Systemic toxicity and limited tumor selectivity remain major obstacles in HER2-positive breast cancer therapy with conventional chemotherapeutics. Extracellular vesicles (EVs) provide a promising delivery platform due to their intrinsic biocompatibility and intercellular communication. Here, we engineered EVs displaying the HER2-binding peptide P51 to create a peptide-mediated targeted delivery system for doxorubicin (Dox), aiming to enhance tumor specificity and apoptotic efficacy. EVs expressing the P51 peptide were generated by transient transfection of HEK-293 cells, followed by Dox loading to produce P51-EVDox. Cellular uptake and antitumor efficacy were evaluated in vitro and in vivo using HER2-overexpressing BT-474 breast cancer cells. RNA sequencing and apoptosis-related protein analyses, including cleaved caspase-3, PARP cleavage, and p53 expression, were performed to elucidate EV-mediated molecular mechanisms. P51-EVDox exhibited significantly enhanced binding and intracellular accumulation in HER2-positive cells compared with non-targeted EV-Dox. Transcriptomic analysis comparing free Dox and P51-EVDox revealed marked upregulation of thioredoxin-interacting protein (TXNIP). Consistently, protein-level analyses demonstrated increased activation of apoptosis-associated markers, including cleaved caspase-3, PARP cleavage, and p53, following P51-mediated EV delivery. These findings suggest activation of a ROS/TXNIP-driven apoptotic cascade. This study demonstrates that peptide-functionalized EVs represent a promising tumor-targeted chemotherapeutic platform for HER2-positive breast cancer. By integrating ligand-mediated cellular recognition with EV-based doxorubicin delivery, P51-EVDox activates the ROS/TXNIP signaling pathway, leading to enhanced tumor suppression and apoptosis.

Lung adenocarcinoma (LUAD) is a major subtype of lung cancer that frequently develops brain metastases. Tumor heterogeneity substantially complicates disease management, and reliable biomarkers for metastatic progression remain lacking. LIM domain kinase 1 (LIMK1), a key regulator of cytoskeletal dynamics, has been implicated in tumor progression; however, its role in regulating brain metastasis in LUAD remains unclear. Single-cell RNA sequencing data from 11 primary LUAD tumors and 10 brain metastases were analyzed to identify malignant epithelial subclusters associated with metastatic dissemination. Tumor evolutionary trajectories and gene regulatory networks were explored using pseudotime trajectory analysis and weighted gene co-expression network analysis. LIMK1 expression was validated using The Cancer Genome Atlas dataset, as well as RT–qPCR and Western blotting in LUAD tissues and cell lines. Functional assays, including cell proliferation, colony formation, migration, and invasion, were performed following LIMK1 knockdown in LUAD cell lines. Six malignant epithelial subclusters were identified, among which clusters 0 and 4 were significantly enriched in brain metastases and associated with poor survival outcomes. LIMK1 was identified as a hub gene within these metastasis-associated clusters. LIMK1 expression was markedly upregulated in LUAD tissues and cell lines, and elevated LIMK1 levels predicted an unfavorable prognosis. Functional experiments demonstrated that LIMK1 depletion significantly inhibited tumor cell proliferation, migration, and invasion, while partially reversing epithelial–mesenchymal transition (EMT). Mechanistically, LIMK1 was found to promote LUAD progression through activation of the PI3K/AKT/mTOR signaling pathway. LIMK1 represents a novel prognostic biomarker and metastasis-associated regulator in LUAD. By driving EMT and activating PI3K/AKT/mTOR signaling, LIMK1 facilitates malignant progression, highlighting its potential as a therapeutic target for LUAD patients with brain metastases.

Despite significant advances in the treatment of clear cell renal cell carcinoma (ccRCC) over the past two decades, therapeutic intervention for patients at advanced or metastatic stages remains a clinical hurdle. Emerging evidence highlights aberrant CD70 expression during tumor progression, supporting its potential as a therapeutic target in advanced ccRCC. However, the molecular mechanisms underlying CD70-mediated metastatic processes remain poorly defined. CD70 was knocked out or overexpressed in three ccRCC cell lines, and its role in tumor progression was assessed using in vitro migration and invasion assays, as well as in vivo tumorigenesis and metastasis models. Mass spectrometry-based proteomics was performed to identify differentially expressed proteins and potential pathway dysregulation following CD70 depletion. Co-immunoprecipitation (Co-IP) assays confirmed the CD70-NF1 interaction, and NF1 knockdown in CD70-knockout cells was conducted to reverse the effects of CD70 depletion on Ras/ERK signaling, migration, invasion, and epithelial-mesenchymal transition (EMT)-related features. Expression of EMT-related marks were evaluated by quantitative real-time PCR (qRT-PCR), Western blot, and immunohistochemistry (IHC). CD70 was significantly upregulated in ccRCC tissues and correlated with poor survival outcomes. CD70 knockout markedly inhibited in vitro cell migration and invasion, as well as in vivo tumor growth and metastasis. Mechanistically, CD70 depletion was associated with increased neurofibromin 1 (NF1) expression in ccRCC cells; CD70 knockout resulted in robust NF1 induction, which in turn suppressed Ras/ERK signaling and downstream EMT-related molecular alterations. Notably, NF1 knockdown largely reversed the inhibitory effects of CD70 knockout on these key molecular and functional phenotype changes. Our findings uncover a novel CD70-NF1-Ras/ERK axis that contributes to ccRCC progression and metastasis, involving suppression of NF1 and subsequent activation of the Ras/ERK signaling pathway, which further promotes EMT and enhances metastatic capacity. These insights provide a mechanistic foundation for targeting CD70 as a potential therapeutic strategy in advanced ccRCC. Notably, we propose that CD70-targeted therapy may be more effective when combined with agents blocking adaptive escape pathways (e.g., Ras/ERK inhibitors), a hypothesis generated by mechanistic rescue and pharmacologic inhibition in vitro, which require further preclinical validation to confirm synergistic efficacy and clinical viability.

