Prostate cancer (PCa) is a type of potentially fatal malignant tumor. Immunotherapy has shown a lot of potential for various types of solid tumors, but the benefits have been less impressive in PCa. Enhancer of zeste homolog 2 (EZH2) is one of the three core subunits of the polycomb repressive complex 2 that has histone methyltransferase activity, and the immune effects of EZH2 in PCa are still unclear. The purpose of this study was to explore the potential of EZH2 as a prognostic factor and an immune therapeutic biomarker for PCa, as well as the expression pattern and biological functions. All analyses in this study were based on publicly available databases, mainly containing Cancer Genome Atlas (TCGA), Gene Expression Omnibus (GEO), UCSCXenaShiny, and TISIDB. We performed differential expression analysis, developed a prognostic model, and explored potential associations between EZH2 and DNA methylation modifications, tumor microenvironment (TME), immune-related genes, tumor mutation burden (TMB), tumor neoantigen burden (TNB), and representative mismatch repair (MMR) genes. We also investigated the molecular and immunological characterizations of EZH2. Finally, we predicted immunotherapeutic responses based on EZH2 expression levels. We found that EZH2 was highly expressed in PCa, was associated with a poor prognosis, and may serve as an independent prognostic factor. EZH2 expression in PCa was associated with DNA methylation modifications, TME, immune-related genes, TMB, TNB, and MMR. By gene set enrichment analysis and gene set variation analysis, we found that multiple functions and pathways related to tumorigenesis, progression, and immune activation were enriched. Finally, we inferred that immunotherapy may be more effective for PCa patients with low EZH2 expression. In conclusion, our study showed that EZH2 could be a potentially efficient predictor of prognosis and immune response in PCa patients.

Objectives: Cancer cells with ‘stemness’ are generally resistant to chemoradiotherapy. This study aims to compare the differences in radiation sensitivity of A549 and CD44⁺A549 stem-like cells to X-rays and carbon ion radiation (C-ions), and to find a target that can kill cancer stem-like cells (CSCs) of non-small cell lung cancer (NSCLC). Methods: The study used two cell lines (A549 and CD44⁺A549). The tumorigenicity of cells was tested with animal experiments. The cells were irradiated with X-rays and C-ions. Cell viability was detected using the CCK-8 and EdU assay. A liquid chromatograph-mass spectrometer (LC–MS) helped detect metabolic differences. Protein and mRNA expression were detected using a Western blot, reverse transcription-quantitative (RT-qPCR), and PCR array. The autophagic activity was monitored with a CYTO-ID^® Autophagy Detection Kit 2.0. Immunofluorescence and co-immunoprecipitation helped to observe the localization and interaction relationships. Results: First, we verified the radio-resistance of CD44⁺A549 stem-like cells. LC-MS indicated the difference in autophagy between the two cells, followed by establishing a correlation between the radio-resistance and autophagy. Subsequently, the PCR array proved that TGM2 is significantly upregulated in CD44⁺A549 stem-like cells. Moreover, the TGM2 knockdown by small interfering RNA could decrease the radio-resistance of CD44⁺A549 cells. Bioinformatic analyses and experiments showed that TGM2 is correlated with the expression of CD44 and LC3B. Additionally, TGM2 could directly interact with LC3B. Conclusions: We established the CD44-TGM2-LC3 axis: CD44 mediates radio-resistance of CD44⁺A549 stem-like cells through TGM2 regulation of autophagy. Our study may provide new biomarkers and strategies to alleviate the radio-resistance of CSCs in NSCLC.

The combination of carbon ion radiotherapy and anti-PD-1 antibody represents a new approach to treating thoracic tumors. However, the lung damage caused by this combination therapy may limit its use, and the potential mechanisms for this are worthy of investigation. The objective of this research was to examine the potential involvement of repulsive guidance molecule b (RGMb) in lung damage promoted by the utilization of carbon ion irradiation combined with an anti-PD-1 antibody. The C57BL/6 mice have been randomly separated into four distinct groups: control, anti-PD-1, whole thorax carbon ion irradiation, and irradiation in combination with anti-PD-1 treatment groups (combination group). Detection of pathological changes in lung tissue using HE staining. Detection of pulmonary fibrosis by Masson staining and the hydroxyproline assay. ELISA to detect TNF-α, TGF-β, IL-6, and IL-1β expression levels within lung homogenates. The expression of RGMb, p38 MAPK, and Erk1/2 pathways was detected using a fully automated digital Western blotting system WES (ProteinSimple, USA). Flow cytometry was employed to analyze tissue-resident memory T cells (TRM) within the lung. Subsequently, the siRNA gene was employed to induce the downregulation of RGMb in mice in order to validate the involvement of RGMb in radiation-immune lung injury. The present study observed a significant increase in both inflammatory and fibrotic indicators within the mice group's lung tissue that received the combination treatment. The combination group exhibited elevated levels of TGF-β, TNF-α, IL-6, and IL-1β in lung homogenates. Anti-PD-1 antibody and carbon ion irradiation, upregulated RGMb, phospho-p38 MAPK and phospho-Erk1/2. The results obtained from the flow cytometry analysis indicated that the combination group was significantly higher in the number of clonal expansion TRMs, which were predominantly characterized by the expression of CD8+CD103+CD69-TRMs. The downregulate of RGMb via siRNA in mice resulted in a decrease in phospho-p38 MAPK and phospho-Erk1/2. The combination group exhibited a reduction in TNF-α, TGF-β, IL-6, and IL-1β in their lung tissues, and the number of CD8+CD103+CD69-TRM was significantly reduced. The combination group exhibited a significant improvement in inflammatory and fibrotic indicators within the lung tissues. Anti-PD-1 antibody and carbon ion irradiation synergistically regulate RGMb, leading to strong clonal expansion of lung TRM through the p38 MAPK and Erk1/2 pathways. The present study offers valuable insights into the treatment of lung injury due to the combined administration of carbon ion radiotherapy and anti-PD-1 antibody therapy.