Triple-negative breast cancer (TNBC) remains an aggressive malignancy with limited therapeutic options and poor prognosis, underscoring the critical need for novel therapeutic targets. This investigation elucidates the functional role of the potassium channel tetramerization domain 15 (KCTD15) in TNBC progression, providing mechanistic insights into its potential as a therapeutic target for this challenging disease. KCTD15 exhibited high expression in TNBC tissues, correlating with advanced grade and unfavorable prognosis. Functionally, KCTD15 knockdown in TNBC cell lines (BT-549/MDA-MB-231) markedly suppressed cellular proliferation, migration, and cancer stem cell properties, while concomitantly enhancing apoptosis. Mechanistically, KCTD15 directly interacted with KLF4, facilitating its nuclear translocation and subsequent activation of the β-catenin signaling cascade. Notably, KLF4 knockdown abrogated KCTD15-mediated stemness maintenance and β-catenin pathway activation. Invivo, KCTD15 silencing reduced xenograft tumor growth and downregulated Ki67, KLF4, and β-catenin protein expression in tumor tissues, confirming its oncogenic role through the KLF4/β-catenin axis. Our findings establish KCTD15 as a pivotal regulator of TNBC stemness through modulation of the KLF4/β-catenin signaling axis. These results provide a robust preclinical rationale for developing therapeutic strategies targeting this molecular axis in TNBC management.

Glycolipids constitute an important component of the plasma membrane based on both abundance as well as function. Gangliosides, being a class of structurally diverse and functionally varied glycolipids, can act both as a receptor as well as a ligand and therefore are established as a crucial player in several normal cellular processes. In certain diseases, and in particular cancer, select gangliosides are over-expressed often leading to disease manifestation. GM2-synthase, the enzyme responsible for the formation of a pro-tumorigenic ganglioside, GM2, is well reported to be over-expressed across various cancer tissues and cell lines. This over-expression of GM2-synthase has been linked with increased migration, invasion, and epithelial to mesenchymal transition (1) as well as induction of a local and systemic host immune suppression in cancer. Despite only a handful of studies demonstrating an epigenetic regulation underlying the transcriptional regulation of the GM2-synthase (B4GalNT1) gene, the detailed mechanism still remains unclear. Here we identified the total proteome associated with the GM2-synthase promoter through a two-step CRISPR-dCas9 based proteome profiling approach by categorizing all the identified proteins leading to a detailed elucidation of the molecular drivers behind GM2-synthase transcription. While the previous study identified an acetylation-dependent de-repression of the transcription factor SP1 causing GM2-synthase activation, the underlying molecular mechanism driving its activation wasn't clear. This study demonstrated that the histone acetyl transferase p300, acts as a pivotal factor which on one hand causes acetylation-mediated degradation of SP1, and on the other hand activates SMAD2/4 to have a direct positive impact on GM2-synthase gene transcription. We identified p300 to have an activator role in GM2-synthase gene transcription through knock out, knock down, and over-expression experiments. Furthermore, SP1 degradation, SMAD activation, and their DNA binding patterns show the reciprocal role of p300 on SP1 and SMAD complexes. Altogether we have identified SMAD2/4 as an activator complex, p300 as a positive regulator, and uncovered a critical p300-SMAD-SP1 regulatory axis in GM2-synthase transcriptional regulation.

