes (EEA1), recycling endosomes (Rab4/Rab11), and lysosomes (LAMP1). EV and I1 cells were also incubated with siRNA targeting both ANXA7 isoforms to determine the impact of ANXA7 loss on endosomal EGFR trafficking. Interestingly, we found drastic differences in endosomal EGFR trafficking in I1 expressing cells. EGFR colocalized with EEA1 in both EV and I1 cells (R2=0.01336, P>0.6275). In EV cells, EGFR was recycled via fast/slow recycling endosomes demonstrated by Rab4/Rab11 colocalization while I1 did not (R2=0.9267, P<0.0001). Furthermore, I1 cells and not EV preferentially degraded EGFR as seen by LAMP1 colocalization (R2=0.9398, P<0.0001). On the other hand, ANXA7 siRNA KD in I1 cells impaired EGFR sorting via a 40% increase (P<0.0010) in total EGFR protein and a 40% reduction (P<0.0001) in colocalization with EEA1 to prefer recycling again while EV cells were unaffected. I1 overexpression significantly reduced EGFR signaling through degradation, which is driven by the phosphorylation of I1-exclusive tyrosine residues in phosphomimetic (p<0.01) and non-phosphorylatable protein mutants. I1-induced reduction in EGFR signaling suggests a new therapeutic vulnerability for GSCs by potentially reducing tumorigenicity or improving the efficacy of EGFR inhibition.

Oncogenic EML4-ALK assemblies suppress growth factor perception and modulate drug tolerance

Drug resistance remains a challenge for targeted therapy of cancers driven by EML4-ALK and related fusion oncogenes. EML4-ALK forms cytoplasmic protein condensates, which result from networks of interactions between oncogene and adapter protein multimers. While these assemblies are associated with oncogenic signaling, their role in drug response is unclear. Here, we use optogenetics and live-cell imaging to find that EML4-ALK assemblies suppress transmembrane receptor tyrosine kinase (RTK) signaling by sequestering RTK adapter proteins including GRB2 and SOS1. Furthermore, ALK inhibition, while suppressing oncogenic signaling, simultaneously releases the sequestered adapters and thereby resensitizes RTK signaling. Resensitized RTKs promote rapid and pulsatile ERK reactivation that originates from paracrine ligands shed by dying cells. Reactivated ERK signaling promotes cell survival, which can be counteracted by combination therapies that block paracrine signaling. Our results identify a regulatory role for RTK fusion assemblies and uncover a mechanism of tolerance to targeted therapies.

SOS1 Inhibition Enhances the Efficacy of KRASG12C Inhibitors and Delays Resistance in Lung Adenocarcinoma.

The clinical effectiveness of KRASG12C inhibitors (G12Ci) is limited both by intrinsic and acquired resistance, necessitating the development of combination approaches. Here, we identified targeting proximal receptor tyrosine kinase (RTK) signaling using the SOS1 inhibitor (SOS1i) BI-3406 as a strategy to improve responses to G12Ci treatment. SOS1i enhanced the efficacy of G12Ci and limited rebound RTK/ERK signaling to overcome intrinsic/adaptive resistance, but this effect was modulated by SOS2 protein levels. G12Ci drug tolerant persister (DTP) cells showed up to a 3-fold enrichment of tumor initiating cells (TIC), suggestive of a sanctuary population of G12Ci resistant cells. SOS1i re-sensitized DTPs to G12Ci and inhibited G12C-induced TIC enrichment. Co-mutation of the tumor suppressor KEAP1 limited the clinical effectiveness of G12Ci, and KEAP1 and STK11 deletion increased TIC frequency and accelerated the development of acquired resistance to G12Ci, consistent with clinical G12Ci resistance seen with these co-mutations. Treatment with SOS1i both delayed acquired G12Ci resistance and limited the total number of resistant colonies regardless of KEAP1 and STK11 mutational status. Together, these data suggest that targeting SOS1 could be an effective strategy to both enhance G12Ci efficacy and prevent G12Ci resistance regardless of co-mutations.

Endocrine dysregulation in COVID-19: molecular mechanisms and insights.

