Cell Communication and Signaling

Ovarian cancer (OC) remains the most lethal gynecological malignancy. Our previous work established that WNK lysine-deficient protein kinase 2 (WNK2) promotes OC cell proliferation and migration. To elucidate how WNK2 drives OC progression, we performed transcriptome sequencing to identify WNK2-regulated mRNAs and noncoding RNAs. Candidate targets were validated via qRT-PCR and Western blot. Functional assays (CCK-8, colony formation, Transwell) assessed the role of POU5F1B and its ability to rescue WNK2 knockdown effects. Given AKTs involvement downstream of POU5F1B, we measured AKT phosphorylation. Additionally, since WNK2 activates RAS (as previously shown), we tested whether RAS inhibition blocks WNK2-mediated POU5F1B regulation. POU5F1B exhibited oncogenic properties in OC cells. WNK2 upregulated POU5F1B mRNA and protein levels, and POU5F1B overexpression reversed tumor-suppressive effects caused by WNK2 knockdown. Mechanistically, WNK2 depletion reduced AKT phosphorylation, which was restored by POU5F1B overexpression. Furthermore, RAS inhibition abolished WNK2-driven POU5F1B upregulation, linking WNK2-RAS signaling to POU5F1B activation. Our study demonstrates that WNK2 promotes OC progression by upregulating POU5F1B, thereby activating AKT signaling. These findings solidify WNK2s oncogenic role and highlight its therapeutic potential in OC.

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 promotes uncontrolled proliferation and malignant progression. Calcium/calmodulin-dependent protein kinase kinase 2 (CAMKK2) is overexpressed in gastric cancer; however, the global phosphorylation networks downstream of CAMKK2 remain incompletely defined. In this study, we investigated the functional and signaling consequences of CAMKK2 inhibition in gastric cancer cells using an integrated phenotypic and quantitative phosphoproteomics approach. Pharmacological inhibition of CAMKK2 using STO-609 in AGS cells significantly suppressed proliferation, clonogenic growth, migration, and invasion, and induced defects in nuclear morphology indicative of impaired cell cycle progression. Tandem mass tag (TMT) based phosphoproteomic profiling identified over 10,500 phosphopeptides and revealed extensive phosphoproteome remodeling following CAMKK2 inhibition, characterized predominantly by hypophosphorylation of proteins involved in nuclear signaling, RNA processing, and cell cycle regulation. Kinase substrate enrichment and motif analyses demonstrated coordinated attenuation of CDK, MAPK, and mitotic kinase-associated signaling pathways, with convergence on E2F regulated transcriptional programs. Collectively, these findings establish CAMKK2 as a central regulator of kinase signaling networks that sustain proliferative and malignant phenotypes in gastric cancer and highlight CAMKK2 inhibition as a potential therapeutic strategy.

S100A4, a metastasis promoting calcium binding protein, drives tumor progression through pleiotropic mechanisms, yet its context dependent functions in gestational malignancies remain elusive. To dynamically decode its role in choriocarcinoma pathogenesis, we leveraged label free real time cell analysis (RTCA) to profile malignant phenotypes in JAR cells following siRNA mediated S100A4 silencing, complemented by apoptosis assessment and targeted signaling profiling. Efficient knockdown (verified by qPCR/Western blotting) significantly attenuated cellular proliferation (96 hr cell index slope decreased vs. scramble control; p<0.01) and suppressed migration capacity (p<0.01). Critically, S100A4 depletion did not induce apoptosis (flow cytometry and cleaved caspase 3/9 blotting confirmed no significant change), and invasion through Matrigel coated membranes remained statistically unaltered despite comparable experimental rigor. Mechanistically, S100A4 silencing triggered adaptive signaling rewiring: IRS1 and PI3K expression were elevated, Akt1 was suppressed, while MEK1/2 remained unchanged suggesting compensatory pathway activation.

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.

