Roles of glucosidase ii in lung cancer cells and its involvement in stimulation of immune response

Abstract

Glucosidase II beta subunit (GluIIβ), encoded by the PRKCSH gene, is an endoplasmic reticulum-resident protein that functions as the beta subunit of glucosidase II. This enzyme plays a crucial role in regulating the post-translation modification of N-linked glycoproteins. Previous studies by our group have shown that suppression of GluIIβ induced autophagy and/or apoptosis, leading to impaired growth behaviors in lung carcinoma-derived cells. This study aimed to investigate the transcriptome expression profile and the impact on immune responses against cancer cells following GluIIβ knockout and in lung carcinoma cell lines. A CRISPR-Cas9 technology was used to knockout the GluIIβ encoding gene (PRKCSH) in lung carcinoma cell lines with different p53 status (wild-type A549 cells and null genotype H1299 cells).The expression profile of genes within non-target transfected cells and GluIIβ knockout cells were analyzed by RNA sequencing. Subsequently, clusterProfiler program was employed for GO (Gene Ontology) KEGG (Kyoto Encyclopedia of Genes and Genomes), and Reactome pathway analyses to define the activities of genes exhibiting notable alterations in response to GluIIβ knockout. Real-time quantitative RT-PCR was employed to validate the RNA sequencing data. The growth behavior of established GluIIβ knockout clones was evaluated using the MTT assay, comparing them to parental cells and non-target transfected controls. Additionally, the influence of GluIIβ suppression on ferroptosis was examined through Western blot analysis. Furthermore, the effects of GluIIβ knockout cancer cells on the survival, proliferation, and function of immune cells were also assessed. Western blot analysis confirmed that GluIIβ encoding gene was successfully knockout from A549 and H1299 cells in the established clones. Profiling the transcriptomics expression compared between non-target transfected cells and GluIIβ knockout A549 cells showed that, of the 23,502 transcripts that were expressed, 1,068 genes were significantly up-regulated, and 807 genes were significantly down-regulated. The KEGG enrichment analysis showed significant down-regulation of genes related to extracellular matrix (ECM), ECM-receptor interaction, cytokines-cytokines receptor interaction and cell adhesion molecules (CAMs) in GluIIβ knockout cells. However, this study was directed towards a specific set of genes encoding cell adhesion molecules (CAMs). Among these genes, nine were selected for verification via quantitative realtime RT-PCR including gene encoded for program death-ligand-1 (PD-L1), a widely recognized CAM that plays critical roles in controlling anti-tumor immunity. The RT-PCR results confirmed significant down-regulation of eight genes in all three different GluIIβ knockout A549 clones, while one gene showed significant down-regulation in only one out of the three knockout clones. These data confirm the reliability of RNA sequencing data. Characterizing the growth behavior of GluIIβ knockout cells revealed their significantly slower growth rate compared to control cells. Furthermore, suppressing GluIIβ induced ferroptosis in wild-type p53 carrying cells (A549), but not in p53-null cell line (H1299), indicating the involvement of p53 in the regulation of ferroptosis cell death influenced by GluIIβ. Comparing Jurkat T cells co-cultured with GluIIβ knockout cells to those co-cultured with non-target transfected cells, the viability of Jurkat T cells was significantly higher upon stimulation with GluIIβ knockout A549 cells. Moreover, the capacity of Jurkat T cells and PBMCs to lyse tumors was significantly greater when stimulated with GluIIβ knockout cells. Additionally, cytokine array and real-time RT-PCR analysis showed a substantial increase in angiogenin levels and a significant decrease in ENA-78 levels, indicating that GluIIβ may influence cytokine secretion from immune and cancer cells. In summary, the results from this thesis indicate that the knockout of GluIIβ from cancer cells resulted in a changed gene expression profile that enhanced the antitumor activities of co-cultured PBMCs and T cells. This suggests that GluIIβ suppression might be an innovative approach to boost immune response against lung cancer and perhaps other malignancies. However, further research is needed to comprehend the underlying mechanisms of GluIIβ function in cancer treatment and tumorigenesis.