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.

Bitter taste receptors (T2Rs), are a subset of G protein-coupled receptors (GPCRs) that play a key role in responding to microbial presence at epithelial surfaces. In epithelia, the activities of ion channels and transporters, and of T2Rs, mutually affect each other. The normal function of one such anion channel, cystic fibrosis transmembrane conductance regulator (CFTR), is essential for the maintenance of healthy epithelia, not just in the respiratory but in the digestive and reproductive system as well. Based on evidence that T2R14 activity is affected upon mutations in CFTR, we explored the possibility that T2R14 and CFTR directly interact in cell membranes. The biophysical interaction between these proteins was mapped to specific regions of the CFTR, and was dependent on agonist stimulation of T2R14. Further, T2R14 was found to couple to Gq, in addition to the canonical Gi, in response to bacterial and fungal quorum sensing molecules. Whether the interaction with CFTR affects T2R14 driven responses to microbial signals is under investigation.

Cancer is a leading cause of death globally, with the majority of cancer-related deaths resulting from cancer metastasis -the process by which cancer cells disseminate to distant sites. To metastasize, cancer cells acquire traits in support of diverse cellular processes that enable dissemination, survival, and colonization. Tumor cell dissemination requires invasion at local and distant sites and this process can be influenced by intrinsic and extrinsic factors. Here, we investigate the role of the neurotransmitter gamma-aminobutyric acid (GABA) in triple-negative breast cancer (TNBC) invasion and metastasis. TNBC cells increased invasion in response to GABA and this was found to be mediated through the GABAA receptor family. TNBC cell lines were found to be responsive to exogenous GABA and also produced endogenous GABA. Pharmacological inhibition of GABAA receptors reduced TNBC invasion and cancer cell dissemination and resulted in inhibition of GSK3 activity. TNBC cell lines were found to express the GABRE subunit and loss of GABRE impaired GABA-mediated invasion and tumor cell dissemination. These findings support a role for GABA signaling through GABAA receptors in mediating TNBC progression.

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 composition of the plasma membrane (PM)-associated proteome of tumor cells determines cell-cell and cell-matrix interactions and the response to environmental cues. Whether the PM-associated proteome impacts the phenotype of Medulloblastoma (MB) tumor cells and how it adapts in response to growth factor cues is poorly understood. Using a spatial proteomics approach, we observed that hepatocyte growth factor (HGF)-induced activation of the receptor tyrosine kinase c-MET in MB cells changes the abundance of transmembrane and membrane-associated proteins. The depletion of MAP4K4, a pro-migratory effector kinase downstream of c-MET, leads to a specific decrease of the adhesion and immunomodulatory receptor CD155 and of components of the fast-endophilin-mediated endocytosis (FEME) machinery in the PM-associated proteome of HGF-activated MB cells. The decreased surface expression of CD155 or of the FEME effector Endophilin A1 reduces growth and invasiveness of MB tumor cells in the tissue context. These data thus describe a novel function of MAP4K4 in the control of the PM-associated proteome of tumor cells and identified two downstream effector mechanisms controlling proliferation and invasiveness of MB cells. Graphical abstractc-MET activation upon HGF stimulation induces c-MET internalization and induces downstream MAP4K4 activity. (1) MAP4K4 is required downstream of activated c-MET for the maintenance of surface presentation of CD155 in activated cells. CD155 expression is required for MB cell migration, invasion and proliferation in the tissue context. (2) MAP4K4 is required downstream of activated c-MET to maintain membrane depolarization, possibly by regulating the surface localization of several ion channels and transporters. (3) MAP4K4 is required downstream of activated c-MET cause PM-proximal localization of FEME effector CIP4, FBP17 and CIN85. The FEME effector endophilin A is necessary for MB cell migration, invasion and dissemination.