Neuroblastoma is a complex paediatric cancer with a spectrum of clinical outcomes ranging from spontaneous regression to aggressive metastatic disease. Low-risk patients achieve over 90% survival with no or minimal treatment, while high-risk patients face less than 50% survival despite intensive multimodal therapy. Half of the high-risk cases harbour amplification of the MYCN oncogene. In addition to MYCN status, Trk receptors have also been linked to prognosis. TrkA expression is seen with low-risk cases while TrkB expression often occurs in high-risk MYCN-amplified NB. While TrkA and TrkB are well studied in NB, the role of TrkC in neuroblastoma genesis is not clear. Therefore, this study investigates the interplay between MYCN status and NT-3/TrkC signalling in neuroblastoma. Using a panel of neuroblastoma cell lines with varying MYCN levels, we found that TrkC activation leads to neuronal differentiation of MYCN non-amplified cells, whereas it promotes proliferation of MYCN-amplified cells. Temporal phosphoproteomics revealed differential activation of the PKA pathway, which was crucial for TrkC-mediated differentiation. Manipulating the PKA pathway altered cell fate outcomes, underscoring its role. In MYCN-amplified cells, MYCN knockdown increased PKA and CREB activity, shifting the phenotype towards differentiation. Analysis of neuroblastoma patient data showed lower expression of PKA pathway genes in MYCN-amplified tumours. Additionally, miR-221, upregulated by MYCN, was identified as a suppressor of the PKA/CREB pathway. These findings highlight the context-dependent nature of NT-3/TrkC signalling influenced by MYCN; and suggest therapeutic potential in targeting the PKA pathway to induce differentiation of high-risk MYCN-amplified neuroblastoma.

Cells continuously experience fluctuating intracellular calcium (Ca{superscript 2}) signals that orchestrate diverse processes such as transcription, proliferation, and apoptosis. Temporal features of Ca{superscript 2} dynamics, including oscillation frequency, are hypothesized to encode information, allowing cells to discriminate between relevant and stochastic signals. However, the mechanisms of frequency decoding and their transcriptional consequences remain incompletely understood. To address this, we investigated how defined Ca{superscript 2} oscillation frequencies are translated into signaling cascades and gene expression programs in human non-excitable cells. Using optogenetic control of melanopsin-mediated Ca{superscript 2} influx, we induced slow (8 mHz) or fast (15 mHz) oscillations with identical single-pulse kinetics to isolate the effect of frequency. We found that TNF and IL8 transcription via NF-{kappa}B displayed sigmoidal frequency dependence, strictly requiring regular periodic stimulation, while random or low-frequency inputs with equal cumulative Ca{superscript 2} exposure were ineffective. Bulk RNA sequencing revealed a MYC-centered transcriptional response, with 116 of 215 differentially expressed genes predicted as MYC targets, despite unchanged MYC mRNA levels. Label-free phosphoproteomics identified PRKDC, CHEK2 and ATM as the top upstream kinases, forming a network linking Ca{superscript 2} oscillations to cell cycle and stress signaling. These findings demonstrate that cells can decode Ca{superscript 2} oscillation frequency through a multi-kinase network that tunes transcription via NF-{kappa}B and MYC, providing mechanistic insight into how temporal dynamics of second messengers shape cellular decision-making.