Liver Hepatocellular Carcinoma (LIHC) is a high-mortality primary liver cancer. Its treatment and prognosis are highly dependent on disease stage and liver function reserve, necessitating novel biomarkers and optimized therapeutic strategies. Sialylation frequently exhibits abnormal elevation (hypersialylation) in cancers and is recognized as both an important malignant marker and a potential therapeutic target. Transcriptomic, mutational, and clinical LIHC data were procured from TCGA/GEO, extracting sialylation-related genes. Single-cell data underwent quality control, clustering, annotation, and risk-cell subpopulation identification using Seurat/Harmony/SCISSOR. AUCell quantified SRGs activity to identify key differentially expressed SRGs. 10 machine learning algorithms (e.g., SVM, Enet, CoxBoost) were integrated; the optimal StepCox + Enet model was selected via cross-validation, stratifying patients by risk score. The model's clinical utility was validated through GSEA, PPI networks, immune infiltration (CIBERSORT/ssGSEA), and drug sensitivity profiling. This integrated study combined single-cell and bulk transcriptomic data to develop an 11-gene sialylation-related prognostic model for LIHC, demonstrating robust predictive accuracy (AUC > 0.74 across cohorts). High-risk patients exhibited myeloid-driven biology, including enhanced SPP1-mediated cell-cell signaling, TP53 mutations, metabolic dysregulation, and an immunosuppressive microenvironment with elevated TIDE scores. In contrast, the low-risk group displayed active anti-tumor immunity and metabolic homeostasis. Drug sensitivity analysis revealed higher sensitivity to chemotherapeutic agents in high-risk patients. Integrated transcriptomics establishes aberrant sialylation as a key LIHC prognostic biomarker and therapeutic target by stratifying risk subgroups and revealing immunosuppressive microenvironment alterations.

Lung adenocarcinoma (LUAD) and squamous cell carcinoma (LUSC) are common histologic subtypes of non-small cell lung cancer (NSCLC), usually diagnosed at advanced stages with a dismal survival rate. A high-resolution SNP array (Affymetrix 250 NspI) analysis of five primary NSCLC tumors and their follow-up mucosal biopsies (n = 25) identified frequent copy neutral loss of heterozygosity (LOH, loss of 59 SNPs) of CACNA1C, a calcium voltage-gated channel subunit. Transcriptomic profiling utilizing The Cancer Genome Atlas (TCGA) revealed frequent loss of mRNA expression of CACNA1C in LUAD and LUSC tumors compared to the normal counterparts. Loss of CACNA1C mRNA expression was significantly associated with stage, grade, lymph node metastasis, P53 gene mutation, and the worst survival of these patients. We also recorded significantly higher promoter methylation of CACNA1C in the tumors compared to the normal counterparts. Increased level of promoter hypermethylation of CACNA1C was significantly associated with stage, grade, lymph node metastasis, P53 gene mutation, and poor survival of the patients. We also identified frequent mutations in CACNA1C among the NSCLC patients, which were associated with immune function modulation. Analysis of an independent cohort of NSCLC subjects revealed significant loss of CACNA1C protein expression in primary tumors (n = 31) and lymph node metastases (n = 10) compared to normal tissues. Loss of CACNA1C protein expression appeared to be an early event in LUAD patients. Further validation of CACNA1C expression in larger cohorts and functional characterization would be beneficial for potential biomarkers and therapeutic development.

Endometriosis is a chronic, estrogen-dependent inflammatory condition that affects approximately 10% of reproductive-age women and is frequently associated with infertility and pelvic pain. Unlike many estrogen-dependent disorders, epidemiologic studies have reported inconsistent associations between obesity, measured by BMI, and endometriosis. Caloric restriction diet (CRD), a dietary regimen that reduces energy intake without malnutrition, has demonstrated anti-inflammatory and anti-proliferative effects in various chronic diseases but remains underexplored in endometriosis. Here, we investigated the effects of CRD on endometriosis using a mouse model with a GFP reporter. Endometriosis was surgically induced by inoculating endometrial fragments, and mice were randomized to receive either adlibitum feeding (regular diet, RD) or CRD. After 90 days, CRD mice exhibited a 24.5% reduction in body weight compared to RD mice, without impairment of female fertility. Notably, both the number and weight of ectopic lesions were significantly reduced in the CRD group compared to the RD group. Immunohistochemical analysis revealed decreased epithelial and stromal cell proliferation, increased apoptosis, and elevated stromal progesterone receptor expression in ectopic lesions from CRD mice. Furthermore, phosphorylation of STAT3 and ERK1/2, key inflammatory and proliferative signaling molecules, was significantly reduced in ectopic lesions from CRD mice. These findings suggest that CRD, without compromising fertility, exerts beneficial effects on endometriosis by targeting cell proliferation, survival, and inflammation. Our results support lifestyle-based dietary interventions as a promising non-hormonal strategy for endometriosis management.

