Cell Communication and Signaling

Cancer metabolism rewiring is one of the hallmarks of cancer, enabling cancer cell survival in a nutrient deprived microenvironment. Key to this is nutrient scavenging where cancer cells rely on extracellular proteins, including extracellular matrix (ECM) components, to sustain their proliferation. ECM uptake is mediated by 2{beta}1 integrin, however it is not clear how this process is controlled by nutrient availability. Here we demonstrated that amino acid starvation promoted ECM internalisation, by inducing the expression of 2 integrin. Mechanistically, starvation-driven RAS/MAPK pathway activation in cells harbouring oncogenic RAS mutations and mTOR inhibition increased 2 integrin, while the GCN2-depedent integrated stress response was not required. Functionally, elevated 2 integrin levels promoted cell adhesion and migration in nutrient starved cells. Finally, 2 integrin was found upregulated in pancreatic tumours and correlated with poor prognosis in pancreatic adenocarcinoma patients. Together, these data indicate that the nutrient- starved pancreatic cancer microenvironment synergises with KRAS mutation to drive pancreatic cancer aggressiveness.

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.

Activated receptor tyrosine kinases (RTKs), such as epidermal growth factor receptor (EGFR) and anaplastic lymphoma kinase (ALK), trigger intracellular signaling while undergoing receptor endocytosis. We recently identified a noncanonical mechanism in which RTK-containing endocytic vesicles deliver extracellular glucose to hexokinases associated with the outer mitochondrial membrane, thereby promoting cellular glucose uptake. Whether this mechanism contributes to cancer metabolism, however, remains unknown. Here, using neuroblastoma cell lines with distinct ALK alterations, we investigated the role of ALK endocytosis in glucose uptake. ALK-amplified, but not ALK-mutant, neuroblastoma cells exhibited a [~]40- 50% reduction in glucose uptake following inhibition of ALK or receptor endocytosis. This process was independent of the ERK MAPK and PI3K-AKT pathways but required dynamin-dependent endocytosis, cytoplasmic dynein, and GLUT1. Overexpressed ALK constitutively co-endocytosed with GLUT1 into vesicles transported to mitochondria. Inhibition of ALK activity or endocytosis suppressed glucose uptake without producing an additive effect, indicating that both function within the same pathway. Furthermore, disruption of the endocytic machinery selectively impaired the growth of ALK-amplified neuroblastoma cells. These findings identify ALK endocytosis as a major regulator of glucose uptake in ALK-amplified neuroblastoma and suggest that RTK endocytosis represents a previously unrecognized metabolic vulnerability that may be therapeutically exploitable in RTK-driven cancers.

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.

G protein-coupled receptor (GPCR) signaling represents a critical interface between oral bacteria and host cellular regulation in oral squamous cell carcinoma (OSCC). Here, we integrated systems biology, exploratory machine learning, and structure-based drug design to characterize potential associations between bacteria-related signaling and autophagy and to identify candidate therapeutic targets. Taste-associated signaling genes belonging to the GPCR superfamily were curated from KEGG, while OSCC- and autophagy-associated proteins were obtained from STRING, Reactome, UniProt, KEGG, and HMDB. Ten bacteria-associated host-interaction datasets were integrated using NetworkAnalyst to construct protein- protein interaction networks, and key hub nodes were identified through degree and betweenness centrality. Feature matrices derived from network topology were analyzed using exploratory dimensionality reduction (PCA), hierarchical clustering, and supervised models (SVM and Gradient Boosting) to assess whether network-derived features showed separability according to literature-informed bacterial reference categories; a Dysbiosis Index was additionally calculated. Results suggested that bacterial sensing through taste-associated GPCR signaling may converge on a MAPK1-centered axis linking calcium signaling, autophagy, and oncogenic pathways. Pathobiont-associated networks showed greater representation of inflammatory and terminal-autophagy-related signaling through MAPK1-STAT3, whereas commensal-associated networks were more closely aligned with cytoprotective autophagy through balanced MAPK1-TP53/PTEN networks. Exploratory machine learning analyses highlighted MDM2 and AKT3 as high-contribution, network-associated candidate features linked to group separability within the current dataset. A dual-target MTDL (SG101) was designed to target downstream nodes (MDM2 and JAK2), showing favorable predicted docking interactions and computationally predicted ADMET properties. In conclusion, bacteria-associated host taste signaling may be linked to differing autophagy-related network states in OSCC, and targeting downstream regulatory hubs with multi-target ligands represents a hypothesis-generating strategy that warrants experimental validation for pathway-oriented therapy.

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.

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.

