Cell Communication and Signaling

I.Understanding the mechanisms governing neuronal differentiation is essential for elucidating neurodevelopmental processes and identifying therapeutic targets for neurological disorders. In this study, we optimized serum-dependent induction conditions and benchmarked multiple RNA-seq pipelines to establish a robust in-vitro model of neurogenesis using P19 embryonal carcinoma cells. Retinoic acid (RA, 0.5 {micro}M) was used to induce neuronal differentiation under varying concentrations (1%, 2%, and 5%) of fetal bovine serum (FBS) obtained from three suppliers. Morphological observation and marker gene analysis (MAP2, OCT4) revealed that serum concentration strongly influenced aggregation, survival, and neuronal commitment, with 2-5% FBS yielding optimal neurogenic differentiation. Total RNA extracted on day 10 of differentiation was subjected to RNA sequencing, and the resulting datasets were analyzed using four independent bioinformatics workflows: a Linux-based R pipeline (HISAT2 + featureCounts + DESeq2), nf-core, Galaxy, and BGIs Dr. Tom platform. Differential gene expression analysis identified 9,943 differentially expressed genes (DEGs) (FDR < 0.05, |log2FC| > 1), enriched in synaptic assembly and axon development among upregulated genes, and in ribosome biogenesis and RNA processing among downregulated genes. Comparison across all pipelines revealed 62 consistently upregulated and 63 downregulated genes, representing a robust core signature of P19 neurogenesis. Together, these findings establish an optimized and reproducible framework for in-vitro neuronal differentiation and transcriptomic analysis, providing a foundation for mechanistic and disease-modeling studies in neurodevelopmental biology.

Gastric cancer is driven by aberrant kinase signaling that supports uncontrolled proliferation and metabolic adaptation. Calcium/calmodulin dependent protein kinase kinase 2 (CAMKK2) is overexpressed in gastric cancer; however, its role in regulating metabolic programs that sustain tumor growth remains incompletely understood. In this study, we employed an integrated multi-omics approach with a primary focus on untargeted metabolomics to investigate the consequences of CAMKK2 inhibition in gastric cancer cells. Pharmacological inhibition of CAMKK2 using STO-609 in AGS cells resulted in significant suppression of proliferation, clonogenic growth, migration, and invasion, accompanied by pronounced nuclear abnormalities and multinucleation indicative of mitotic defects. Global metabolomic profiling revealed extensive and time-dependent metabolic reprogramming following CAMKK2 inhibition, characterized by a marked depletion of nucleotide intermediates, including purine and pyrimidine metabolites required for DNA synthesis. Pathway enrichment analysis highlighted suppression of nucleotide metabolism, lipid metabolism, and central carbon metabolic pathways, indicating a broad impairment of biosynthetic capacity. Integration with proteomic and phosphoproteomic datasets demonstrated that metabolic alterations were accompanied by downregulation of DNA replication machinery and attenuation of kinase signaling pathways governing cell cycle progression. Protein metabolite interaction and docking analyses further supported functional coupling between nucleotide metabolites and key replication-associated enzymes, revealing disruption of metabolite enzyme interactions upon CAMKK2 inhibition. Collectively, these findings identify CAMKK2 as a critical regulator of metabolic programs that support DNA replication and cell cycle progression. Its inhibition induces replication stress through coordinated depletion of nucleotide pools and disruption of replication-associated signaling, leading to impaired proliferation and mitotic failure. These results highlight CAMKK2 as a potential therapeutic target for exploiting metabolic vulnerabilities in gastric cancer.