Loss of function G-protein coupled receptor 75 (GPR75) variants in humans are associated with leanness, and Gpr75 null mice are protected from diet-induced obesity (DIO). However, the mechanisms underlying this protection are largely unknown. Here, we investigated the contribution of adipocyte-derived Gpr75 to DIO. Adipocyte-specific Gpr75 knockout (adipo-Gpr75-/-) male and female mice and their wild-type (WT) littermates were placed on a high-fat diet (HFD) for 14 weeks. Metabolic parameters including body weight, energy intake and expenditure, activity, and glucose metabolism were monitored before and after diet feeding. While WT mice obtained a diabetogenic phenotype on HFD, the adipo-Gpr75-/- counterparts were protected. This protection showed sexual dimorphism. Female adipo-Gpr75-/- mice displayed a 50% (p < 0.001) decrease in weight gain and adiposity compared to WT, whereas male adipo-Gpr75-/- gained weight like WT mice. Interestingly, both male and female adipo-Gpr75-/- mice exhibited improved glucose handling compared to WT, which was correlated to decreased adiposity, abrogated adipose tissue inflammation, and increased insulin sensitivity in skeletal muscle. Importantly, no differences in food intake were observed; however, adipo-Gpr75-/- mice exhibited increased activity and energy expenditure, regardless of sex. Taken together, these findings demonstrate that deletion of GPR75 specifically in adipocytes is sufficient to confer protection against DIO and suggest that adipocyte-derived GPR75 contributes importantly to the pathogenesis of DIO potentially by mechanisms that may include promotion of inflammation, impairment of insulin signaling, and disruption of metabolic homeostasis.

Elevated circulating low-density lipoprotein cholesterol (LDL-C) is a key risk factor for coronary artery disease (CAD). The pathogenesis of CAD is multifactorial, driven by heritable and lifestyle-related risk factors. Although CD4+ T cells are one of the main cell types in atherosclerotic lesions, their interaction with atherogenic oxidized LDL (ox-LDL) remains poorly understood. Therefore, we sought to characterize the transcriptomic and epigenomic consequences of ox-LDL on activated human CD4+ T cells. We find that ox-LDL causes a shift towards a pro-inflammatory, cytokine-producing CD4+ T cell transcriptomic state. Concurrently, ox-LDL induces genome-wide changes in chromatin accessibility, notably in promoter regions. By integrating our multiomic data, we identify the NRF1 and SP1 transcription factors as likely mediators of ox-LDL-induced changes in gene expression. In contrast, the influence of AP-1 related factors over CD4+ T cell gene expression decreases following ox-LDL stimulation. We leveraged our multiomic data to investigate the disease relevance of ox-LDL exposure, by investigating genomic locations where CAD-associated single nucleotide polymorphisms were found within dynamic ox-LDL-regulated accessible chromatin regions. Together, we demonstrate a disease-relevant role for ox-LDL in atherogenic conditioning of CD4+ T cells. Understanding such cell-type specific interactions with CAD risk factors may facilitate the development of targeted therapies for CAD.

Cadmium (Cd) exposure is an emerging risk factor for neurodegeneration, yet the contribution of necroptosis to Cd-induced neuronal loss and its tractability for pharmacologic intervention remain unclear. Here, we identify an AMP-activated protein kinase (AMPK)-mitochondrial reactive oxygen species (mtROS)-RIPK1/RIPK3-MLKL axis as a central driver of Cd neurotoxicity and a critical target of metformin. In a chronic Cd-exposed mouse model and in primary hippocampal neurons, Cd provoked prominent neuronal injury characterized by the activation of necroptotic signaling (elevated p-RIPK1, p-RIPK3, and p-MLKL), excessive mtROS production, and mitochondrial injury. Oral metformin preserved hippocampal neuronal integrity and suppressed necroptotic markers invivo, while in cultured neurons and SH-SY5Y cells, it reduced mtROS, maintained mitochondrial morphology and membrane potential, and prevented necrosome assembly. Pharmacologic inhibition of RIPK1 (necrostatin-1) or RIPK3 (GSK-872), as well as CRISPR/Cas9-mediated deletion of RIPK1, RIPK3, or MLKL, attenuated Cd-induced necrosis and phenocopied or enhanced the anti-necroptotic effects of metformin. Mechanistically, metformin restored Cd-suppressed AMPK activity, whereas blockade of AMPK with compound C or expression of a dominant-negative AMPKα1 mutant abolished metformin-mediated suppression of mtROS, RIPK1/RIPK3-MLKL signaling, and necroptosis. Scavenging mtROS with Mito-TEMPO or MitoQ reduced necroptosis and synergized with metformin to disrupt RIPK3-MLKL complex formation. Collectively, these findings demonstrate that Cd triggers neuronal necroptosis via mtROS-driven activation of RIPK1/RIPK3-MLKL, and that metformin confers neuroprotection by activating AMPK to restore mitochondrial homeostasis and restrain necroptotic signaling. This work positions the AMPK-mtROS-necroptosis axis as a therapeutically actionable pathway in heavy-metal neurotoxicity and supports repurposing metformin for necroptosis-associated neurodegenerative conditions.

