T-cell acute lymphoblastic leukemia (T-ALL) is a highly aggressive hematological malignancy with limited treatment options. Zinc finger protein X-linked (ZFX) and programmed cell death protein 1 (PD1) have been implicated in tumor progression, but their roles in T-ALL and their regulatory interplay with RNA methylation (m5C) remain unclear. Clinical specimens and T-ALL cell lines (Jurkat, MOLT4) were analyzed using immunohistochemistry, reverse transcription-polymerase chain reaction (RT-qPCR), and western blot to assess ZFX/PD1 expression and m5C levels. Functional consequences of ZFX knockdown (sh-ZFX) were investigated through cell counting kit-8(CCK-8) proliferation, apoptosis assay, Transwell migration, and reactive oxygen species (ROS) assays. Mechanistic studies integrated Chromatin Immunoprecipitation (ChIP)-qPCR assay and luciferase reporter assay. Rescue experiments were performed by co-transfecting sh-ZFX with NSUN2 overexpression plasmids. In vivo validation utilized a syngeneic C57BL/6 Mouse T-ALL tumor model. ZFX and PD1 were significantly upregulated in T-ALL samples and cell lines, correlating with elevated global m5C levels. ZFX knockdown suppressed cell proliferation, migration, and m5C levels while increasing apoptosis and ROS. Mechanistically, ZFX bound to the NSUN2 (m5C methyltransferase) promoter (-1719 -1706) and transcriptionally activated NSUN2 expression, further promoting the m5C-dependent stabilization of PD1 mRNA. NSUN2 overexpression reversed ZFX knockdown effects, restoring PD1 expression, m5C modification, and tumor aggressiveness in vitro and in vivo. Our study demonstrated that ZFX promoted the progression of T-ALL by regulating NSUN2-mediated m5C modification of PD1, suggesting ZFX and NSUN2 as potential therapeutic targets for T-ALL.T-cell acute lymphoblastic leukemia (T-ALL) is a highly aggressive hematological malignancy with limited treatment options. Zinc finger protein X-linked (ZFX) and programmed cell death protein 1 (PD1) have been implicated in tumor progression, but their roles in T-ALL and their regulatory interplay with RNA methylation (m5C) remain unclear. Clinical specimens and T-ALL cell lines (Jurkat, MOLT4) were analyzed using immunohistochemistry, reverse transcription-polymerase chain reaction (RT-qPCR), and western blot to assess ZFX/PD1 expression and m5C levels. Functional consequences of ZFX knockdown (sh-ZFX) were investigated through cell counting kit-8(CCK-8) proliferation, apoptosis assay, Transwell migration, and reactive oxygen species (ROS) assays. Mechanistic studies integrated Chromatin Immunoprecipitation (ChIP)-qPCR assay and luciferase reporter assay. Rescue experiments were performed by co-transfecting sh-ZFX with NSUN2 overexpression plasmids. In vivo validation utilized a syngeneic C57BL/6 Mouse T-ALL tumor model. ZFX and PD1 were significantly upregulated in T-ALL samples and cell lines, correlating with elevated global m5C levels. ZFX knockdown suppressed cell proliferation, migration, and m5C levels while increasing apoptosis and ROS. Mechanistically, ZFX bound to the NSUN2 (m5C methyltransferase) promoter (-1719 -1706) and transcriptionally activated NSUN2 expression, further promoting the m5C-dependent stabilization of PD1 mRNA. NSUN2 overexpression reversed ZFX knockdown effects, restoring PD1 expression, m5C modification, and tumor aggressiveness in vitro and in vivo. Our study demonstrated that ZFX promoted the progression of T-ALL by regulating NSUN2-mediated m5C modification of PD1, suggesting ZFX and NSUN2 as potential therapeutic targets for T-ALL.