The extracellular matrix (ECM) is a meshwork of proteins that orchestrates a broad range of cellular phenotypes, including proliferation, adhesion, migration, and differentiation. SNED1 is a newly characterized ECM glycoprotein that promotes cell adhesion and is essential for embryonic development. Its upregulation is also associated with breast cancer metastasis and poor prognosis for breast cancer patients. We recently showed that SNED1 assembles into fibrillar structures, but the mechanisms guiding its incorporation into the ECM scaffold remain unknown. Combining biochemical assays and confocal immunofluorescence imaging, we found that SNED1 assembly in the ECM occurs early in the process of ECM building and is concomitant and overlaps with the deposition of fibronectin and collagen I, two major ECM proteins. By knocking down fibronectin or destabilizing collagen I fibers, we further demonstrate that SNED1 requires the presence of these proteins for its assembly. Last, using biolayer interferometry, we identify collagen I as the first direct binding partner of SNED1. Altogether, our results lay the foundation for future studies aimed at determining the mechanisms by which SNED1 fibers contribute to SNED1 pathophysiological functions. SUMMARY STATEMENTThe novel protein SNED1 requires the presence of fibronectin and collagen I to assemble into fibrillar structures in the extracellular matrix scaffold.

Extracellular matrix (ECM) protein expression/deposition within and stiffening of the breast cancer microenvironment facilitates disease progression and correlates with poor patient survival. However, the mechanisms by which ECM components control tumorigenic behaviors and responses to therapeutic intervention remain poorly understood. Fibronectin (FN) is a major ECM protein controlling multiple processes. In this regard, we previously reported that DHPS-dependent hypusination of eIF5A1/2 is necessary for fibronectin-mediated breast cancer metastasis and epithelial to mesenchymal transition (EMT). Here, we explored the clinical significance of an interactome generated using hypusination pathway components and markers of intratumoral heterogeneity. Solute carrier 3A2 (SLC3A2 or CD98hc) stood out as an indicator of poor overall survival among patients with basal-like breast cancers that express elevated levels of DHPS. We subsequently discovered that blockade of DHPS or SLC3A2 reduced triple negative breast cancer (TNBC) spheroid growth. Interestingly, spheroids stimulated with exogenous fibronectin were less sensitive to inhibition of either DHPS or SLC3A2 - an effect that could be abrogated by dual DHPS/SLC3A2 blockade. We further discovered that a subset of TNBC cells responded to fibronectin by increasing cytoplasmic localization of eIF5A1/2. Notably, these fibronectin-induced subcellular localization phenotypes correlated with a G0/G1 cell cycle arrest. Fibronectin-treated TNBC cells responded to dual DHPS/SLC3A2 blockade by shifting eIF5A1/2 localization back to a nucleus-dominant state, suppressing proliferation and further arresting cells in the G2/M phase of the cell cycle. Finally, we observed that dual DHPS/SLC3A2 inhibition increased the sensitivity of both Rb-negative and -positive TNBC cells to the CDK4/6 inhibitor palbociclib. Taken together, these data identify a previously unrecognized mechanism through which extracellular fibronectin controls cancer cell tumorigenicity by modulating subcellular eIF5A1/2 localization and provides prognostic/therapeutic utility for targeting the cooperative DHPS/SLC3A2 signaling axis to improve breast cancer treatment responses.

Human embryo implantation, occurring approximately one week after fertilization, remains poorly understood due to ethical and technical limitations of in vivo investigation. To overcome these barriers, and model this critical developmental event, encompassing peri- and early post-implantation stages, we used an in vitro embryo attachment model composed of donor-derived endometrial epithelial cells forming an open-faced endometrial layer (OFEL) and human stem cell-derived blastoids recapitulating human day 5 blastocysts in peri-implantation model. Following attachment, developmental progression was further investigated on laminin-coated substrates to capture early post-implantation dynamics. Despite its central role as the primary endocrine signal of early pregnancy, human chorionic gonadotropin (hCG) remains largely uncharacterized in this context. Here, we describe the transcriptomic profile of blastoid-endometrial co-cultures relative to OFEL alone, identifying CGA and CGB3/5/8 as among the most strongly upregulated genes following blastoid attachment to hormonally stimulated OFEL. Consistent with these findings, immunoassays and luteinizing hormone/choriogonadotropin receptor (LHCGR) activation assays of conditioned media confirmed the secretion of heterodimeric, biologically active hCG and its free subunits in co-cultures, but not in endometrial layers alone. Notably, the hyperglycosylated hCG heterodimer was the predominant isoform detected. Co-culture with the endometrial component significantly increased hCG secretion compared with blastoids cultured alone, an effect further enhanced by hormonal priming in the peri-implantation model. Collectively, these findings indicate that a hormonally primed endometrial environment not only promotes blastoid attachment but also amplifies embryonic hCG production and bioactivity, underscoring the importance of maternal endocrine cues in early embryo-endometrium communication. Furthermore, our peri- and early post-implantation models recapitulate key aspects of reciprocal endocrine signaling between embryonic and endometrial tissues, providing a tractable experimental framework to investigate embryo-endometrium crosstalk.

