Tumor recurrence, the chief reason for poor prognosis of glioma, is largely attributed to glioma stem cells (GSCs) and epithelial-mesenchymal transition (EMT). However, the mechanisms among them remain unknown. Here, we determined whether leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5), known as a stem cell marker for colon cancer and gastric cancer, can serve as a novel GSC marker involved in EMT and a therapeutic target in glioma. Stemness properties were examined in FACS-isolated LGR5⁺/LGR5⁻ cells. Reported stem cell markers, EMT and the Wnt/β-catenin pathway were examined in stable LGR5 knockdown or overexpressed GSCs by Western Blot. The treatment experiment was performed in an intracranial orthotopic xenograft model by knockdown of LGR5 or by using the Wnt/β-catenin pathway inhibitor Wnt-C59. LGR5 expression was determined in 268 glioma specimens by immunohistochemistry. LGR5⁺ cells possessed stronger stemness properties compared to LGR5⁻ cells. The expression of SOX2, Nanog, CD133, CD44, CD24 and EpCAM was modulated by LGR5. Both LGR5 knockdown and Wnt-C59 reduced tumor invasion and migration and blocked EMT by inhibiting the Wnt/β-catenin pathway in vitro and suppressed the intracranial orthotopic xenograft growth and prolonged the survival of xenograft mice in vivo. Moreover, LGR5 was positively correlated with Ki67, N-cadherin and WHO grade and negatively correlated with IDH1. Glioma patients with high expression of LGR5 showed significantly poorer prognosis. LGR5 is a new functional GSC marker and prognostic indicator that can promote EMT by activating the Wnt/β-catenin pathway and would thus be a novel therapeutic target for glioma.

Background: Identification of promising targeted antigens that exhibited cancer-specific expression is a crucial step in the development of novel antibody-targeted therapies. We here aimed to investigate the anti-tumor activity of a novel monoclonal antibody (mAb) 11C9 and identify the antibody tractable target in the hepatocellular cancer stem cells (HCSCs). Methods: The identification of the targeted antigen was conducted using SDS-PAGE, western blot, mass spectrometry, and co-immunoprecipitation. Silence of HSP90 was induced by siRNA interference. Positive cells were sorted by fluorescence-activated cell sorting. Double-immunofluorescent (IF) staining and two-color flow cytometry detected the co-expression. Self-renewal, invasion, and drug resistance were assessed by sphere formation, matrigel-coated Transwell assay, and CCK-8 assay, respectively. Tumorigenicity was evaluated in mouse xenograft models. RNA-seq and bioinformatics analysis were performed to explore the mechanism of mAb 11C9 and potential targets. Results: MAb 11C9 inhibited invasion and self-renewal abilities of HCC cell lines and reversed the cisplatin resistance. HSP90 (~ 95 kDa) was identified as a targeted antigen of mAb 11C9. Tissue microarrays and online databases revealed that HSP90 was overexpressed in HCC and associated with a poor prognosis. FACS and double-IF staining showed the co-expression of HSP90 and CSCs markers (CD90 and ESA). In vitro and in vivo demonstrated the tumorigenic potentials of HSP90. The inhibition of HSP90 by siRNA interference or 17-AAG inhibitor both decreased the number of invasion, sphere cells, and CD90⁺ or ESA⁺ cells, as well as reversed the resistance. Bioinformatics analysis and western blot verified that HSP90 activated Wnt/β-catenin signaling. Conclusions: The study preliminarily revealed the anti-tumor activity of mAb 11C9. More importantly, we identified HSP90 as a targeted antigen of mAb 11C9, which functions as an oncogene in phenotype shaping, stemness maintenance, and therapeutic resistance by activating Wnt/β-catenin signaling.

Resistance to treatment is a critical factor contributing to poor prognosis in gastric cancer patients. HSP90 has emerged as a promising therapeutic target; however, its role in regulating tumor metabolic pathways, particularly glycolysis, remains poorly understood, which limits its clinical application. We identified proteins that directly interact with HSP90 using immunoprecipitation (IP) followed by mass spectrometry. The relationship between HSP90 and glycolysis was further investigated through transcriptomic analyses and in vitro experiments. Mechanistic insights were obtained through mass spectrometry, co-immunoprecipitation (Co-IP) assays, drug sensitivity tests, and bioinformatics analyses. Additionally, we developed a scoring system based on transcriptomic data to evaluate its prognostic significance and association with treatment resistance in gastric cancer patients. Our multi-omics and in vitro studies revealed that HSP90 regulates glycolysis and influences the stemness properties of gastric cancer cells. Mechanistically, HSP90 facilitates the assembly of a glycolytic multi-enzyme complex, termed the HGEO complex, which enhances glycolytic metabolism. Mechanistically, HSP90 facilitates the formation of a multienzyme complex comprising key enzymes including PGK1, PKM2, ENO1, and LDHA, thereby facilitating the production of the final glycolytic products. We refer to this as the "HSP90-Glycolytic Output Complex" (HGEO Complex). We quantified this phenomenon with a scoring system (HGScore), finding that patients with a high HGScore exhibited more malignant signatures, increased resistance to treatment, and poorer prognoses. Furthermore, we demonstrated that the HGEO complex is localized in the nucleus, regulated by the nuclear lamina protein LMNA, which further contributes to treatment resistance and adverse outcomes. In vitro experiments indicated that inhibiting the formation of this complex sensitizes gastric cancer cells to chemotherapy. Our findings suggest that HSP90 and LMNA mediated the formation and nuclear localization of the HGEO complex, thereby enhancing the malignant traits and resistance mechanisms in gastric cancer. Targeting this pathway may offer a novel therapeutic strategy to improve treatment outcomes.