This review describes the impact of COVID-19 on the endocrine system, focusing on cortisol signaling and growth factor-induced endocrine resistance. As expected, SARS-CoV-2 infection induces systemic inflammation, resulting in stimulation of the adrenal glands leading to elevated cortisol levels with normal adrenocorticotropic hormone (ACTH) levels. The cytokine storm could also stimulate cortisol production. However, in some instances, cortisol levels rise independently of ACTH due to a phenomenon known as "pseudo-Cushing's syndrome," where adrenal glands become less responsive to ACTH. Plasma proteomic analyses showed that this pattern was variably observed among COVID-19 patients, potentially involving calcium dysregulation and GNAS-regulated activities, ultimately impacting the regulation of microvascular permeability. COVID-19 also exhibited a syndrome resembling endocrine resistance, governed by receptor tyrosine kinase signaling pathways. Mild cases displayed elevated activity of EGFR and MMP9, along with increased expression of survival factors like Bax and Bcl2. In contrast, more severe cases involved IGFR-I and enhanced NOTCH signaling, with altered expression of Bcl2, AKT1, and MAPK8. In summary, these findings describe the complex interplay between COVID-19 and endocrine pathology, particularly endocrine resistance. These insights suggest potential endocrine targets for therapeutic interventions to improve short- and long-term outcomes for COVID-19 patients.

SPROUTY2, a Negative Feedback Regulator of Receptor Tyrosine Kinase Signaling, Associated with Neurodevelopmental Disorders: Current Knowledge and Future Perspectives

Growth-factor-induced cell signaling plays a crucial role in development; however, negative regulation of this signaling pathway is important for sustaining homeostasis and preventing diseases. SPROUTY2 (SPRY2) is a potent negative regulator of receptor tyrosine kinase (RTK) signaling that binds to GRB2 during RTK activation and inhibits the GRB2-SOS complex, which inhibits RAS activation and attenuates the downstream RAS/ERK signaling cascade. SPRY was formerly discovered in Drosophila but was later discovered in higher eukaryotes and was found to be connected to many developmental abnormalities. In several experimental scenarios, increased SPRY2 protein levels have been observed to be involved in both peripheral and central nervous system neuronal regeneration and degeneration. SPRY2 is a desirable pharmaceutical target for improving intracellular signaling activity, particularly in the RAS/ERK pathway, in targeted cells because of its increased expression under pathological conditions. However, the role of SPRY2 in brain-derived neurotrophic factor (BDNF) signaling, a major signaling pathway involved in nervous system development, has not been well studied yet. Recent research using a variety of small-animal models suggests that SPRY2 has substantial therapeutic promise for treating a range of neurological conditions. This is explained by its function as an intracellular ERK signaling pathway inhibitor, which is connected to a variety of neuronal activities. By modifying this route, SPRY2 may open the door to novel therapeutic approaches for these difficult-to-treat illnesses. This review integrates an in-depth analysis of the structure of SPRY2, the role of its major interactive partners in RTK signaling cascades, and their possible mechanisms of action. Furthermore, this review highlights the possible role of SPRY2 in neurodevelopmental disorders, as well as its future therapeutic implications.

Spatially Informed Graph Structure Learning Extracts Insights from Spatial Transcriptomics.

Embeddings derived from cell graphs hold significant potential for exploring spatial transcriptomics (ST) datasets. Nevertheless, existing methodologies rely on a graph structure defined by spatial proximity, which inadequately represents the diversity inherent in cell-cell interactions (CCIs). This study introduces STAGUE, an innovative framework that concurrently learns a cell graph structure and a low-dimensional embedding from ST data. STAGUE employs graph structure learning to parameterize and refine a cell graph adjacency matrix, enabling the generation of learnable graph views for effective contrastive learning. The derived embeddings and cell graph improve spatial clustering accuracy and facilitate the discovery of novel CCIs. Experimental benchmarks across 86 real and simulated ST datasets show that STAGUE outperforms 15 comparison methods in clustering performance. Additionally, STAGUE delineates the heterogeneity in human breast cancer tissues, revealing the activation of epithelial-to-mesenchymal transition and PI3K/AKT signaling in specific sub-regions. Furthermore, STAGUE identifies CCIs with greater alignment to established biological knowledge than those ascertained by existing graph autoencoder-based methods. STAGUE also reveals the regulatory genes that participate in these CCIs, including those enriched in neuropeptide signaling and receptor tyrosine kinase signaling pathways, thereby providing insights into the underlying biologicalprocesses.