The Ras/PI3K/ERK signaling network is frequently mutated in various human cancers including cervical cancer and pancreatic cancer. Previous studies showed that the Ras/PI3K/ERK signaling network displays features of excitable systems including propagation of activity waves, all-or-none responses, and refractoriness. Oncogenic mutations lead to enhanced excitability of the network. A positive feedback loop between Ras, PI3K, the cytoskeleton, and FAK was identified as a driver of excitability. In this study, we investigated the effectiveness of targeting signaling excitability by inhibiting both FAK and PI3K in cervical and pancreatic cancer cells. We found that the combination of FAK and PI3K inhibitors synergistically suppressed the growth of select cervical and pancreatic cancer cell lines through increased apoptosis and decreased mitosis. In particular, FAK inhibition caused downregulation of PI3K and ERK signaling in cervical cancer but not pancreatic cancer cells. Interestingly, PI3K inhibitors activated multiple receptor tyrosine kinases (RTKs), including insulin receptor and IGF-1R in cervical cancer cells, as well as EGFR, Her2, Her3, Axl, and EphA2 in pancreatic cancer cells. Our results highlight the potential of combining FAK and PI3K inhibition for treating cervical and pancreatic cancer, although appropriate biomarkers for drug sensitivity are needed, and concurrent targeting of RTKs may be required for resistant cells.

The mechanisms by which environmental signals induce permanent developmental changes remain a fundamental biological problem. We investigated sea star metamorphosis, where microbial biofilms induce a total body plan reorganization. Using systems biology and functional assays, we identified a three-tiered signaling cascade: sensing, conversion, and execution. The immune adaptor MyD88 senses microbes, while MAPK proteins convert this signal into a retinoic acid developmental cue. An amyloid precursor protein (APP)-centered module acts as the irrevocable commitment gateway, stabilized by a positive feedback loop to ensure irreversibility. Remarkably, the genes driving this transition overlap with human pathways for Alzheimers disease and ADHD/Autism. By defining this ancient neuro-immune axis in an echinoderm adult body plan similar to the chordate head, our study establishes sea star metamorphosis as a model for understanding the evolutionary origins of human neurological disorders from the evolutionary developmental pathology perspective.

One of the main glioblastoma (GB) features is the diffuse migration of the tumor cells within the surrounding brain parenchyma, rendering almost impossible the complete tumor resection and irradiation, leading to inexorable lethal relapse of the disease. In the past years, we demonstrated that IRE1 (hereafter IRE1), one of the Endoplasmic Reticulum (ER) stress sensors, plays a key role in GB biology by impacting on immune infiltration, angiogenesis and tumor cell migration/invasion, all these features being linked to an alteration of protein secretion. In the present study, we investigated if and how IRE1 could regulate the functionality of the secretory machinery in GB cells and identified GOLIM4, a Golgi-associated molecule whose expression is regulated downstream of IRE1 through the transcription of XBP1s. Interestingly, GOLIM4 silencing led to decreased surface expression of multiple molecules including MHC class I molecules, growth factor receptors (PDGFRA and IL13RA2) and proteins involved in cell-cell adhesion (CD44, CD54, NCAM1), adhesion to matrix (ITGB1) or cell migration (CD90) without alteration of their encoding transcripts expression levels. Moreover, GOLIM4 silencing phenotypically affected GB cell-cell adhesion and cell migration in multiple models. Overall, we have described a novel IRE1/XBP1s/GOLIM4 operon that controls the secretion of specific proteins and impacts the tumor aggressiveness. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=100 SRC="FIGDIR/small/619629v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@511636org.highwire.dtl.DTLVardef@18971f6org.highwire.dtl.DTLVardef@98ff81org.highwire.dtl.DTLVardef@ae7189_HPS_FORMAT_FIGEXP M_FIG C_FIG