To facilitate survival, migration and evasion of immune surveillance, cancer cells tightly coordinate the synthesis and trafficking of a diverse repertoire of proteins to their cell surface and the surrounding tumor microenvironment. A key mechanism underlying this process is the intracellular membrane trafficking pathways, including vesicular transport systems. There remains a paucity of mechanistic insight into the regulatory components that mediate nascent protein trafficking and their dysregulation in cancer. Herein, we investigate Tumor Protein D54 (TPD54) as a central regulator of intracellular protein transport that is exploited by melanoma cells to promote disease progression. Integrative analyses of patient-derived tumor tissue specimens show that the expression of TPD52L2 (the gene encoding TPD54) is frequently overexpressed in melanoma and correlates with adverse clinical outcomes, including reduced responses to immune checkpoint blockade. Mechanistic investigations further revealed that TPD54 maintains Golgi integrity and orchestrates trafficking of early endosomes, anterograde vesicles and extracellular vesicles. Functionally, TPD54 augments the secretion of pro-cancerous cytokines, increases the cell surface expression of adhesion-signaling receptors (e.g. integrin-{beta}1 and desmoglein-2), promotes melanoma cell migration and elevates their capability to undergo vasculogenic mimicry. Targeting TPD52L2 in two mouse models of melanoma (B16-F10 and HCmel12) showed significant attenuation of tumor growth, disrupted tumor vasculature, enhanced anti-tumor immunity with infiltration of CD8+ T cells and reduced metastatic disease. Collectively, these findings establish TPD54 as a critical and previously underappreciated regulator of protein trafficking in cancer cells that directly contributes to disease progression and highlights its potential as a novel therapeutic target to combat melanoma.

Thyroid eye disease (TED) is a debilitating condition characterized by orbital fibroblast (OF) activation and excessive hyaluronic acid (HA) accumulation within the retro-ocular space. While IGF-1R blockade with teprotumumab has significantly advanced TED management, incomplete clinical responses and disease relapse underscore the need to identify alternative targets. In this study, we used high-throughput RNA sequencing to map the transcriptomic landscape in TED OFs compared with non-TED OF controls. Our analysis identified robust enrichment of pathways critical to the TED phenotype, including PI3K-AKT signaling, the platelet-derived growth factor (PDGF) pathway, and extracellular matrix remodeling. We validated several key upregulated mediators that may contribute to orbital remodeling, including FOXC2, HGF, MET, and HMGA2, alongside the downregulation of the Wnt antagonist SFRP2. By employing a computational drug-repositioning approach, we identified the multi-kinase inhibitor sorafenib, which targets VEGFR, PDGFR, and RAF, as a potent candidate to neutralize the TED-specific gene signature. Functional assays demonstrated that sorafenib dose-dependently inhibited PDGF-induced AKT phosphorylation and significantly attenuated HA synthesis in primary TED OFs. These results define a persistent, receptor tyrosine kinase-driven program in the TED orbit and suggest that multi-kinase inhibition represents a viable therapeutic strategy for refractory TED. HighlightsO_LIThyroid eye disease (TED) orbital fibroblasts exhibit a transcriptomic signature characterized by elevated PI3K/AKT, angiogenic, and growth factor signaling. C_LIO_LIComputational drug prediction identifies sorafenib as a candidate to reverse the TED gene signature. C_LIO_LISorafenib dose-dependently inhibits AKT activation and hyaluronic acid production in TED orbital fibroblasts. C_LI

The most prevalent RNA alteration in the mammalian genome is N-6-methylenediosine (m6A). There is mounting evidence linking dysregulation of m6A regulatory factors and alterations in m6A levels to the development, course, or prognosis of ovarian cancer. Genes having prognostic value were screened using the univariate, multifactorial, and Least Absolute Shrinkage Selection Operator (LASSO) Cox regression analyses. Important genes' m6A expression in clinical material was verified by real-time fluorescent quantitative polymerase chain reaction (RT-qPCR). In present study, all 23 regulators were significantly differentially expressed in ovarian cancer tissues. LASSO regression analysis screened for 10 key genes associ-ated with both autophagy and m6A. A risk score was constructed and nomogram was developed to forecast the prognosis of ovarian cancer patients. Additionally, individuals with ovarian cancer were classified as high-risk or low-risk; and the low-risk group might be more likely to benefit from im-munotherapy. RT-qPCR was used for the bioinformatics study of human ovarian cancer and normal tissues. Lastly, PLK2 and LEPR were confirmed to be associated with tumorigenesis in scRNA-seq. The risk score established by m6A and autophagy can be used to predict prognosis and susceptibility to anticancer drugs in patients with ovarian cancer.

