High-grade serous ovarian cancer (HGSOC) is one of the most deadly and heterogenic cancers. We have recently shown that ZIP4 (gene name SLC39A4), a zinc transporter, is functionally involved in cancer stem cell (CSC)-related cellular activities in HGSOC. Here, we identified ZIP4 as a novel CSC marker in HGSOC. Fluorescence-activated cell sorter (FACS)-sorted ZIP4⁺, but not ZIP4⁻ cells, formed spheroids and displayed self-renewing and differentiation abilities. Over-expression of ZIP4 conferred drug resistance properties in vitro. ZIP4⁺, but not ZIP4⁻ cells, formed tumors/ascites in vivo. We conducted limiting dilution experiments and showed that 100–200 ZIP4⁺ cells from both PE04 and PEA2 cells formed larger tumors than those from 100–200 ALDH⁺ cells in mice. Mechanistically, we found that ZIP4 was an upstream regulator of another CSC-marker, NOTCH3, in HGSOC cells. NOTCH3 was functionally involved in spheroid formation in vitro and tumorigenesis in vivo in HGSOC. Genetic compensation studies showed that NOTCH3, but not NOTCH1, was a critical downstream mediator of ZIP4. Furthermore, NOTCH3, but not NOTCH1, physically bound to ZIP4. Collectively, our data suggest that ZIP4 is a novel CSC marker and the new ZIP4-NOTCH3 axis represents important therapeutic targets in HGSOC.

Background: Ceramide, a bioactive lipid, plays an essential role in the development of several pulmonary inflammatory diseases. Matrix metallopeptidase 9 (MMP-9) regulates the synthesis and degradation of extracellular matrix, and is associated with airway remodeling and tissue injury. This study was conducted to investigate the effects and underlying mechanisms of ceramide on MMP-9 expression in airway epithelium. Methods: BEAS-2B cells, normal human bronchial epithelium cell lines, were pretreated with AG490, a selective janus tyrosine kinase 2 (JAK2) inhibitor, or Stattic, a selective signal transducer and activator of transcription 3 (STAT3) inhibitor. The cells were then stimulated with C6-ceramide. The levels of MMP-9 were determined by ELISA and real-time quantitative PCR (RT-qPCR). JAK2, phosphorylated JAK2 (p-JAK2), STAT3, and phosphorylated STAT3 (p-STAT3) expression was examined by Western blotting. BALB/c mice were pretreated with AG490 or Stattic before intratracheally instillated with C6-ceramide. Pathological changes in lung tissues were examined by Hematoxylin and Eosin staining, Periodic-acid Schiff staining, and Masson’s trichrome staining. MMP-9, JAK2, p-JAK2, STAT3, and p-STAT3 expression in the lung tissues was examined by Western blotting. Results: The expression of MMP-9, p-JAK2 and p-STAT3 in BEAS-2B cells was significantly increased after the treatment of C6-ceramide. Furthermore, the increased expression of MMP-9 induced by C6-ceramide was inhibited by AG490 and Stattic. Similar results were obtained in the lung tissues of C6-ceramide-exposed mice which were treated with AG490 or Stattic. Conclusions: Ceramide could up-regulate MMP-9 expression through the activation of the JAK2/STAT3 pathway in airway epithelium. Targeted modulation of the ceramide signaling pathway may offer a potential therapeutic approach for inhibiting MMP-9 expression. This study points to a potentially novel approach to alleviating airway remodeling in inflammatory airway diseases.

Asthma, a chronic inflammatory disorder, imposes significant economic and social burdens despite advances in understanding its pathophysiology. PANoptosis, a newly identified form of programmed cell death involving pyroptosis, apoptosis, and necroptosis, has been linked to various respiratory diseases, but its role in asthma exacerbation remains unclear. This study aimed to investigate PANoptosis variations in asthma induced by different stimuli and identify potential therapeutic targets during acute asthma exacerbations. We generated a murine ovalbumin (OVA)-induced asthma model and subsequently challenged the mice with LPS or Poly(I:C) to assess the impact on airway inflammation. Results showed that LPS and Poly(I:C) significantly exacerbated PANoptosis in asthma models, with elevated levels of phospho-MLKL, NLRP3, and cleaved Caspase-3 and their colocalization. We retrieved asthma exacerbation-related dataset GSE256534 from the GEO database and identified 68 PANoptosis-associated differentially expressed genes (DEGs), including key genes CASP1 and CASP8. Functional enrichment analysis revealed their involvement in inflammation and immune response pathways. Immune cell infiltration analysis showed significant correlations between key genes and infiltrating immune cells. Validation in murine asthma models confirmed elevated Caspase-1 and Caspase-8 expression in OVA-induced asthma, further increased by LPS and Poly(I:C). Our study provides novel insights into PANoptosis in asthma exacerbation, highlighting the aggravating role of LPS and Poly(I:C). The identification of key genes such as CASP1 and CASP8 suggests potential therapeutic targets for managing acute asthma exacerbations.