Osteosarcoma is the most common primary malignant bone tumor in children and adolescents, characterized by high invasiveness, early metastasis, and frequent recurrence, which are largely driven by genomic copy number variations (CNVs). However, existing prognostic models based on bulk RNA-seq are constrained by intratumoral heterogeneity and fail to reflect intrinsic malignant cell characteristics. Based on single-cell transcriptomic data, osteoblastic cells were stratified by CNV burden into high- and low-malignancy subpopulations, and malignant feature-related genes (MFGs) were identified. An integrative machine-learning algorithm was used to construct the malignant feature-based prognostic signature (MFPS) prognostic model, validated in independent cohorts. We then analyzed the MFPS-associated immune microenvironment, drug sensitivity, and biological mechanisms, and screened key genes. Finally, knockdown experiments in osteosarcoma cell lines assessed proliferation, cell cycle, migration, invasion, and transcriptomic changes. The high-grade malignant osteoblastic cell (HG-MOB) subpopulation exhibited high CNV burden, enhanced proliferation and intercellular communication, and low differentiation. The MFPS model, built from 11 MFGs, performed robustly across multiple cohorts and outperformed most reported models. High-MFPS patients had shorter overall survival and higher metastasis and recurrence risks. The high-MFPS group showed an immunosuppressive microenvironment, with reduced CD8⁺ T and NK cells, M2 macrophage enrichment, immunomodulatory molecule downregulation, and activation of mTORC1, hypoxia, and glycolysis. OPN3 was prioritized among MFGs. OPN3 knockdown modestly reduced proliferation but significantly arrested G₁/S transition and suppressed migration and invasion. RNA-seq revealed perturbations in PI3K-Akt, TNF, and cytokine-cytokine receptor pathways, with downregulation of angiogenesis- and hypoxia-related gene sets. This study identifies a CNV-defined HG-MOB subpopulation and establishes MFPS as a valuable prognostic tool reflecting tumor aggressiveness and immune heterogeneity. OPN3 is validated as a key driver of osteosarcoma malignancy, potentially via the PI3K-Akt axis and downstream angiogenic and metabolic pathways, representing a novel candidate therapeutic target.
Treatment of colorectal cancer (CRC) is limited by drug resistance and an immunosuppressive microenvironment. Tumor-associated macrophages (TAMs), especially M2-polarized TAMs, promote CRC progression. This study investigates the effects of formononetin (FN), a candidate bioactive constituent identified from Jianpi Tongluo Formula (JPTLF), on M2-TAM polarization and CRC progression. Multi-omics integration (network pharmacology, transcriptomics, weighted gene co-expression network analysis and single-cell sequencing) was used to identify key targets. Absorbed components were analyzed by ultra-performance liquid chromatography–quadrupole time-of-flight tandem mass spectrometry. Molecular docking and dynamics simulations predicted compound-target interactions, and cellular thermal shift assays (CETSA) were performed to assess AXL thermal stability following FN treatment. In vitro assays, MC-38 xenograft models and patient-derived CRC organoids were employed for validation. AXL was identified as the sole overlapping target. FN, present in Astragalus membranaceus, Sophora flavescens and Glycyrrhiza uralensis, increased AXL thermal stability in CETSA, supporting FN–AXL target engagement. FN co-treatment with tumor-conditioned medium reduced M2 markers (CD206, arginase 1) and suppressed AXL, Janus kinase 2 (JAK2) and signal transducer and activator of transcription 3 (STAT3) phosphorylation. The AXL activator recombinant mouse Gas6 (rmGas6) partially reversed these effects, while the AXL inhibitor R428 mimicked FN’s action. In MC-38 xenografts, FN (100 mg/kg) inhibited tumor growth, reduced intratumoral M2-TAMs without observed body weight loss or histological injury in the major organs examined. Patient-derived CRC organoids were sensitive to FN. Formononetin suppresses CRC progression, with inhibition of M2-TAM polarization through AXL/JAK2/STAT3 signaling contributing to its antitumor activity. These findings provide preclinical support for further investigation of FN as a candidate compound for CRC treatment.