Unexplained Infertility (UI) is a complex condition of elusive etiology, where the interplay between immune dysregulation and metabolic disturbances remains poorly understood. We hypothesized that gut microbiota-derived metabolites act as central modulators of the systemic immune and metabolic balance in UI patients. We employed an integrated multiomics approach, combining metabolomics, gut microbiome analysis, and immune profiling, in a cross sectional discovery cohort (47 UI patients and 53 healthy controls), and validated key findings in an independent cohort (37 UI patients and 39 healthy controls). Our findings demonstrated that UI patients exhibited a proinflammatory Th1-dominant immune profile, marked by elevated proinflammatory cytokines and reduced anti-inflammatory IL-10. This immune imbalance was accompanied by a deficiency in protective gut-derived secondary metabolites, notably secondary bile acids and phenylpropanoic acid. Furthermore, gut microbiota analysis revealed significant dysbiosis (increased pathogenic taxa and decreased beneficial microbes) and a functional deficiency in the aromatic amino acid metabolism gene cluster, explaining the observed metabolite scarcity. Mechanistically, invitro assays and network pharmacology indicated that these metabolites directly modulate the Th1/Th2 immune balance by regulating a core host network centered on TNF, PPARG, and PTGS2. In summary, our data reveal the role of a novel gut microbiota-metabolite-immune axis in UI pathophysiology, where a deficiency in protective gut-derived secondary metabolites contributes directly to systemic immune dysregulation and a proinflammatory state. These metabolites serve as potential candidates for future evaluation and represent promising therapeutic targets for interventions to restore immune homeostasis in UI patients.

Phospholipids are important components of the bilayer of biological membranes. Alterations of phospholipids are associated with metabolic disorders, including insulin resistance. However, how impaired insulin signaling impacts phospholipids has not been well established. Disruption of hepatic insulin signaling is achieved by insulin receptor substrate 1 (IRS1) and IRS2 double deletion (DKO) in the liver. Further deletion of TGF-β1 or Foxo1 in the liver of DKO mice was used to examine the role of TGF-β1 or Foxo1 in contributing to the alterations of phospholipid metabolism in DKO mice. Disruption of hepatic insulin signaling led to the dysregulation of phospholipids, including phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), phosphatidylserine (PS), sphingomyelin (SM), cardiolipin (CL), and lysophospholipids in the liver. Mechanistically, disruption of hepatic insulin signaling dysregulated the expression of genes related to phospholipid metabolism. Interestingly, further deletion of Tgfb1 in the liver of DKO mice (TKObeta1) attenuated the alterations of phospholipids and rescued the abnormal expression of genes related to phospholipid metabolism. Moreover, deletion of transcription factor Foxo1, a key mediator of insulin signaling, achieved similar beneficial effects as Tgfb1 deletion in DKO mice. Our study suggests that insulin signaling plays a crucial role in maintaining phospholipids balance in the liver via TGF-β1 or Foxo1. Targeting TGF-β1 or Foxo1 could be promising strategies to combat phospholipids alterations and related metabolic dysfunctions.