7862 The Paradoxical Role Of ERFFI1 In Breast Cancer Progression Is Supported By Its Dynamic Protein Interactome

Abstract Disclosure: A. Ocampo: None. P.D. Bagamasbad: None. In breast cancer (BCa), glucocorticoid (GC) therapy is used in anti-emetic and palliative care. However, the effects of GC therapy in BCa are conflicting as it is beneficial for hormone-responsive luminal subtypes while it promotes tumor progression in more aggressive triple-negative BCa (TNBCs). A factor implicated in this paradoxical shift is the feedback inhibitor of epidermal growth factor receptor (EGFR) signaling, ERBB receptor feedback inhibitor 1 (ERRFI1). In BCa cell lines, ERRFI1 expression is directly regulated by GC receptor (GR). More interestingly, ERRFI1 was found to restrict the GC-enhanced proliferation in normal breast epithelia but enhances the GC-mediated pro-tumorigenic effects in highly aggressive TNBC. To identify the mechanism behind the shift in ERFFI1 function through BCa progression, ERRFI1 protein interactions in the luminal, HER2, and TNBC subtypes were analyzed by identifying differentially expressed proteins from each subtype using available proteome and interactome datasets from Integrated Interactions Database and followed by network and clustering analyses through STRING. The ERRFI1 interactome varied across the subtypes, and gene ontology analysis of the protein networks revealed progressive tumorigenic effects from the least aggressive luminal subtype to the most aggressive TNBC. The luminal BCa-ERRFI1 interactome showed metastatic potential that is countered by negative regulation of receptor tyrosine kinase (RTK) signaling. The HER2-ERRFI1 interactome highlighted the amplification of RTK signaling and its possible modulation through SRC while the TNBC-ERRFI1 interactome presents pro-oncogenic proteins implicated in drug resistance, angiogenesis, and EMT. Interestingly, the interactome also revealed a link between EGFR signaling and GR signaling possibly through ERRFI1, EGFR, and SFN. To determine the function of ERFFI1 in EGFR signaling in BCa, we generated stable ERRFI1 knockdown in different breast epithelial cell models through lentiviral transduction. Cells were then treated with EGF followed by gene expression analysis of known EGF-upregulated genes such as MYC, GSK3B, MCL1, and MMP9. We found that ERRFI1 knockdown enhanced the EGF-dependent induction of MYC and MMP9 in normal MCF10A cells and this effect was not observed in the TNBC MDA-MB-468 line, suggesting the loss of ERRFI1-mediated inhibition of EGFR signaling with BCa tumorigenic potential. Taken together, our findings suggest that the progressive loss of anti-tumorigenic function of ERRFI1 may be explained by its changing interactome in BCa molecular subtypes. Presentation: 6/3/2024