Lenvatinib has been approved as a treatment for advanced hepatocellular carcinoma (HCC), but drug resistance limits its clinical efficacy. Tumor-associated macrophages (TAMs) are abundant in the tumor microenvironment and critically contribute to HCC progression. However, the mechanism of interaction between HCC cells and TAMs in the context of lenvatinib resistance remains unclear. Lenvatinib-resistant HCC (HCC-LR) cells were established by gradually exposing the cells to lenvatinib. Western blot analysis was conducted to assess the expression levels of phosphorylated EGFR and its downstream pathway-associated proteins in HCC cells. Flow cytometry and colony formation assays were utilized to evaluate apoptosis and cell proliferation, respectively. Bioinformatic analyses were performed to investigate the expression of relevant genes, pathway activation, and immune cell infiltration. Additionally, TAMs were generated from THP-1 cells to investigate their role in mediating lenvatinib resistance in HCC. Our results indicated that phosphorylated EGFR and interleukin-8 (IL-8) were overexpressed in HCC-LR cells. Pharmacological inhibition of EGFR in HCC-LR cells increased apoptosis and attenuated cell proliferation upon lenvatinib treatment in vitro. Mechanistically, EGFR activation promoted IL-8 secretion, which recruited macrophages and enhanced lenvatinib resistance in HCC by activating the EGFR-Akt/JNK signaling axis in vitro. Notably, these recruited macrophages differentiated into TAMs, which in turn secreted HB-EGF, a prominent ligand responsible for reactivating EGFR signaling in HCC cells, thus establishing a resistance-promoting feedback loop. Our findings reveal a key tumor cell-TAM crosstalk that mediates lenvatinib resistance in HCC, providing a rational basis for developing improved combination therapies.
Bladder cancer (BCa) remains a therapeutically challenging malignancy with limited targeted treatment options. Natural products from food–medicine homology resources offer a promising reservoir for discovering bioactive compounds with favorable safety profiles, yet their mechanisms of action often remain incompletely characterized. A multi‑omics strategy integrating computational ADMET profiling, network pharmacology, single‑cell transcriptomics, and multi‑layer experimental validation was employed to systematically evaluate the anti‑BCa potential of twelve metabolites from Myrica rubra. Target identification was performed through reverse virtual screening, protein–protein interaction network analysis, and molecular docking. The core target was validated by gene dependency analysis, and its cell‑type‑specific expression pattern was examined using public scRNA‑seq datasets. Anti‑proliferative, pro‑apoptotic, and anti‑metastatic effects were assessed in UM‑UC‑3 and T24 cells in vitro, and tumor growth inhibition was evaluated in a subcutaneous xenograft model in vivo. Myricetin was identified as the top candidate compound, directly binding to the ATP‑binding pocket of HSP90AA1 with a Vina score of − 8.9 kcal/mol through hydrogen bonds with TYR‑139, ASP‑93, and ASN‑51. Myricetin treatment significantly suppressed BCa cell viability, reduced colony formation, inhibited migration and invasion, and induced apoptosis in a concentration‑dependent manner, accompanied by downregulation of CCND1 and BCL‑2, upregulation of BAX, and activation of caspase‑3. Flow cytometric analysis further confirmed that myricetin induces G1 phase cell cycle arrest in BCa cells. Mechanistically, myricetin reduced HSP90AA1 expression and selectively suppressed PI3K and AKT phosphorylation without affecting total protein levels. Rescue experiments using tamoxifen confirmed that these effects were mediated at least in part through HSP90AA1 engagement. In vivo, myricetin (60 mg/kg) significantly suppressed UM‑UC‑3 xenograft growth without causing body weight loss. Single‑cell transcriptomic analysis revealed that HSP90AA1 is preferentially enriched in BC_3 and BC_4 subpopulations, which occupy the root of the developmental trajectory and exhibit elevated PI3K–Akt pathway activity, suggesting a potential subpopulation‑specific vulnerability that warrants further investigation. Myricetin directly targets HSP90AA1 and suppresses PI3K–Akt signaling to inhibit BCa progression. These findings provide a mechanistic framework for myricetin as a lead compound from food–medicine homology resources, warranting further pharmacokinetic optimization and clinical investigation.