Glucocorticoids are steroid hormones well-known for their potent anti-inflammatory effects. However, their immunomodulatory properties are multifaceted. Increasing evidence suggests that glucocorticoid signaling promotes effective immunity and that disruption of glucocorticoid signaling impairs immune function. In this study, we conditionally deleted the glucocorticoid receptor (GR) in the myeloid lineage using the LysM-Cre driver (myGRKO). We examined the impact on macrophage activation and gastric immune responses to Helicobacter pylori, the best-known risk factor of gastric cancer. Our results indicate that compared to WT, GRKO macrophages exhibited higher expression of proinflammatory genes in steroid-free conditions. However, when challenged in vivo, GRKO macrophages exhibited aberrant chromatin landscapes and impaired proinflammatory gene expression profiles. Moreover, gastric colonization with Helicobacter revealed impaired gastric immune responses and reduced T cell recruitment in myGRKO mice. As a result, myGRKO mice were protected from atrophic gastritis and pyloric metaplasia development. These results demonstrate a dual role for glucocorticoid signaling in preparing macrophages to respond to bacterial infection but limiting their pathogenic activation. In addition, our results support that macrophages are critical for gastric anti- Helicobacter immunity. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=159 SRC="FIGDIR/small/575574v1_ufig1.gif" ALT="Figure 1"> View larger version (34K): org.highwire.dtl.DTLVardef@7d5281org.highwire.dtl.DTLVardef@1b39ca2org.highwire.dtl.DTLVardef@f081caorg.highwire.dtl.DTLVardef@1d9215d_HPS_FORMAT_FIGEXP M_FIG C_FIG

Sigma 1 Receptor (S1R) is a therapeutic target for a wide spectrum of pathological conditions ranging from neurodegenerative diseases to cancer and COVID-19. S1R is ubiquitously expressed throughout the visceral organs, nervous, immune and cardiovascular systems. It is proposed to function as a ligand-dependent molecular chaperone that modulates multiple intracellular signaling pathways. The purpose of this study was to define the S1R interactome under native conditions and upon binding to well-characterized ligands. This was accomplished by fusing the biotin ligase, Apex2, to the C terminus of S1R. Cells stably expressing S1R-Apex or a GFP-Apex control were used to map specific protein interactions. Biotinylated proteins were labeled under native conditions and in a ligand dependent manner, then purified and identified using quantitative mass spectrometry. Under native conditions, S1R biotinylates over 200 novel proteins, many of which localize within the endomembrane system (ER, Golgi, secretory vesicles) and function within the secretory pathway. Under conditions of cellular exposure to either S1R agonist or antagonist, results show enrichment of proteins integral to secretion, extracellular matrix formation, and cholesterol biosynthesis. Notably, Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9) displays increased binding to S1R under conditions of treatment with Haloperidol, a well-known S1R antagonist; whereas Low density lipoprotein receptor (LDLR) binds more efficiently to S1R upon treatment with (+)-Pentazocine ((+)-PTZ), a classical S1R agonist. Our results are consistent with the postulated role of S1R as an intracellular chaperone and further suggest important and novel functionalities related to cholesterol metabolism and biosynthesis.

Stress granules (SGs) are dynamic cytoplasmic structures assembled in response to various stress stimuli that enhance cell survival under adverse environmental conditions. Here we show that SGs contribute to breast cancer progression by enhancing the survival of cells subjected to anoikis stress. SG assembly is triggered by inhibition of Focal Adhesion Kinase (FAK) or loss of adhesion signals. Combined proteomic analysis and functional studies reveal that SG formation enhances cancer cell proliferation, resistance to metabolic stress, anoikis resistance, and migration. Importantly, inhibiting SG formation promotes the sensitivity of cancer cells to FAK inhibitors being developed as cancer therapeutics. Furthermore, we identify the Rho-ROCK- PERK-eIF2 axis as a critical signaling pathway activated by loss of adhesion signals and inhibition of the FAK-mTOR-eIF4F complex in breast cancer cells. By triggering SG assembly and AKT activation in response to anoikis stress, PERK functions as an oncoprotein in breast cancer cells. Overall, our study highlights the significance of SG formation in breast cancer progression and suggests that therapeutic inhibition of SG assembly may reverse anoikis resistance in treatment-resistant cancers such as triple-negative breast cancer (TNBC). HighlightsO_LIEither anoikis stress or loss of adhesion induce stress granule (SG) formation C_LIO_LIThe Rho-ROCK-PERK-eIF2 axis is a crucial signaling pathway triggered by the absence of adhesion signals, leading to the promotion of SG formation along with the inhibition of the FAK- AKT/mTOR-eIF4F complex under anoikis stress. C_LIO_LIPERK functions as an oncogene in breast cancer cells, initiating SG formation and activating AKT under anoikis stress. C_LIO_LIInhibiting SG formation significantly enhances the sensitivity to Focal Adhesion Kinase (FAK) inhibitors, suggesting a potential for combined therapy to improve cancer treatment efficacy. C_LI