7862 The Paradoxical Role of ERFFI1 in Breast Cancer Progression Is Supported by Its Dynamic Protein Interactome

Abstract Disclosure: A. Ocampo: None. P.D. Bagamasbad: None. In breast cancer (BCa), glucocorticoid (GC) therapy is used in anti-emetic and palliative care. However, the effects of GC therapy in BCa are conflicting as it is beneficial for hormone-responsive luminal subtypes while it promotes tumor progression in more aggressive triple-negative BCa (TNBCs). A factor implicated in this paradoxical shift is the feedback inhibitor of epidermal growth factor receptor (EGFR) signaling, ERBB receptor feedback inhibitor 1 (ERRFI1). In BCa cell lines, ERRFI1 expression is directly regulated by GC receptor (GR). More interestingly, ERRFI1 was found to restrict the GC-enhanced proliferation in normal breast epithelia but enhances the GC-mediated pro-tumorigenic effects in highly aggressive TNBC. To identify the mechanism behind the shift in ERFFI1 function through BCa progression, ERRFI1 protein interactions in the luminal, HER2, and TNBC subtypes were analyzed by identifying differentially expressed proteins from each subtype using available proteome and interactome datasets from Integrated Interactions Database and followed by network and clustering analyses through STRING. The ERRFI1 interactome varied across the subtypes, and gene ontology analysis of the protein networks revealed progressive tumorigenic effects from the least aggressive luminal subtype to the most aggressive TNBC. The luminal BCa-ERRFI1 interactome showed metastatic potential that is countered by negative regulation of receptor tyrosine kinase (RTK) signaling. The HER2-ERRFI1 interactome highlighted the amplification of RTK signaling and its possible modulation through SRC while the TNBC-ERRFI1 interactome presents pro-oncogenic proteins implicated in drug resistance, angiogenesis, and EMT. Interestingly, the interactome also revealed a link between EGFR signaling and GR signaling possibly through ERRFI1, EGFR, and SFN. To determine the function of ERFFI1 in EGFR signaling in BCa, we generated stable ERRFI1 knockdown in different breast epithelial cell models through lentiviral transduction. Cells were then treated with EGF followed by gene expression analysis of known EGF-upregulated genes such as MYC, GSK3B, MCL1, and MMP9. We found that ERRFI1 knockdown enhanced the EGF-dependent induction of MYC and MMP9 in normal MCF10A cells and this effect was not observed in the TNBC MDA-MB-468 line, suggesting the loss of ERRFI1-mediated inhibition of EGFR signaling with BCa tumorigenic potential. Taken together, our findings suggest that the progressive loss of anti-tumorigenic function of ERRFI1 may be explained by its changing interactome in BCa molecular subtypes. Presentation: 6/3/2024

383 (PB371): Characterization of cancer cell lines selected for resistance to growth factor receptor tyrosine kinase inhibitors and signal transduction therapies

383 (PB371): Characterization of cancer cell lines selected for resistance to growth factor receptor tyrosine kinase inhibitors and signal transduction therapies

A heterozygous SPRY4 variant identified in female infertility characterized by reduced oocyte potential and early embryonic arrest.

Can novel genetic factors contributing to early embryonic arrest in infertile patients be identified, along with the underlying mechanisms of the pathogenic variant? We identified a heterozygous variant in the SPRY4 (sprouty RTK signaling antagonist 4) in infertile patients and conducted in vitro and in vivo studies to investigate the effects of the variant/deletion, highlighting its critical role in female reproductive health. SPRY4 acts as a negative regulator of receptor tyrosine kinases (RTKs) and functions as a tumor suppressor. Its abnormal expression can lead to recurrent miscarriage by affecting trophoblast function. In mice, Spry4 knockout (KO) leads to craniofacial anomalies and growth defects. A human study links the SPRY4 variant to a male patient with isolated hypogonadotropic hypogonadism (IHH), hypothetically impacting gonadotropin-releasing hormone (GnRH) neurons, and causing reproductive dysfunctions. SPRY4 is thus potentially integral in regulating endocrine homeostasis and reproductive function. To date, no study has reported SPRY4 variants associated with female fertility, and a causal relationship has not been established with functional evidence. Whole-exome sequencing (WES) was performed in 392 infertile women who suffered from primary infertility of unknown reason, and the heterozygous SPRY4 variant were identified in one independent family. The infertile patients presenting were recruited from July 2017 to November 2023. Women diagnosed with primary infertility were recruited from the Reproduction Center of Zhongshan Hospital, Fudan University. Genomic DNA was extracted from peripheral blood for WES analysis. The SPRY4 variant were identified through WES, in silico analysis, and variant screening. All variants were confirmed by Sanger sequencing. The effects of the variants were investigated in human embryonic kidney (HEK) 293T (HEK293T) cells via western blotting, and in mouse oocytes and embryos through complementary RNA (cRNA) injection, RNA sequencing, fluorescence, absorbance, and RT-qPCR assays. Gene function was further examined in Spry4 KO mice via histology, western blotting, ELISA, and RT-qPCR assays. We identified a missense heterozygous pathogenic variant in SPRY4 (GRCh38, GenBank: NM_030964.5, c.157C>T p.(Arg53Trp), rs200531302) that reduces SPRY4 protein levels in HEK293T cells and disrupts the redox system and mitochondrial function in mouse oocyte, and perturbs developmental potential in mouse embryos. These phenotypes could be partially reversed by the exogenous addition of Nrf1 cRNA. Additionally, Spry4-/- mice exhibit ovarian oxidative stress and decreased ovarian function. Due to the limited WES data and population, we identified only one family with a SPRY4 mutation. The deeper mechanism and therapeutic strategy should be further investigated through mutant mice and recovery experiment. Our study has identified a pathogenic variant in SPRY4 associated with early embryonic arrest in humans. These findings enhance our understanding of the role of SPRY4 in early embryonic development and present a new genetic marker for female infertility. This work was supported by the National Natural Science Foundation of China (82071643 and 82171655) and Natural Science Foundation of Shanghai (22ZR1456200). None of the authors have any competing interests. N/A.