Melanoma, one of the most metastatic and multidrug resistant cancer, is the first leading cause of death from skin cancer. This complex disease requires identification of additional cooperating events that contribute to progression, invasion and metastasis to reinforce therapeutics. RhoGTPases play key roles in cancer development and metastasis. Rhophilin-2 (RHPN2), a Rho effector, is amplified in various human cancers and its role in melanoma remains unexplored. Here, we combined knock-down experiments in human melanoma cells, with knock-out and overexpression experiments in zebrafish to uncover the roles of RHPN2 in melanoma development. We show that in human melanoma cells RHPN2 contributes to growth, and to clonogenic, migratory and invasive properties of the cells. Using NRASQ61L and BRAFV600E zebrafish models, we provide the first in vivo evidence that Rhpn2 promotes melanoma onset and development. Histological analysis of the Rhpn2 deficient tumors showed decreased cellular density and absence of primary cilia structures at the invasive tumor/stroma borders. Transcriptomic profiling of the Rhpn2-KO melanoma revealed increased expression of the IFN1-responsive genes and modulation of genes involved in lipid metabolism and cilia function. Together these findings position RHPN2 as a modulator of melanoma, offering new perspectives in considering it as a target to impair the development of the tumor.

Cell polarity complexes are essential for embryogenesis, but their regulatory mechanisms during early developmental transitions remain incompletely understood. Here, we individually deleted the Crumbs, Par, and Scrib polarity complexes in mouse embryonic stem cells (mESCs). While loss of any single complex did not affect pluripotency or proliferation, deletion of Par complex disrupted the naive-to-primed transition and impaired subsequent differentiation, particularly lumen formation in neural tube organoids. Mechanistically, Par complex deficiency led to hyperphosphorylation of focal adhesion kinase (FAK) at the primed stage, driving a morphological shift from flat monolayer clusters to dome-shaped colonies. FAK inhibition rescued the aberrant morphology. Upstream, Par complex loss increased AKT phosphorylation, which remodeled extracellular matrix (ECM) and regulated integrin signaling via FURIN-LEFTY, ultimately modulating FAK activity. In addition, conditioned medium from wild-type cells partially rescued differentiation defects in Par knockout cells in a LEFTY-dependent manner. These phenotypes were consistently observed in naive-to-primed transition, neural stem cell differentiation, embryoid body formation, teratoma assays, and neural tube organoid differentiation. Together, these findings establish a Par complex-AKT-FURIN-LEFTY-ECM-integrin-FAK signaling cascade that links apical-basal polarity to early lineage specification and morphogenesis, providing a mechanistic framework for how polarity cues are translated into developmental outcomes. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/722465v1_ufig1.gif" ALT="Figure 1"> View larger version (64K): org.highwire.dtl.DTLVardef@1c62c46org.highwire.dtl.DTLVardef@184d5b5org.highwire.dtl.DTLVardef@1ea9017org.highwire.dtl.DTLVardef@9a0318_HPS_FORMAT_FIGEXP M_FIG C_FIG Significance StatementThis study elucidates the molecular mechanism by which the Par complex regulates the establishment of cell polarity. The authors demonstrate that the Par complex promotes the expression of the protein convertase FURIN via AKT signaling, thereby enhancing the maturation and secretion of LEFTY protein. This process remodels the ECM and modulates integrin signaling, ultimately regulating FAK activity and controlling the establishment of cell polarity. These findings reveal how polarity cues govern early lineage specification and morphogenesis, with implications across multiple developmental contexts.