Breast cancer is the leading cause of cancer mortality in women worldwide and commonly metastasizes to the bone marrow, drastically reducing patient prognosis and survival. In the bone marrow niche, metastatic cells can enter into a dormant state, thereby evading immune surveillance and treatment, and can be reactivated to enter a proliferative state due to poorly understood cues. Mesenchymal stromal cells (MSCs) maintain cells in this niche partly by secreting extracellular matrix and paracrine factors and by responding to regenerative cues. MSCs also produce extracellular vesicles (EVs) that carry a range of cargoes, some of which are implicated in cell signalling. Here, we investigate if the changing metabolic state of MSCs alters the cargoes they package into EVs, and how these changing cargoes act on dormant breast cancer cells (BCCs) using an in vitro BCC spheroid model and a scratch assay to create a regenerative demand on MSCs. Our findings show that EVs produced by standard MSCs contain glycolytic metabolites that maintain BCC dormancy. When MSCs are placed under a regenerative demand and increase their respiration to fuel differentiation, these metabolites disappear from the EV cargo and their absence encourages rapid growth in the BCC spheroids. This work implicates EVs in cancer cell dormancy in the bone marrow niche and indicates that pressures on the niche, such as regeneration, can be a driver of BCC activation.

Over 10 million women worldwide have breast implants for breast cancer/prophylactic reconstruction or cosmetic augmentation. In recent years, a number of patients have described a constellation of symptoms that are believed to be related to their breast implants. This constellation of symptoms has been named Breast Implant Illness (BII). The symptoms described include chronic fatigue, joint pain, muscle pain and a host of other manifestations often associated with autoimmune illnesses. In this work, we report that bacterial biofilm is associated with BII. We postulate that the pathogenesis of BII is mediated via a host-pathogen interaction whereby the biofilm bacteria Staphylococcus epidermidis interacts with breast lipids to form the oxylipin 10-HOME. The oxylipin 10-HOME was found to activate CD4+ T cells to Th1 subtype. An increased abundance of CD4+Th1 was observed in the breast tissue of BII subjects. The identification of a mechanism of immune activation associated with BII via a biofilm enabled pathway provides insight into the pathogenesis for implant-associated autoimmune symptoms.

Background and ObjectiveFOLR3 serves as an important member of the folate metabolic pathway and plays a crucial role in various malignant tumors. However, the expression pattern, diagnostic value, and regulatory mechanism of FOLR3 in endometrial cancer (EC) remain unclear. This study aimed to explore the expression characteristics, clinical significance, and related molecular regulatory networks of FOLR3 in EC through bioinformatics analysis. MethodsTCGA datasets served as training sets, while GSE17025 from GEO served as validation sets. FOLR3 differential expression was analyzed using Wilcoxon rank-sum test, diagnostic efficacy evaluated by ROC curves, and prognostic value assessed via Kaplan-Meier survival analysis. Candidate genes were identified through WGCNA, univariate Cox regression, and differential expression analysis. Key genes were screened using machine learning algorithms (RF, LASSO, SVM-RFE) and PPI network analysis. ceRNA regulatory networks were constructed, and immune infiltration was analyzed using CIBERSORT. ResultsFOLR3 was significantly overexpressed in EC (P < 0.01) with diagnostic AUC values > 0.6 in both datasets. High FOLR3 expression indicated poor prognosis (HR=2.5, P < 0.05). Among 5,539 differentially expressed genes, 3 key genes (AURKA, POLQ, CDKN2A) were identified via multi-algorithm screening. Enrichment analysis showed involvement in cell division, p53 signaling, and cellular senescence. The ceRNA network comprised 72 nodes and 169 relationships, with KCNQ1OT1 and XIST as key lncRNA regulators. FOLR3 positively correlated with memory B cells and M0 macrophages, negatively with naive B cells and resting mast cells, with significant immune score differences (P < 0.05). ConclusionFOLR3 was significantly upregulated in EC, serving as an effective diagnostic biomarker and independent prognostic predictor. FOLR3 participated in tumorigenesis via complex ceRNA networks and regulated the tumor immune microenvironment. This study provides novel molecular targets and theoretical foundation for precision diagnosis and therapy of EC.