TYRO3 and EPHA2 Expression Are Dysregulated in Breast Cancer.

Receptor tyrosine kinases (RTKs) are involved in cell growth, motility, and differentiation. Deregulation of RTKs signaling is associated with tumor development and therapy resistance. Potential RTKs like TAM (TYRO3, AXL, MERTK), RON, EPH, and MET have been evaluated in many cancers like lung, prostate, and colorectal, but little is known in breast tumors. In this study, 51 luminal breast cancer tissue and 8 triple negative breast cancer (TNBC) subtypes were evaluated by qPCR for the expression of TAM, RON, EPHA2, and MET genes. Statistical analysis was performed to determine the correlation to clinical data. TYRO3 is related to tumor subtype and stage, patient's age, smoking habits, and obesity. MET expression is correlated to EPHA2 and TAM gene expression. EPHA2 expression is also related to aging and smoking habits. The expression levels of the TAM and EPHA2 genes seem to play an important role in breast cancer, being also influenced by the patient's lifestyle.

MEK Inhibitors Lead to PDGFR Pathway Upregulation and Sensitize Tumors to RAF Dimer Inhibitors in NF1-Deficient Malignant Peripheral Nerve Sheath Tumor.

Malignant peripheral nerve sheath tumor (MPNST) is a highly aggressive subtype of soft-tissue sarcoma with a high propensity to metastasize and extremely limited treatment options. Loss of the RAS-GAP NF1 leads to sustained RAF/MEK/ERK signaling in MPNST. However, single-agent MEK inhibitors (MEKi) have failed to elicit a sustained inhibition of the MAPK signaling pathway in MPNST. We used pharmacological, biochemical, and genetic perturbations of the receptor tyrosine kinase and MAPK signaling pathway regulators to investigate the mechanisms of MEKi resistance and evaluated combination therapeutic strategies in various preclinical MPNST models in vitro and in vivo. Here, we report that MEKi treatment resistance in MPNST involves two adaptive pathways: direct transcriptional upregulation of the receptor tyrosine kinase PDGFRβ and MEKi-induced increase in RAF dimer formation and activation of downstream signaling. Although the pharmacologic combination of a MEKi with a PDGFRβ-specific inhibitor was more effective than treatment with the MEKi alone, the combination of the MEKi and RAF dimer inhibitors led to a robust inhibition of MAPK pathway signaling. This combination treatment was effective in vitro and in vivo, as demonstrated by the significant increase in drug synergism and its high effectiveness in decreasing MPNST viability. Our findings suggest that the combination of MEKis and PDGFR and/or RAF dimer inhibitors can overcome MEKi resistance and may serve as a novel targeted therapeutic strategy for patients with NF1-deficient MPNST, which in turn could impact future clinical investigations for this patient population.

The LCLAT1/LYCAT acyltransferase is required for EGF-mediated phosphatidylinositol-3,4,5-trisphosphate generation and Akt signaling.