Macrophage-melanoma interactions critically shape the tumor microenvironment, yet the cytokine networks driving this process remain incompletely understood. Among these, colony-stimulating factors--particularly granulocyte-macrophage colony-stimulating factor (GM-CSF) and granulocyte colony-stimulating factor (G-CSF or CSF3)--regulate myeloid cell behavior during cancer progression, although their specific roles in melanoma remain unclear. Using co-culture systems of melanoma and monocyte-derived macrophages, we found that GM-CSF-primed macrophages induced robust G-CSF secretion and concurrent CSF3 upregulation in both cell compartments. Moreover, transcriptomic profiling of patient-derived tumor-associated macrophages (TAMs) confirmed elevated CSF3 and CSF3R expression in metastatic melanoma. Multiplex immunofluorescence analysis of a stage II-IV primary melanoma cohort (n=84) revealed increased G-CSF and CD114 expression in TAMs and tumor cells from patients who subsequently developed metastasis. High G-CSF levels in either TAMs or melanoma cells emerged as an independent prognostic factor for shorter disease-free and overall survival (p < 0.001). Mechanistically, G-CSF activated STAT1/STAT3 signaling in melanoma cells and promoted proliferation and invasion in a CD114-dependent manner. Of note, G-CSF drove monocyte differentiation toward a distinct inflammatory macrophage state characterized by STAT1/STAT3 activation and a pro-invasive secretory profile. Furthermore, melanoma cells conditioned by G-CSF-differentiated macrophages displayed increased in vivo lung colonization and enriched transcriptional programs linked to invasion and proliferation. Collectively, these findings establish the G-CSF/CD114 axis as a potential clinically relevant driver of melanoma metastasis and identify G-CSF as a novel independent prognostic biomarker. HIGHLIGHTSO_LIDespite its extended clinical use, G-CSF role in human melanoma remains undefined. C_LIO_LIHigh G-CSF expression in primary melanomas correlates with poor patient survival. C_LIO_LIG-CSF promotes melanoma cell invasion and proliferation through CD114 signaling. C_LIO_LIG-CSF drives macrophage differentiation toward a pro-tumoral phenotype. C_LIO_LIMelanoma cells conditioned by G-CSF-primed macrophages are more metastatic. C_LI

Breast cancer is the most common malignancy in women, with triple-negative breast cancer (TNBC) representing the most aggressive subtype and carrying a poor metastatic prognosis. Metastasis requires tumor cells to cross the endothelial barrier, a process facilitated by tumor-derived extracellular vesicles (EVs), which can disrupt vascular integrity. Fluid shear stress (FSS), generated by blood flow, shapes endothelial physiology and may influence EV uptake, yet the mechanisms underlying TNBC-derived small EV (sEV) internalization remain unclear. Here, we investigated TNBC sEV-endothelial interactions using combined in silico and in vitro approaches. Human umbilical vein endothelial cells (HUVECs) were cultured under static or FSS conditions (20 dyn/cm{superscript 2}), followed by proteomic profiling and protein-protein interaction analyses with sEV proteomes. Uptake assays employed pharmacological inhibition (Dynasore, M{beta}CD, Pitstop2), Caveolin-1 (CAV-1) and Clathrin Heavy Chain (CLHC), siRNA-mediated knockdown, and junctional interaction analyses via confocal microscopy and co-immunoprecipitation. FSS downregulated proliferation- and angiogenesis-associated proteins while upregulating adhesion and cytoskeletal regulators assessed by proteomics. Network analysis identified clathrin- and caveolin-mediated endocytosis (CME and CavME), integrins, and early endosomes as central mediators of sEV uptake. Functionally, uptake was reduced by Pitstop2, M{beta}CD, and CAV-1/CLHC knockdown under static conditions, but silencing paradoxically enhanced uptake under FSS, suggesting compensatory flow-dependent pathways. Notably, under FSS, sEVs accumulated at endothelial junctions, colocalizing with VE-CAD and associating with CLDN5, indicating a potential disruption mechanism of adherens and tight junctions and consequent endothelial permeability. These findings identify CME and CavME as key uptake routes while underscoring FSS as a critical determinant of endothelial-tumor EV interactions. By revealing junctional targeting of sEVs, this work provides new mechanistic insight into vascular remodeling during metastasis and highlights EV pathways as potential therapeutic targets in TNBC. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=104 SRC="FIGDIR/small/721946v1_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@f91c5org.highwire.dtl.DTLVardef@2b4dc8org.highwire.dtl.DTLVardef@ff94f1org.highwire.dtl.DTLVardef@18b714b_HPS_FORMAT_FIGEXP M_FIG C_FIG Uptake and localization of sEVs on HUVEC under (a) static and (b) fluid shear-stress conditions. sEVs: Small Extracellular Vesicles. CME: Clathrin-mediated Endocytosis. CavME: Caveolin-mediated Endocytosis. CLDN5: Claudin-5. VE-CAD: Vascular Endothelial Cadherin. FSS: Fluid shear-stress.