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

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

Head and neck squamous cell carcinoma (HNSCC) remains clinically challenging, with limited molecularly targeted options and a strong dependence on the tumor microenvironment. Cancer-associated fibroblasts (CAFs) are major stromal regulators that shape tumor progression, extracellular matrix remodeling, invasion, and therapeutic response. However, how CAF heterogeneity and plasticity translate into distinct tumor-promoting functions and targetable vulnerabilities remains insufficiently defined. Here, we integrated patient-matched primary CAFs and normal fibroblasts with 3D functional assays, tumor-stroma co-culture models, quantitative extracellular matrix analysis, whole-proteome profiling, and pharmacological perturbation. Primary fibroblast populations displayed marked interpatient heterogeneity and context-dependent plasticity in invasion, contractility, and responsiveness to tumor-derived signals, whereas enhanced fibronectin-rich matrix deposition and disorganization emerged as a conserved CAF-associated feature. Both normal fibroblasts and CAFs promoted HNSCC cell invasion in a population-dependent manner, whereas CAFs consistently induced less compact and more dispersed tumor nest architectures. Integrative functional analyses identified distinct CAF phenotypes characterized by either invasive and matrix-remodeling activity or high responsiveness to tumor-derived cues. Proteomic profiling revealed recurrent enrichment of adhesion, cytoskeletal, and extracellular matrix programs and guided the selection of pharmacological inhibitors aimed at modulating specific CAF-mediated pro-tumoral functions. Pharmacological targeting selectively altered these functions: CHI3L1 inhibition disrupted fibronectin matrix deposition, broad phosphodiesterase inhibition increased matrix alignment, and FZD7 inhibition consistently blocked tumor-induced CAF invasion across all tested populations. These findings define functionally distinct and pharmacologically targetable CAF programs in HNSCC and support stromal-directed interventions as a rational component of future combination treatment 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.

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.

The eIF4F translation initiation complex controls ERK MAPK signaling in malignant melanomas with BRAF and NRAS mutations. It also contributes to the development of melanoma resistance to therapies targeting BRAF and MEK kinases. Here, we uncovered a critical role for eIF4F in regulating the main cellular metabolic sensor, AMP-activated protein kinase (AMPK). In melanoma cells with the most common BRAF V600E mutation, ERK and AMPK pathway activities were reported as mutually exclusive. This is because BRAF-driven ERK activity negatively impacts LKB1-mediated canonical AMPK activation. However, we observed that eIF4F inhibition can stimulate AMPK activity in melanoma cells, both in vitro and in vivo, despite concomitant ERK hyperactivation. Notably, the protein levels of LKB1 and its co-factor MO25 were sensitive to eIF4F inhibition, indicating a non-canonical LKB1-independent mechanism of AMPK activation. In a proteomic screen, we aimed to identify eIF4F roles in melanoma cell physiology beyond the MAPK pathway. We found that the eIF4F function is essential for maintaining cellular levels of key cell cycle and metabolic regulators, including CDK1, CDK2, TYMS, and UHRF1. Importantly, we also identified the protein phosphatase PP2A as a new eIF4F target. Our subsequent analyses showed that inhibition or siRNA-mediated knockdown of PP2A increases AMPK activity in melanoma cells, independent of LKB1. This data shows that PP2A plays a significant role in regulating AMPK activity in melanoma. Thus, eIF4F inhibition not only impairs canonical AMPK activators but also downregulates PP2A, which negatively regulates AMPK dynamics. Collectively, our data highlight a dual role of eIF4F in the control of AMPK in BRAF-mutant melanoma cells. It maintains the canonical AMPK signaling pathway while simultaneously limiting the extent of AMPK activation via the eIF4F-PP2A-AMPK axis. Pharmacological inhibition of this axis can overcome the negative control of AMPK signaling by the ERK pathway. This suggests new therapeutic opportunities to disrupt melanoma growth.

Because ErbB receptors play distinct roles in regulating diverse cellular functions, the mechanisms governing ErbB receptor activation are likely to be more diverse than previously recognized. Phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] positively regulates ErbB1 kinase activity, but the role of PI(4,5)P2 in regulating other ErbB family members in living cells remains poorly understood. We show that disruption of PI(4,5)P2 binding enhances ErbB4 oligomerization and kinase activity while reducing both processes in ErbB1. Analysis of chimeric receptors identified the juxtamembrane (JM) regions of ErbB1 and ErbB4 as key determinants of their distinct responses to PI(4,5)P2 during receptor oligomerization and kinase activation. Furthermore, the JM-kinase module of ErbB1 is more active in the presence of PI(4,5)P2, whereas that of ErbB4 is activated by the disruption of PI(4,5)P2 binding. In contrast, the JM-kinase module of ErbB2 exhibits weak dependence on PI(4,5)P2. ErbB2 preferentially promotes ErbB4 oligomerization over ErbB1 oligomerization, thereby enhancing ErbB4 activation. Collectively, these findings identify plasma membrane PI(4,5)P2 availability and ErbB2 abundance as two factors that jointly govern ErbB receptor oligomerization and activation.