Receptor tyrosine kinases such as EGF receptor (EGFR) stimulate phosphoinositide 3 kinases to convert phosphatidylinositol-4,5-bisphosophate [PtdIns(4,5)P2] into phosphatidylinositol-3,4,5-trisphosphate [PtdIns(3,4,5)P3]. PtdIns(3,4,5)P3 then remodels actin and gene expression, and boosts cell survival and proliferation. PtdIns(3,4,5)P3 partly achieves these functions by triggering activation of the kinase Akt, which phosphorylates targets like Tsc2 and GSK3β. Consequently, unchecked upregulation of PtdIns(3,4,5)P3-Akt signaling promotes tumor progression. Interestingly, 50-70% of PtdIns and PtdInsPs have stearate and arachidonate at sn-1 and sn-2 positions of glycerol, respectively, forming a species known as 38:4-PtdIns/PtdInsPs. LCLAT1 and MBOAT7 acyltransferases partly enrich PtdIns in this acyl format. We previously showed that disruption of LCLAT1 lowered PtdIns(4,5)P2 levels and perturbed endocytosis and endocytic trafficking. However, the role of LCLAT1 in receptor tyrosine kinase and PtdIns(3,4,5)P3 signaling was not explored. Here, we show that LCLAT1 silencing in MDA-MB-231 and ARPE-19 cells abated the levels of PtdIns(3,4,5)P3 in response to EGF signaling. Importantly, LCLAT1-silenced cells were also impaired for EGF-driven and insulin-driven Akt activation and downstream signaling. Thus, our work provides first evidence that the LCLAT1 acyltransferase is required for receptor tyrosine kinase signaling.

A novel m6A reader RBFOX2 expression is increased in oral squamous cell carcinoma and promotes tumorigenesis

Background and ObjectiveOral squamous cell carcinoma (OSCC) is a significant global health concern due to its aggressive nature and poor prognosis. Recent research has highlighted the important role of RNA modifications in cancer biology, especially N6-methyladenosine (m6A) modifications, which are controlled by a complex interplay of m6A regulators. This study specifically investigates RBFOX2, a new m6A reader, and its involvement in OSCC. MethodsOur study primarily utilized OSCC tissue, adjacent normal tissue samples, OSCC cell lines, and normal healthy oral keratinocytes to validate RBFOX2 mRNA expression. Additionally, we used the TCGA-HNSCC dataset for large cohort analysis and clinicopathological characterization. Furthermore, we visualized the RBFOX2 network to identify the primary functions of the protein. ResultsOur research shows a noticeable increase in RBFOX2 expression in OSCC tissues compared to adjacent non-tumorous tissues, as determined by quantitative PCR and immunohistochemistry analyses. Functional pathway enrichment analysis revealed that RBFOX2 is involved in the receptor tyrosine kinase signaling pathway and the Hippo signaling pathway, which plays a critical role in oral cancer development and progression. Clinically, elevated RBFOX2 expression correlated with advanced tumor stages and poorer patient outcomes, demonstrating its prognostic value. These findings indicate that RBFOX2 acts as an oncogenic driver in OSCC as an m6A reader, facilitating the expression of m6A-modified oncogenes. ConclusionOur study identifies RBFOX2 as a critical player in OSCC pathogenesis and opens avenues for novel therapeutic strategies targeting the m6A regulatory machinery in this malignancy.

Genetic code expansion, click chemistry, and light-activated PI3K reveal details of membrane protein trafficking downstream of receptor tyrosine kinases

Ligands such as insulin, epidermal growth factor, platelet-derived growth factor, and nerve growth factor (NGF) initiate signals at the cell membrane by binding to receptor tyrosine kinases (RTKs). Along with G-protein-coupled receptors, RTKs are the main platforms for transducing extracellular signals into intracellular signals. Studying RTK signaling has been a challenge, however, due to the multiple signaling pathways to which RTKs typically are coupled, including MAP/ERK, PLCγ, and Class 1A phosphoinositide 3-kinases (PI3K). The multi-pronged RTK signaling has been a barrier to isolating the effects of any one downstream pathway. Here, we used optogenetic activation of PI3K to decouple its activation from other RTK signaling pathways. In this context, we used genetic code expansion to introduce a click chemistry noncanonical amino acid into the extracellular side of membrane proteins. Applying a cell-impermeant click chemistry fluorophore allowed us to visualize delivery of membrane proteins to the plasma membrane in real time. Using these approaches, we demonstrate that activation of PI3K, without activating other pathways downstream of RTK signaling, is sufficient to traffic the TRPV1 ion channels and insulin receptors to the plasma membrane.

Genetic code expansion, click chemistry, and light-activated PI3K reveal details of membrane protein trafficking downstream of receptor tyrosine kinases.