Triggering receptor expressed on myeloid cells 2 (TREM2) plays a crucial role in regulating various microglial functions, including phagocytosis, inflammation, chemotaxis, and proliferation. Recent studies have demonstrated that TREM2 cooperates with DAP12 to mediate intracellular signaling essential for these processes. Despite the importance of the TREM2-DAP12 complex in microglial physiology, the mechanisms controlling its expression and activity remain poorly understood. In this study, we report that the soluble ectodomain of TREM2 (sTREM2) regulates microglial phagocytic activity by attenuating the surface expression of DAP12. We found that stimulation of the microglial cell line BV2 with recombinant sTREM2 reduces the membrane expression of DAP12, but not that of TREM2. In addition, sTREM2 binds to full-length TREM2, leading to the uncoupling of TREM2 from DAP12. Furthermore, pre-treatment of BV2 cells with sTREM2 significantly inhibited amyloid-{beta} incorporation. These findings suggest that sTREM2 negatively regulates TREM2 signaling through the destabilization of the TREM2-DAP12 complex, and act as a novel bioactive molecule that modulates TREM2 signaling under physiological and pathological conditions.

Resistance to targeted therapy in HER2-positive breast cancer remains a clinical challenge, especially for patients with relapsed or metastatic disease. Particularly, persistent activation of hypoxia-inducible factor 1 (HIF-1) signalling is well documented in the context of trastuzumab and trastuzumab emtansine resistance. To achieve a deeper understanding of how HIF-1 activity modulates the response to anti-HER2 treatment, we functionally characterized a cellular model of hypoxia-induced drug resistance for HER2-positive breast cancer using shotgun proteomics. By global phosphoproteomics profiling, the Rac1 pathway was identified as one of the most enriched signalling networks under hypoxia. Furthermore, the selective Rac1 blockade with the 1A-116 small-molecule inhibitor sensitised HER2-positive cells to trastuzumab in both 2D and 3D culture systems. Altogether, our findings demonstrate that hypoxic conditions induce the resistance of HER2-positive breast cancer cells to targeted therapy and suggest the therapeutic potential of Rac1 inhibition to enhance trastuzumab efficacy. HighlightsO_LIHypoxic conditions induce trastuzumab resistance in HER2-positive breast cancer. C_LIO_LIRac1 signalling was mapped under hypoxia by phosphoproteomics profiling. C_LIO_LIRac1 inhibition sensitises HER2-positive cells to trastuzumab. C_LI

The scaffold protein FRS2 is central to FGFR signaling, linking receptor activation to MAPK/ERK and PI3K/AKT pathways. Elevated FRS2 expression correlates with aggressive tumor phenotypes and poor prognosis across multiple cancers, including the pediatric cerebellar tumor medulloblastoma (MB). Here, we characterized FRS2s subcellular localization and interactome in MB cells, employing live-cell imaging, phosphoproteomics, immunoprecipitation, and APEX2-based proximity labeling. We found that increased FRS2 expression is associated with increased motile and invasive behavior in MB tumor cells. We furthermore identified novel candidate FRS2-associated proteins involved in actin cytoskeleton remodeling, cell junction assembly, and translation initiation, which indicate a growth factor-dependent reorganization of the FRS2 signalosome. Our data furthermore indicate a regulatory role of FRS2 in directing subcellular distribution of the cell junction and cell motility regulator TJP1. Our findings highlight the relevance of FRS2 as a mediator of cell motility and invasiveness and provide candidate proteins associated with FRS2 that are involved in cellular processes governing migration and invasion. This study thus provides a framework for exploring the FRS2 interactome as a possible target to attenuate FGFR-driven oncogenic processes with next-generation therapeutic strategies.