Cancer cells exposed to nutrient deprivation activate adaptive programs to survive metabolic stress, often acquiring enhanced plasticity and motility. We have previously reported that colon cancer cell lines that survived nutrient depletion underwent partial epithelial-mesenchymal transition (pEMT), which was further exacerbated when these cells also underwent lysosomal alkalinization. Here, we have attempted to dissect the molecular mechanisms that drive the motility and shape change from cobblestone to elongated in subpopulations of cells. Using RNA-seq-based bioinformatic analyses integrated with pathway scoring, protein-protein interaction networks, probabilistic modeling and confirmatory experimental data, we have identified the coordinated activation of sublethal apoptotic signaling, fatty acid oxidation, mitochondrial ROS generation, and Ca{superscript 2}-dependent lysosomal exocytosis in the nutrient-depleted cells. Among these phenotypes, the cells undergoing starvation and lysosomal alkalinization exclusively mediated lysosomal exocytosis and cell motility. Probabilistic modeling further revealed non-linear relationships between metabolic stress signals and cell fate transitions, highlighting heterogeneous lysosomal functions as a key determinant of the altered phenotype of cells under nutrient depletion. Overall, our study has identified that aberrant lysosomal functioning in cells under nutrient depletion can specifically select for a subpopulation of cells that are highly viable, metabolically plastic and capable of motility.

The extracellular matrix (ECM) is a complex scaffold of proteins that supports multicellular structures. Interactions between cells and the ECM via receptors, like integrins, govern cellular phenotypes (e.g., proliferation, adhesion), but also contribute to ECM assembly. Understanding how ECM-receptor interactions regulate matrix assembly is critical to uncover how alterations of the ECM cause or accompany congenital diseases, cancer, or fibrosis. SNED1 is a novel ECM protein with roles in development and metastasis. However, the mechanisms governing its assembly and signaling functions remain largely unknown. SNED1 contains two integrin-binding motifs, RGD and LDV, and we recently showed that its interaction with RGD-integrins mediates cell adhesion. Here, we investigated the role of SNED1/integrin interactions in SNED1 ECM assembly. While SNED1/integrin interactions were not necessary for its initial incorporation in the ECM, interaction with LDV-, but not RGD-, integrins, was required for ECM build-up and the patterning of SNED1 and the fibrillar proteins fibronectin and collagen I. Moreover, SNED1/LDV-integrin interaction promoted ECM alignment, cell alignment, and cell proliferation, processes essential to SNED1-driven neural crest cell migration during craniofacial development and breast cancer invasion. SUMMARY STATEMENTInteraction of SNED1 with LDV-binding integrins, but not RGD-binding integrins, mediates ECM remodeling and controls cytoskeletal rearrangement and cell proliferation.