The mammalian/mechanistic Target of Rapamycin Complex 1 (mTORC1) orchestrates cell growth and metabolism in response to diverse external cues. mTORC1 has a myriad of phosphorylation substrates, each playing important physiological roles. Emerging evidence suggests that mTORC1 can respond to upstream signals in a nuanced manner, enabling differential regulation of individual substrates and downstream biological processes. However, the nature of signals that determine the signaling selectivity of mTORC1 remains incompletely understood. Here, we studied mTORC1 regulation by G protein-coupled receptors (GPCRs). We found that phosphorylation of the Transcription Factor EB (TFEB), a non-canonical mTORC1 substrate that controls lysosome biogenesis, responds to GPCRs differently, compared to canonical mTORC1 substrates controlling protein synthesis such as S6K1 and 4EBP1. In particular, the muscarinic acetylcholine receptor M5 (M5R) promoted phosphorylation of S6K1 and 4EBP1, while triggering TFEB dephosphorylation. Consequently, M5R stimulated protein synthesis without inhibiting lysosome biogenesis. The regulations of an anabolic process and a catabolic process, albeit both governed by mTORC1, are thus decoupled downstream of M5R. This study highlights the importance of reassessing the effects of GPCRs on mTORC1 by concurrently monitoring individual substrates, a critical consideration to be made when evaluating GPCR ligands as therapeutic agents targeting the mTORC1 pathway.

In clinical practice, the co-existence of endometriosis and gastrointestinal symptoms is often observed; however, the factors driving this link remain largely unknown. Here, using large-scale multifaceted data including observational, genetic, and pharmaceutical datasets, we report a positive phenotypic and genetic association of endometriosis with peptic ulcer disease (PUD), gastro-oesophageal reflux disease (GORD), a combined GORD/PUD Medicated (GPM) phenotype and irritable bowel syndrome (IBS), but not with inflammatory bowel disease (IBD). Mendelian randomization analysis identified a causal effect of the GPM phenotype on endometriosis and a bidirectional causal association between endometriosis and IBS. Cross-trait meta-analysis and colocalization along with comprehensive functional annotation confirmed two shared genetic loci (FN1, TACSTD2) for endometriosis with IBS and twelve loci (ETAA1, HOXC4, RERG, SEMA3F, SPAG16, HIST1H2BC, RAB5B, CCKBR and PDE4B) with GORD and PUD. Shared genetic loci may contribute to risk of both endometriosis and digestive disorders through the involvement of DNA damage, estrogen regulated cell-proliferation and inflammation, and barrier dysfunction. Analyses of medication usage identified a higher use of drugs for IBS, GORD and PUD in women diagnosed with endometriosis as well as a higher use of hormone therapies in women diagnosed with IBS, GORD and PUD but not for IBD, which strongly supports the co-occurrence of these conditions and highlights the potential for drug repositioning and caution around drug contraindications in clinical practice. Taken together, the combined evidence robustly suggests a shared disease aetiology and provides important clinical implications for diagnostic and treatment decisions for endometriosis and digestive disorders. WHAT IS ALREADY KNOWN ON THIS TOPIC?O_LIBoth endometriosis and gastrointestinal disorders affect a large proportion of people worldwide and the co-existence of endometriosis and gastrointestinal symptoms (eg, abnormal pain, bloating, constipation) is often observed in clinical practice. C_LIO_LIThe association of these two diseases was supported but also limited to previous observational evidence which highlights a three-fold increase in the prevalence of irritable bowel syndrome (IBS) in women with endometriosis. C_LIO_LIObservational study is easily subject to measurement error, confounding and reverse causation. Therefore, it is important to assess the association using multidimensional datasets and more accurate approaches, such as the use of genetic data in a mendelian randomisation framework as well as the analysis from a perspective of medication usage. C_LI WHAT THIS STUDY ADDSO_LIGenetic risk factors for endometriosis and gastrointestinal disorders, two leading causes of discomfort and chronic pelvic pain, are correlated. C_LIO_LIMendelian randomisation analyses supported a causal relationship between genetic predisposition to gastrointestinal disorders (gastro-oesophageal reflux disease (GORD) and peptic ulcer diseases (PUD)) and endometriosis risk, and evidence for a bidirectional causal relationship between endometriosis and IBS, which might explain in part the co-occurrence of these diseases. C_LIO_LIThe identification of shared risk loci highlighted biological pathways that may contribute to the pathogenesis of both diseases, including estrogen regulation and inflammation, as well as potential therapeutic drug targets such as CCKBR and PDE4B. C_LIO_LIThe higher use of drugs for IBS, GORD and PUD in women diagnosed with endometriosis as well as the higher use of hormone therapies in women diagnosed with IBS, GORD and PUD, support the co-occurrence of these conditions and shared disease aetiology but also highlights the potential for drug repositioning and caution around drug contraindications. C_LI Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=132 SRC="FIGDIR/small/22281201v1_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@1a85d4forg.highwire.dtl.DTLVardef@8623baorg.highwire.dtl.DTLVardef@939f2corg.highwire.dtl.DTLVardef@1b70f55_HPS_FORMAT_FIGEXP M_FIG C_FIG