Heterogeneous nuclear ribonucleoprotein U (HNRNPU) deficiency is a rare genetic cause of neurodevelopmental disorders (NDDs) lacking targeted therapies. Here, we developed a transcriptomic-guided compound prioritization pipeline using Connectivity Map (CMap) analysis on multi-model transcriptomic signatures from HNRNPU-deficient human cells and mouse models. Ten compounds were selected through manual curation and functionally screened in patient-derived HNRNPU-deficient neuroepithelial stem (NES) cells with earlier observed cellular phenotypes. Two of the compounds, AS601245 and Lenalidomide, significantly reduced the elevated neural progenitor population during differentiation, and their combination further decreased primary cilia incidence, indicating partial rescue of the patient-specific cellular phenotypes. To understand the mechanisms underlying the partial rescue, we employed proteome integral solubility alteration (PISA) and expression proteomics. PISA assay identified TMEM150C and GSK3A as proximal targets of combined treatment. Additionally, we observed reversal of multiple biological pathways including downregulation of Wnt signalling and upregulation of mitochondrial pathways and transmembrane proteins. Altogether, we established a computational-experimental pipeline for transcriptomic-guided drug repurposing for a monogenic NDD, and demonstrated that the network-level modulation partially rescues the delayed neural differentiation in HNRNPU-deficient neural cells.

Extracellular vesicles (EVs) are emerging mediators of oncogenic communication within the neuroblastoma (NB) tumour microenvironment (TEM). Here, we investigated how constitutive MYCN overexpression influences the proteomic and functional properties of small EVs (sEVs) derived from SKNAS-MYCN-GFP (SK-M) cells and assessed their impact on non-cancerous immune cells. SK-M cells exhibited robust MYCN upregulation at both the mRNA and protein levels and produced sEVs that were selectively enriched in MYCN. Transwell co-culture revealed transfer of MYCN-GFP to recipient DC2.4 nuclei, indicating intercellular transport of functional transcription factor cargo. LC-MS/MS profiling showed that SK-M sEVs incorporated oncogenic cargo non-randomly, displaying significant enrichment of metabolic and MYC/MYCN-regulated pathways, including glycolysis, mTORC1 signalling, and suppression of oxidative phosphorylation (OXPHOS). These observations are consistent with emerging evidence that MYC family proteins can regulate metabolism through vesicular transfer of glycolytic kinases to neighbouring cells. Functionally, SK-M cells displayed elevated lactate secretion and reduced acetyl-CoA, and their sEVs induced a glycolytic shift in recipient immune cells, increasing lactate output in DC2.4, RAW264.7, BMDCs, and splenocytes. sEV-treated BMDCs and splenocytes acquired immunoregulatory phenotypes characterised by increased IL-10, reduced IL-12, expansion of regulatory T cells (Tregs), and macrophage polarization toward an M2-like state. These findings demonstrate that MYCN-driven NB cells disseminate metabolic and immunosuppressive cues via sEVs, reshaping the local immune landscape to favour tumour tolerance. This study provides mechanistic insight into how MYCN-amplified NB cells exploit EV-based communication to coordinate metabolic rewiring and immune escape.

Zebrafish (Danio rerio) possess remarkable regenerative capacity, making them an ideal model for studying the molecular mechanisms underlying tissue regeneration. In this article we report the identification of blastema linked exosome like extracellular vesicles (EVs) in zebrafish, that to the vesicles were plausibly being translocated in the proximo-distal axis through filipodia. We further thoroughly examined the exosome like EVs isolated from regenerating tissues of zebrafish caudal fins to characterize their nucleic acid cargo and evaluate their potential regulatory functions in regeneration. Caudal fins were amputated and allowed to regenerate and exosome like EVs isolated from blastema tissues displayed increased abundance compared to non-amputated controls. RNA sequencing identified a dynamic cluster of EV linked microRNAs (miRs). These differentially expressed miRs, including dre-miR-21, dre-miR-200b, dre-miR-218a and dre-let-7e were upregulated and associated with promoting proliferation, migration, differentiation, and tumour suppression pathways. Moreover, dre-miR-100, dre-miR-146a and dre-miR-200c regulated osteogenic differentiation, inflammatory signalling, epithelial-mesenchymal transition, and cell adhesion. Regeneration is generally believed to be coordinated only by local morphogen diffusion. Through this study it is indicative that filipodia bound EVs might have a pivotal role in long-range communication between blastema and the proximal tissues during the regeneration process. A detailed analyses of the miR targets and their validation could potentially lead to novel advancement and solutions in the field of regeneration and regenerative medicine in the near future.