1,4-Dinitrosopiperazine (DNP), a nitrite-derived compound widely found in Cantonese-style salted fish, induces malignant transformation of nasopharyngeal epithelial cells, but its tissue specificity and carcinogenic mechanisms remain unclear. In this study, tree shrews were used to investigate the tissue specificity and carcinogenic mechanisms of DNP. A pathological atlas of tree shrew nasopharyngeal tissue was constructed via anatomical and pathological examination. Pharmacokinetics and tissue affinity were assessed through single-dose administration, quantifying blood/tissue distribution via liquid chromatography-tandem mass spectrometry‌ (LC‒MS/MS). Chronic intervention was established via 12-month DNP exposure, with micro positron emission tomography/computed tomography (PET-CT) imaging and pathology confirming malignant transformation. Network toxicology analyzed DNP-associated head/neck carcinomas, followed by qPCR validation of key mechanisms. A three-dimensional reconstruction model and tissue atlas of the tree shrew nasopharynx were developed. LC-MS/MS analysis revealed peak DNP accumulation in the nasopharynx at 24 h post-injection (with significantly higher retention compared to other tissues), followed by complete systemic clearance within 72 h. After 12-month chronic DNP intervention, microPET/CT imaging and pathological examination confirmed precancerous lesions or malignant transformation in nasopharyngeal tissues of all tree shrews. Network toxicology highlighted cytochrome P450 (CYP450) enzyme activation as closely associated with malignant progression, with CYP19A1 (HR = 1.52) and CYP2D6 (HR = 0.745) expression showing significant prognostic relevance. qPCR validation demonstrated marked CYP19A1 upregulation and CYP2D6 downregulation in animal nasopharyngeal tissues, indicating their role as key pathogenic biomarkers. These findings confirm DNP’s selective nasopharyngeal accumulation drives carcinogenesis via CYP450 activation, with CYP19A1 overexpression and CYP2D6 suppression may serve as key pathogenic biomarkers and potential regulatory factors in DNP-induced malignant nasopharyngeal transformation.

Gallbladder stones (GBS) and gallbladder cancer (GBC) are prevalent gallbladder diseases that, while presenting distinct clinical manifestations, may share common regulatory mechanisms at the molecular level. This study aimed to identify and validate common differentially expressed genes (C-DEGs) in GBS and GBC through comprehensive bioinformatics analysis and experimental validation to elucidate potential molecular pathological mechanisms linking these two conditions. We analyzed gene expression data from publicly available databases for GBS and GBC, identifying 94 common differentially expressed genes (C-DEGs) through differential expression analysis. Gene ontology (GO) functional analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were performed to characterize the functions and pathways associated with these genes. Furthermore, we examined the differential expression of transcription factors (TFs) in both GBS and GBC groups and experimentally validated the functions of these genes. Our analysis identified 94 C-DEGs, including 10 hub genes (HC-DEGs): SOCS3, GADD45B, SGK1, MYC, HBEGF, KLF10, EGF, IL6, NR4A3, and CDKN1A. GO functional analysis revealed that these genes primarily regulate smooth muscle cell proliferation, animal organ regeneration, peptidyl-tyrosine phosphorylation, epidermal growth factor-activated receptor activity, and the JAK-STAT signaling pathway. KEGG pathway analysis demonstrated their involvement in multiple cancers (bladder, endometrial, colorectal, thyroid, breast, and gastric), the FoxO signaling pathway, the JAK-STAT signaling pathway, the ErbB signaling pathway, and transcriptional dysregulation in cancer. Additionally, we identified 14 differentially expressed TFs in the GBS group and 11 in the GBC group. Four transcription factors—E2F1, ETS2, EZH2, and MYC—showed differential co-expression in both conditions and were jointly involved in regulating two hub genes (CDKN1A and MYC). Through comprehensive bioinformatics analysis and experimental validation, this study revealed common molecular mechanisms between GBS and GBC. We identified and validated 10 hub genes that exhibited significant differential expression in both conditions and were primarily involved in multiple cancer-related signaling pathways. Furthermore, we identified four transcription factors (E2F1, ETS2, EZH2, and MYC) showing differential co-expression in both GBS and GBC, which jointly regulated two hub genes (CDKN1A and MYC). These findings illuminate shared molecular mechanisms between GBS and GBC, providing novel insights for further investigation of their molecular pathological mechanisms and establishing an important theoretical foundation for future preventive and therapeutic strategies.