AbstractRegulation of translation initiation is essential for maintenance of protein homeostasis and typically involves the phosphorylation of translation initiation factor eIF2 in eukaryotes. In response to stressors, cells employ eIF2-dependent signaling to control translation initiation, which regulates multiple biological and physiological processes. However, the precise regulatory mechanism remains unclear. In this study, we focus on the role of TRIM21 in the regulation of protein translation mediated by protein kinase R (PKR), one of the classic kinases that phosphorylates eIF2. TRIM21 deficiency enhances the activation of PKR under different types of stress. TRIM21 interacts with PKR, and the E3 ligase activity of TRIM21 is crucial for stress-triggered PKR inactivation. TRIM21 interacts with the PKR phosphatase PP1 and promotes K6-linked polyubiquitination of PP1 under stresses. Ubiquitination of PP1 augments its interaction with PKR, causing PKR inactivation and subsequent dephosphorylation of eIF2, initiating protein synthesis. Moreover, TRIM21 constitutively restricts viral infection by reversing PKR-mediated inhibition of the protein synthesis of intrinsic antiviral genes. The TRIM21-PP1 axis acts as a newly discovered program that regulates PKR-associated protein synthesis and broadens our knowledge of antiviral genes. Moreover, TRIM21-mediated regulation of PKR activation provides evidence that TRIM21 may anticipate the interferon-dependent immunotherapy. Our study highlights the essential role of TRIM21 in regulating protein translation and may provide a novel target for the treatment of translation-associated diseases.

BackgroundPDLIM2, a PDZ-LIM domain-containing protein expressed highest in the lung and immune cells, serves as a unique tumor suppressor and immune modulator, mainly by turning off the activation of the master transcription factors NF-{kappa}B and STAT3. While its role in cancer is established, the involvement of PDLIM2 in viral infection remains unclear. ResultsHere, we analyzed public gene expression data of blood leukocytes, bronchoalveolar lavage cells, and lung tissues from uninfected healthy humans and those infected with the respiratory virus SARS-CoV-2 or influenza. We found that PDLIM2 expression was repressed by viral infection, and notably, this repression correlated with the severity of infectious diseases. Consistently, the expression level of PDLIM2 was negatively associated with NF-{kappa}B and STAT3 activity across a diverse range of cell types, such as macrophages, monocytes, neutrophils, T cells, alveolar type 1 and 2 epithelial cells, airway epithelial cells, and fibroblasts. Accordingly, cells with low PDLIM2 expression exhibited aberrant activation of signaling pathways essential for cellular functions and immune responses. ConclusionsThese findings highlight PDLIM2 repression as a common mechanism underlying human viral infectious diseases and suggest PDLIM2 as a potential biomarker and therapeutic target for disease prognosis, prevention, and treatment.