BackgroundDysregulated cell migration leading to metastasis remains the primary cause of cancer-related mortality. It has been challenging to understand how cells regulate migration. We have previously created the first selective inhibitor of cell migration, KBU2046. Here, we use it as a probe to identify regulatory processes. MethodsMetastatic and primary human prostate cancer cells were treated for different times and at different concentrations with KBU2046. Immunofluorescent microscopy examined protein localization in cells. Label-free mass spectrometry (MS) was performed on total cell proteins, Tandem Mass Tag (TMT) labeling MS was used on membrane fractions, and temporal phosphoproteomic profiling. Results were analyzed with a suite of bioinformatic tools. ResultsKBU2046-induced migrastasis is associated with the accumulation of activated integrin {beta}1 into focal adhesions. Whole-cell proteomics demonstrated suppression of processes that mediate intracellular protein trafficking and increases in mitochondrial energy-generation signatures. Evaluation of the membrane fraction identified increases in membrane repair and maintenance processes and decreases in those that drive motility. Temporal- and concentration-dependent phosphoproteomic profiling revealed that KBU2046 initiates a dynamic, cascading sequence of transient signaling waves rather than a static block. ConclusionsKBU2046-induced migrastasis appears to operate through spatial decoupling rather than structural degradation. By restricting the intracellular trafficking machinery required for receptor recycling, KBU2046 limits focal adhesion turnover, providing a correlative framework to inhibit metastatic dissemination independent of direct cytotoxicity. O_FIG O_LINKSMALLFIG WIDTH=122 HEIGHT=200 SRC="FIGDIR/small/736165v1_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@1cc69d3org.highwire.dtl.DTLVardef@137b843org.highwire.dtl.DTLVardef@1225e50org.highwire.dtl.DTLVardef@15dd8d2_HPS_FORMAT_FIGEXP M_FIG Graphic Abstract C_FIG

Lymphocyte-extracellular matrix (ECM) interactions occur intermittently throughout the lymphocytes life cycle. Alterations in blood insulin levels following feeding modulates naive lymphocyte trafficking and adhesion to fibronectin via a pathway involving insulin-like growth factor-1 receptor (IGF-1R), phospholipase C gamma 1 (PLC-{gamma}1) and {beta}2 integrin activation. Lymphocytes exert traction forces, on the ECM during the process of extravasation. While these forces are essential for several homeostatic processes, the role of insulin in modulating lymphocyte-derived traction forces upon ECM adhesion is unknown. The aim of the current study was to investigate the effect of insulin on the traction generated by lymphocytes when adhered onto a fibronectin-coated substrate. Jurkat T-cells were placed on a fibronectin layer (50{micro}g/ml, 100{micro}m thickness) coated on polyacrylamide gels of stiffness 400Pa with red fluorescence beads as fiduciary markers. The cellular force generated by Jurkat T-cells was mapped using traction force microscopy. To elucidate the role of PLC-{gamma}1 in cellular force generation, the traction of Jurkat T-cells lacking PLC-{gamma}1, as well as those of a knockout cell where PLC-{gamma}1 was restored were quantified and compared with wild-type Jurkat T-cells. Lack of PLC-{gamma}1 attenuated adhesion when compared to wild-type Jurkat T-cells. Additionally, the traction force generated by each cell type decreased with increasing concentration of extracellular calcium. Treatment of adherent Jurkat T-cells with insulin increased traction in lower extracellular calcium condition while a dip was observed when a high extracellular calcium was present, in comparison to the untreated cells. However, the effect of insulin treatment was lost in the case of Jurkat T-cells lacking PLC-{gamma}1. Together these results indicate that insulin regulates traction force generated by adherent Jurkat T-cells via a process involving PLC-{gamma}1, in a calcium dependent manner.