PurposePancreatic ductal adenocarcinoma (PDAC) is characterized by a nutrient-deprived and hypoxic tumor microenvironment (TME) that imposes severe metabolic stress on cancer cells. Under these conditions, tumor cells frequently activate the integrated stress response (ISR) to adapt to TME and develop resistance to therapies. However, how TME components support tumor adaptation to mitochondrial metabolic stress remains incompletely understood. Here, we aimed to identify key metabolite involved in ISR adaptation under oxidative phosphorylation (OXPHOS) inhibition and to elucidate the metabolic symbiosis between cancer-associated fibroblasts (CAFs) and PDAC cells. MethodsWe integrated transcriptomic and metabolomic analyses with functional assays. ISR activation was evaluated by assessing the phosphorylation of eIF2 (p-eIF2) following treatment with carboxyamidotriazole orotate (CTO), an Complex I inhibitor. Metabolomic profiling was used to identify metabolites involved in ISR activation alleviation. Mouse models were used to assess therapeutic responses following depletion of the identified metabolite under CTO treatment. Genetic perturbation of Slc38a4 was performed to assess its functional role in tumor cell adaptation to metabolic stress. ResultsWe identified asparagine (ASN) as a critical metabolite supplied by CAFs to PDAC cells under OXPHOS inhibition. A minimum level of ASN is required for PDAC cells to execute ISR downstream adaptation. ASN depletion significantly enhanced the anti-tumor efficacy of OXPHOS inhibition both in vitro and in vivo. SLC38A4 emerged as a potential mediator of this interaction. SLC38A4 expression was associated with c-Myc, and its loss increased the sensitivity of PDAC cells to CTO-induced metabolic stress. ConclusionOur findings reveal a CAF-tumor metabolic crosstalk in which stromal-derived ASN supports PDAC cell adaptation to mitochondrial metabolic stress. Adaptive outcome of ISR signaling depends on the availability of key metabolic substrates such as ASN. When extracellular ASN supply is limited, the ATF4-dependent adaptive program collapses, converting ISR from a pro-survival response into a therapeutic vulnerability. SLC38A4 may function as a key mediator of this metabolic coupling and represents a potential target for enhancing the efficacy of OXPHOS inhibition in PDAC.

IntroductionThe insulin-like growth factor (IGF-1/IGF1R) pathway has been implicated in breast cancer aggressiveness; however, inhibition of this pathway has not been successful in clinical trials, indicating a lack of understanding about its role in TNBC metastasis. Recent studies have explored IGF1R involvement in integrin function and cancer cell adhesion dynamics. The goal of this study was to test the hypothesis that IGF1R itself regulates cancer cell adhesion. MethodsWe use MDA-MB-231 and Hs578T TNBC cell lines, siRNA-mediated knockdown, and adhesion assays to assess how IGF1R and integrin knockdowns impact cancer cell adhesion. Using xCELLigence E-plates, we quantify the effect of IGF-1 ligand stimulation versus IGF1R knockdown on functional cell adhesion. We also use HUVEC human endothelial cells to determine how IGF1R regulates adhesion to the endothelium. ResultsWe found that IGF-1 stimulation increased MDA-MB-231 TNBC adhesion, which was reversed by the IGF1R tyrosine kinase inhibitor BMS-754807 and the ligand-dependent receptor internalization inhibitor dansylcadaverine. Unexpectedly, IGF1R knockdown also potently stimulated cell adhesion. Concomitant {beta}1 integrin knockdown reversed the increased cell adhesion after both IGF-1 stimulation or IGF1R knockdown, indicating that the increased adhesion is {beta}1 integrin dependent. This was also seen via immunocytochemistry when cells were seeded on fibronectin. Finally, inhibiting IGF1R signaling also reduced MDA-MB-231 cell adhesion to HUVEC endothelial cells. DiscussionBoth IGF-1 stimulation and IGF1R knockdown in TNBC cells promote cell adhesion, which seems paradoxical. However, the commonality of both interventions is removal of IGF1R from the cell surface, since IGF-1 stimulation causes IGF1R internalization and intracellular trafficking. Blocking IGF1R signaling using a tyrosine kinase IGF1R inhibitor preserves IGF1R on the cell surface. Thus, we propose a model whereby surface-bound IGF1R inhibits {beta}1 integrin function and blocks cell adhesion. This model is supported further by our finding that treatment of MDA-MB-231 cells with dansylcadaverine, which inhibits ligand-mediated receptor internalization, blocked the effect of IGF-1 on adhesion. These findings may explain why selective IGF1R receptor antagonists, which downregulate IGF1R protein upon chronic administration, were unsuccessful in the clinical setting.