Ligands such as insulin, epidermal growth factor, platelet-derived growth factor, and nerve growth factor (NGF) initiate signals at the cell membrane by binding to receptor tyrosine kinases (RTKs). Along with G-protein-coupled receptors, RTKs are the main platforms for transducing extracellular signals into intracellular signals. Studying RTK signaling has been a challenge, however, due to the multiple signaling pathways to which RTKs typically are coupled, including MAP/ERK, PLCγ, and Class 1A phosphoinositide 3-kinases (PI3K). The multi-pronged RTK signaling has been a barrier to isolating the effects of any one downstream pathway. Here, we used optogenetic activation of PI3K to decouple its activation from other RTK signaling pathways. In this context, we used genetic code expansion to introduce a click chemistry noncanonical amino acid into the extracellular side of membrane proteins. Applying a cell-impermeant click chemistry fluorophore allowed us to visualize delivery of membrane proteins to the plasma membrane in real time. Using these approaches, we demonstrate that activation of PI3K, without activating other pathways downstream of RTK signaling, is sufficient to traffic the TRPV1 ion channels and insulin receptors to the plasma membrane.

Receptor Tyrosine Kinase Signaling Involves Echinococcus-Host Intercommunication: A Potential Therapeutic Target in Hepatic Echinococcosis.

Echinococcosis, one of the most serious and life-threatening parasitic forms of zoonosis worldwide, is caused by the larvae of Echinococcus granulosus (E. granulosus) and Echinococcus multilocularis (E. multilocularis). Various drugs are being applied clinically to treat zoonosis; however, their therapeutic efficacy remains a great challenge, especially with albendazole as the preferred drug of choice. Receptor tyrosine kinase (RTK) signaling controls normal cellular proliferation, differentiation, and metabolism in humans and mammals, which are intermediate hosts of E. granulosus and E. multilocularis. Disruption of RTK signaling can cause various forms of carcinogenesis and exacerbate the progression of certain forms of parasitic disease. As a result, a significant number of studies on tyrosine kinase inhibitors (TKIs) have been conducted for the treatment of cancer and parasitic infection, with some TKIs already approved for clinical use for cancer. Notably, RTK signaling has been identified in the parasites E. granulosus and E. multilocularis; however, the mechanisms of RTK signaling response in Echinococcus-host intercommunication are not fully understood. Thus, understanding the RTK signaling response in Echinococcus-host intercommunication and the potential effect of RTK signaling is crucial for identifying new drug targets for echinococcosis. The present review illustrates that RTK signaling in the host is over-activated following infection by E. granulosus or E. multilocularis and can further facilitate the development of metacestodes in vitro. In addition, some TKIs exert strong parasitostatic effects on E. granulosus or E. multilocularis, both in vitro and/or in vivo, through downregulation of RTK signaling molecules. The summarized findings suggest that RTK signaling may be a promising drug target and that TKIs could be potential anti-Echinococcus drugs warranting further research.

Effects of heterologous kinase domains on growth factor receptor specificity

The kinase domains of receptor tyrosine kinases (RTKs) are highly conserved, yet they are able to discriminate among potential substrates to selectively activate downstream signaling pathways. In this study, we tested the importance of catalytic domain specificity by creating two series of chimeric RTKs. In one set, the kinase domain of insulin-like growth factor I receptor (IGF1R) was replaced by the kinase domains from insulin receptor (IR), macrophage stimulating protein 1 receptor/Ron (Ron) or Src. In the other set of chimeras, the kinase domain of epidermal growth factor receptor (EGFR) was similarly replaced by the kinase domains of IR, Ron, or Src. We expressed the wild-type and chimeric forms of the receptors in mammalian cells. For some signaling events, such as recognition of IRS1, the identity of the tyrosine kinase catalytic domain did not appear to be crucial. In contrast, recognition of some sites, such as the C-terminal autophosphorylation sites on EGFR, did depend on the identity of the kinase domain. Our data also showed that ligand dependence was lost when the native kinase domains were replaced by Src, suggesting that the identity of the kinase domains could be important for proper receptor regulation. Overall, the results are consistent with the idea that the fidelity of RTK signaling depends on co-localization and targeting with substrates, as well as on the intrinsic specificity of the kinase domain.