Psychoneuroimmunology suggests that positive physiological states, including laughter, could affect anti-tumor immunity, but the underlying mechanisms remain unclear. Here, we investigated whether {beta}-endorphin (BE), an endogenous opioid peptide associated with positive physiological stimuli, modulates Natural Killer (NK) cell cytotoxicity and the anti-tumor activity of NK-derived extracellular vesicles (EVs). Using NK-92 cells, we assessed cytotoxicity against JIMT1 breast cancer cells, CD107a mobilization, cytotoxic activity of conditioned medium (CM), and EV yield, cargo, and function. BE enhanced NK-92-mediated killing of JIMT1 cells without increasing CD107a mobilization, suggesting that improved cytotoxicity was not driven by classical degranulation. Consistently, CM from BE-treated NK cells retained contact-independent cytotoxicity. NK-EVs were enriched in granzyme B and perforin following BE treatment exhibiting enhanced cytotoxicity against JIMT1 and BW tumor cells. BE also increased the cytotoxic activity of primary human NK cells, and BE-conditioned NK-EVs primed naive NK-92 cells for enhanced tumor killing. These findings indicate that BE enhances NK anti-tumor immunity by remodeling the cytotoxic secretome and generating EVs that act as both direct cytotoxic effectors and mediators of NK cell priming. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=119 HEIGHT=200 SRC="FIGDIR/small/741417v1_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@16d4026org.highwire.dtl.DTLVardef@18e91fcorg.highwire.dtl.DTLVardef@1124baeorg.highwire.dtl.DTLVardef@268d0e_HPS_FORMAT_FIGEXP M_FIG C_FIG Proposed neuroendocrine-immune model linking positive physiological stimuli, NK cell-derived extracellular vesicles (EVs), and anti-tumor activity. Laughter is depicted as a conceptual upstream trigger of hypothalamic-pituitary signaling leading to {beta}-endorphin (BE) release. BE conditioning enhanced NK-92 cytotoxicity and the anti-tumor activity of NK-derived EVs, consistent with granzyme B and perforin enrichment and supporting EV-mediated contact-independent cancer cell killing.

Brain metastases (BrM) occur in 10-15% of patients with estrogen receptor (ER)-positive breast cancer, and remain a significant clinical challenge. While current therapeutic paradigms, including surgical resection and systemic therapy, have efficacy in the management of BrM from ER+BC, median overall survival following the diagnosis of BrM is approximately 18 months. The limited efficacy of current therapies, along with a relative paucity of therapeutic options, highlight the urgent clinical need to identify druggable targets for BrM from ER+BC. We previously identified recurrent overexpression of the receptor tyrosine kinase RET in breast cancer BrM relative to patient-matched primary tumors. The principal ligand for RET, Glial Cell-Derived Neurotrophic Factor (GDNF), is predominately expressed in the brain, where it functions in the maintenance of neurons and multiple glial cell types. Here, we show that increased RET expression and activation correlates with brain metastasis in breast cancer patients. Further, we confirm the role of RET signaling in the promotion of metastatic properties in vitro, including tumor spheroid formation, migration, invasion, and transformation. Using RET-overexpressing and RET-deficient models in four ER+BC cell lines, we demonstrate that RET overexpression enhances activation of RET downstream targets, such as ERK and AKT, and promotes metastatic phenotypes in in vitro, ex vivo and in vivo assays. Importantly, we demonstrate that the phenotypic effects of GDNF-mediated RET signaling can be abrogated by administration of Pralsetinib, a highly potent RET-selective kinase inhibitor. Using an ex vivo mouse brain slice co-culture model, we further demonstrate that RET overexpression in breast cancer cell lines potentiates their colonization and invasion into the brain tissues, which are reduced in RET-knockdown cell lines. RET overexpression facilitates brain colonization of breast cancer cells in vivo and is associated with reduced survival. Using proteomic analyses, we identify novel GDNF-mediated signaling pathways in breast cancer cell line models, including PLC{gamma}, P70S6K, STAT3 and CREB. RET-mediated activation of this range of downstream targets is sensitive to inhibition by Pralsetinib. In sum, this study identifies and characterizes RET as a targetable driver of breast cancer brain metastasis, and further describes the action of Pralsetinib in ER-positive breast cancer cell lines, providing insights to improve current targeted therapies for patients with breast cancer brain metastasis. Bullet pointsO_LIRET expression and activation are increased in breast cancer brain metastases. C_LIO_LIPralsetinib potently inhibits RET activation in RET overexpressing and in non-overexpressing breast cancer cell line models. C_LIO_LIRET overexpression and activation confers pro-metastatic properties including cell proliferation, colonization, migration and invasion in vitro and in ex vivo brain co-culture and enhances brain colonization of breast cancer cells in vivo. C_LIO_LIGDNF increases activation of ERK1/2, AKT, PCL{gamma}, P70S6K, STAT3, and CREB in RET overexpressing MCF-7 cells. C_LIO_LICREB or P70S6K knockdown attenuates GDNF stimulated activation of RET downstream targets and cell migration. C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=193 HEIGHT=200 SRC="FIGDIR/small/647085v1_ufig1.gif" ALT="Figure 1"> View larger version (57K): org.highwire.dtl.DTLVardef@1628735org.highwire.dtl.DTLVardef@19ad565org.highwire.dtl.DTLVardef@2fdcc2org.highwire.dtl.DTLVardef@468233_HPS_FORMAT_FIGEXP M_FIG C_FIG