BackgroundEndometriosis is a chronic, inflammatory disease affecting 1 in 10 women of reproductive age worldwide. It is characterized by a range of debilitating symptoms which collectively impair patients quality of life. While stress is a well-documented factor known to exacerbate endometriosis symptoms, the impact of extreme and prolonged external stressors, such as ongoing war, on disease progression and patient well-being hasnt been thoroughly studied. On October 7, 2023, Israel faced a severe terror attack impacting Jewish and non-Jewish victims alike. Since then, Israel has been involved in an on-going conflict on multiple fronts. This study explores the compounded effects of ongoing war on women with endometriosis, focusing on symptom severity, physical and mental health, and illness perception. Materials & MethodsA cross-sectional survey study was conducted in Israel in August 2024, recruiting 841 women with confirmed diagnosis of endometriosis and/or adenomyosis through social media platforms and endometriosis clinics. The survey assessed the impact of the war on participants lives, physical, and mental health. It consisted of several validated measures as well as sociodemographic and clinical questions. Statistical analyses included descriptive statistics, univariate statistics for assessing temporal changes in symptoms severity and perceptions across three timepoints (pre-war, 2 months post-war, [~]1 year post-war), and correlation between war-related stressors, mental status, and health outcomes. Univariate and multivariable logistic regression models were employed to identify factors associated with symptom worsening, adjusting for sociodemographic characteristics, medical conditions, and war-related factors. Results82.8% of participants were directly affected by war-related stressors. Mental health deteriorated substantially, with anxiety rates increasing from 34.7% to 94.1% and depression from 39.6% to 89.3% (p<0.001). Physical health was also affected, with 77.4% reporting worsening endometriosis symptoms and 62.5% indicating overall medical decline. All specific symptoms, including pelvic pain, digestive and urinary symptoms, dyspareunia, fatigue and more, showed significant deterioration (p<0.001). Additionally, 38.9% and 14.4% of participants reported increased usage of pain and hormonal medications, respectively, indicating higher symptom-management needs. Multivariable analysis revealed significant associations between worsening in symptom severity to war-related stressors (aOR=1.24, 95%CI=1.07-1.45), war-related stress levels (aOR=1.83, 95%CI=1.50-2.22), depression and anxiety levels (aOR=2.00, 95%CI=1.52-2.63), and impaired healthcare accessibility (aOR=1.64, 95%CI=1.04-2.57). Negative illness personification was associated with worse outcomes (aOR=1.37, 95%CI=1.12-1.67), while positive illness personification showed protective effects (aOR=0.77, 95%CI=0.59-0.99). ConclusionsThis study demonstrates the profound impact of war and war-related stress on both the physical and mental health of endometriosis patients, highlighting the critical need for targeted healthcare interventions and psychological support during times of conflict.

The most devastating feature of cancer cells is their ability to metastasize to distant sites in the body. HER2+ and TN breast cancers frequently metastasize to the brain and stay potentially dormant for years until favorable conditions support their proliferation. The sheltered and delicate nature of the brain prevents, however, early disease detection and effective delivery of therapeutic drugs. Moreover, the challenges associated with the acquisition of brain biopsies add compounding difficulties to exploring the mechanistic aspects of tumor development, leading to slow progress in understanding the drivers of disease progression and response to therapy. To provide insights into the determinants of cancer cell behavior at the brain metastatic site, this study was aimed at exploring the early response of HER2+ breast cancer cells (SKBR3) to factors present in the brain perivascular niche. The neural microenvironment was simulated by using the secretome of a set of brain cells that come first in contact with the cancer cells upon crossing the blood brain barrier, i.e., endothelial cells (HBEC5i), astrocytes (NHA), and microglia (HMC3). Cytokine microarrays were used to investigate the secretome mediators of intercellular communication, and proteomic technologies for assessing the changes in the behavior of cancer cells upon exposure to the brain cell-secreted factors. The cytokines, growth factors and enzymes detected in the brain secretomes were supportive of inflammatory conditions, indicating a collective functional contribution to cell activation, defense, inflammatory responses, chemotaxis, adhesion, angiogenesis, and ECM organization. The SKBR3 cells, on the other hand, secreted numerous cancer-promoting growth factors that were either absent or present in lower abundance in the brain cell cultures, indicating that upon exposure the SKBR3 cells may have been deprived of favorable conditions for optimal growth. Altogether, the results suggest that the subjection of SKBR3 cells to the brain cell-secreted factors altered their growth potential and drove them toward a state of quiescence, with broader overall outcomes that affected cellular metabolism, adhesion and immune response processes. The findings of this study underscore the key role played by the neural niche in shaping the behavior of metastasized cancer cells, provide insights into the cellular cross-talk that may lead cancer cells into dormancy, and highlight novel opportunities for the development of metastatic breast cancer therapeutic strategies.

Toll-like receptor 9 (TLR9), primarily expressed in human dendritic and B cells, recognizes double-stranded DNA motifs from pathogens, initiating an inflammatory response. Recent studies have revealed TLR9s involvement beyond its conventional role in immune response, notably during the tumorigenesis of various cancers such as head and neck, cervical, and ovarian cancers. Here we show by immunohistochemistry analysis demonstrated significantly lower TLR9 levels in breast cancer tumors compared to normal breast tissue epithelium. Similarly, TLR9 downregulation was also observed in several transformed breast cancer cell lines versus untransformed breast epithelial cell lines. Furthermore, overexpression of TLR9 led to reduced proliferation potential of breast cancer cell associated with activation of senescence as was evident by upregulation of proinflammatory cytokine IL-6, chemokines IL-8 and CXCL1; and growth factor GM-CSF. These findings support TLR9s regulatory role in mitigating breast cancer and highlight its critical connection between the inflammatory response and tumor immunity.