Deregulation of sphingolipid (SL) metabolism is well-established across many cancers, yet the underlying mechanisms that drive changes in SLs are poorly understood. We previously identified dihydroceramide desaturase 1 (DES1) as a downstream target of HER2 and implicated DES1 as a driver of anchorage-independent survival in breast cancer. In this study, we expand on these results to establish the oncogenic PI3K pathway as a driver of post-translational DES1 activity following cell detachment from the extracellular matrix. PI3K activation of DES1 required glucose uptake and metabolism through both glycolysis and the pentose phosphate pathway. However, it did not require glucose flux into the TCA cycle and was independent of antioxidant capacity of the cell. Moreover, Instead, results identify GAPDH - a point of convergence between glycolysis and PPP - as important for oncogene-driven DES1 activity. Overall, this study defines a novel pathway of DES1 regulation and establishes DES1 as a point of crosstalk between glucose and SL metabolic pathways.

BackgroundMesothelioma (Me) is an aggressive cancer with limited response to conventional therapies. The tumors harsh microenvironment contributes to immune escape and therapy resistance and the effects of ICIs on Me are still unclear. Adenosine, an immunosuppressive molecule produced from AMP by the enzyme CD73, accumulates in hypoxic tumor areas. Elevated CD73 and adenosine receptor A2B (A2Br) levels on Me cells are linked to worse patient outcomes, indicating their important role in disease progression and potential as targets for treatment. AimThis study characterizes the Me-ME (micro environment) and evaluates the efficacy of TT-4 (A2B inibitor) and AB680 (CD73 inibitor), alone or with aPD-1, using 3D models in vitro and in vivo. MethodsCD73 and A2B receptor levels were quantified in tumor and normal samples using qRT-PCR and IHC. Cells lines were treated with CoCl2 to mimic hypoxia, then CD73, A2Br and related markers were analyzed. MSTO-211H and REN cells were silenced for CD73, grown as spheroids and adenosine release was measured. Co-culture spheroids of MSTO-211H and Jurkat cells were treated with AMP and CD73 inhibitor, then analyzed for viability and immune markers. An orthotopic Me model was established by injecting AB1-B/c-LUC cells and monitored by in vivo imaging. Proteomic analysis of spheroids was conducted to identify proteins and pathways involved. ResultsHypoxia boosts CD73 and A2Br expression in Me cells, leading to adenosine production via CD73. In 3D co-cultures, AB680 lowered Me cell viability and enhanced activation of Jurkat T cells. In mice, combining aPD-1 therapy with A2Br or CD73 inhibitors strongly reduced tumor growth. Proteomics identified 93 proteins influenced by adenosine signaling through A2B. ConclusionTargeting the adenosine pathway alongside PD-1 blockade offers a promising new immunotherapy strategy for Me.

Tumours reshape their surrounding extracellular matrix (ECM), creating a microenvironment with altered chemical and mechanical properties. Integrins detect these changes, linking the ECM to the intracellular cytoskeleton and promoting cell survival, motility, invasion and differentiation, and further ECM remodelling. However, the molecular mechanisms by which tumours remodel their ECM are not well understood. Here, we found that the cytokine oncostatin M (OSM) promotes breast cancer progression by activating ECM remodelling and integrin signalling in cancer cells, as shown by combining complementary in vitro, in ovo and in vivo models, and transcriptomic and proteomic analyses. We demonstrated that OSM induces fibrosis, characterized by increased collagen deposition and hydroxylation, together with activation of ECM and ECM-associated proteins and modifiers such as fibronectin, tenascin C, LOX, PLOD2 and collagen prolyl hydroxylases. OSM also promoted the expression of integrins. Integrin alpha 5 (ITGA5) was identified as an important mediator of OSM-effects. ITGA5 blockade, by means of small interference RNA and therapeutic inhibition with a blocking peptide, abrogated OSM-induced cancer cell migration, invasion and in vivo tumour growth. In addition, OSM blockade with a specific inhibitor reduced tumour growth in an immunocompetent mouse model. Our results are clinically relevant as the expression of integrins and matrisome genes strongly correlated with OSM and its receptor OSMR in breast cancer clinical samples; and co-expression of OSMR and ITGA5 associated with decreased survival in basal breast cancer patients. Collectively, our data reinforce the potential of the OSM-ITGA5 axis as a therapeutic target in this breast cancer subtype, which shows the highest mortality rates.