Here we report that a steroidal lactone withaferin A (WFA) can inhibit T-cell motility, which is crucial for adaptive immune responses as well as autoimmune reactions. Tandem mass spectrometry identified WFA-interactome in human T-cells that were stimulated to migrate via cross-linking of the lymphocyte function-associated antigen-1 (LFA-1) integrin with the ligand intercellular adhesion receptor 1 (ICAM-1). Data revealed significant enrichment of the zeta-chain-associated protein kinase 70 (ZAP70) and cytoskeletal actin protein interaction networks. Phospho-peptide mapping and kinome analysis substantiated kinase signaling downstream of ZAP70 and cytoskeletal kinase pathways as key WFA targets, which was further confirmed by in silico analysis and molecular assays. The WFA-ZAP70 complex was disrupted by a redox agent dithiothreitol, suggesting a covalent binding interface. Moreover, WFA ablated the phosphorylation of the myosin light chain, further constraining T-cell motility. These studies identify a mechanism whereby WFA can impact T-cell motility. WFA can therefore be exploited to pharmacologically controlling host immune responses and preventing autoimmune-mediated pathologies.

OBJETIVEFood intake, energy expenditure, and metabolic homeostasis depend on hypothalamic neurons responses to peripheral signals, such as leptin, involving the primary cilium (PC). The PC is crucial for signal transduction and is dynamically regulated by assembly/disassembly or reabsorption of its microtubules-based axoneme. Absence or reduction in the length of PC is associated with obesity and type-2 diabetes (T2D). In other cellular systems, PC reabsorption is primarily regulated by calcium-mediated activation of the Aurora kinase A (AurkA)/histone deacetylase C6 (HDAC6) axis, which promotes axonemal disassembly. Here, we explore the role of Galectin-8 (Gal-8), a glycan-binding protein, in regulating PC structure and signaling related to metabolic parameters in hypothalamic neurons. METHODSGal-8 effects were assessed in hypothalamic Clu-177 cells by analyzing the PC presence and length by immunofluorescence, PC dynamics, and intracellular calcium changes by in vivo cell imaging, activation of FAK, Src, AurkA, HDAC6 and STAT3 by immunoblot, and Gal-8 interactions with {beta}1-integrins by pull-down assays. Gal-8-KO mice were used to evaluate PC length in hypothalamic neurons, metabolic phenotype, and responses to Gal-8 intranasal administration. RESULTSIn Clu-177 cells, Gal-8 induced PC reabsorption and reduced responsiveness to leptin signaling towards STAT3 activation. PC reabsorption involves glycan-mediated Gal-8 interactions with a5b1 and a3b1 integrins, activation of FAK and Src leading to calcium influx through L-type calcium channels (LTCC), and subsequent AurkA/HDAC6 axis activation. Gal-8-KO mice showed longer PC in hypothalamic neurons, higher STAT3 activation, decreased body weight and food intake, improved glucose tolerance, higher locomotor activity, and a glycolytic respiratory exchange rate (RER). Daily intranasal Gal-8 administration for 4 days restored hypothalamic PC length and STAT3 signaling, as well as RER in Gal-8-KO mice to the level of WT mice. CONCLUSIONSEndogenous Gal-8 is required to maintain PC structure and leptin signaling in hypothalamic neurons, impacting body weight, energy balance, and glucose homeostasis. The mechanism involves calcium influx via LTCC downstream of b1-integrin/FAK/Src signaling and subsequent AurkA/HDAC6 axis activation. Both Gal-8 and the AurkA/HDAC6 axis may offer new therapeutic opportunities for treating metabolic diseases characterized by ciliogenesis impairment, including obesity and type-2 diabetes.