The PI3K-AKT-MTOR signalling axis is pivotal in regulating cell survival, proliferation, and growth. TSC2 (tuberous sclerosis complex subunit 2) is a well-established negative regulator of this pathway, which primarily acts by suppressing the MTORC1 activity. While the cytoplasmic role of TSC2 is well characterized, emerging evidence suggests its additional nuclear functions. Previous work from our laboratory identified TSC2 as a transcriptional repressor of the EREG (Epiregulin) gene. Building on this foundation, the present study investigates the transcriptional role of TSC2 in miRNA (microRNA) gene regulation. A genome-wide miRNA microarray profiling of TSC2-depleted cells from an oral squamous cell carcinoma (OSCC) cell line, SCC131, identified 19 upregulated and 24 downregulated miRNAs. Of them, miR-514b-3p emerged as one of the most significantly upregulated miRNAs. TSC2 knockdown resulted in robust miR-514b-3p upregulation, whereas TSC2 overexpression suppressed its expression. Moreover, TSC2 negatively regulates MIR514B promoter activity in an NLS-dependent manner. The chromatin immunoprecipitation analysis showed direct binding between TSC2 and MIR514B promoter, establishing miR-514b-3p as a transcriptional target of TSC2. We further identified TSPAN9 (Tetraspanin 9) as a direct downstream target of miR-514b-3p. The dual-luciferase reporter assay and Western blot analysis confirmed direct interaction between miR-514b-3p and TSPAN9 3UTR. Furthermore, TSC2 positively regulates TSPAN9 levels by repressing miR-514b-3p, thereby establishing a novel TSC2-miR-514b-3p-TSPAN9 regulatory axis. Additionally, we uncovered crosstalk between TSC2-miR-514b-3p-TSPAN9 axis and the canonical PI3K-AKT-MTOR signalling, where miR-514b-3p positively, and TSPAN9 negatively regulates the PI3K-AKT-MTOR pathway. Interestingly, AKT functions as an upstream regulator of this axis by modulating TSC2 nuclear localization. Collectively, this study provides new insights into the non-canonical, nucleus-dependent transcriptional functions of TSC2, thus expanding its role beyond cytoplasmic signalling regulation and underscoring its significance in the cellular signalling networks.

Cyclin-Dependent Kinase 4 (CDK4) is a key regulator of cell cycle progression, driving the G0/G1-to-S phase transition through phosphorylation of Retinoblastoma 1 (RB1). Clinically, CDK4/6 inhibitors are under investigation in Triple Negative Breast Cancer (TNBC), a subtype characterized by invasiveness, aggressiveness and poor prognosis. While CDK4 is primarily targeted for its role in proliferation, emerging evidence suggests it may also regulate other cellular processes. In particular, the mechanisms by which CDK4 could influence cancer cell migration, remain largely unexplored, particularly in highly heterogenous cell line like MDA-MB-231. This study investigates whether CDK4 contributes to the regulation of TNBC cells migration and identifies the pathways involved in MDA-MB-231 cells, independently of its role in proliferation. We demonstrate that loss or inhibition of CDK4, using respectively CRISPR/Cas9 mediated CDK4 knockout and pharmacological CDK4/6 inhibitor, leads to enhanced migration capacities and reorganization of actin subcellular networks. Mechanistically, the absence of CDK4 results in decreased phosphorylation of Myo9b at serine 1935 (S1935), which enhances RhoA signaling, a key driver of cytoskeletal dynamics, leading to polarity defects and increased cell migration. These findings reveal a non-canonical function of CDK4 in limiting TNBC cell migration through the CDK4/CyclinD-Myo9b-RhoA signaling axis. This work highlights the broader cellular roles of CDK4 beyond its established function in proliferation and suggest that inhibition of Myo9b-RhoA pathway could reduce metastatic behaviour in TNBC treated with CDK4/6i, thereby informing future co-therapeutic strategies against aggressive cancer subtypes.