Transcriptomic analysis of glucosidase II beta subunit (GluIIß) knockout A549 cells reveals its roles in regulation of cell adhesion molecules (CAMs) and anti-tumor immunity.

Glucosidase II beta subunit (GluIIß), encoded from PRKCSH, is a subunit of the glucosidase II enzyme responsible for quality control of N-linked glycoprotein folding and suppression of GluIIß led to inhibitory effect of the receptor tyrosine kinase (RTKs) activities known to be critical for survival and development of cancer. In this study, we investigated the effect of GluIIß knockout on the global gene expression of cancer cells and its impact on functions of immune cells. GluIIß knockout lung adenocarcinoma A549 cell line was generated using CRISPR/Cas9-based genome editing system and subjected to transcriptomic analysis. Among 23,502 expressed transcripts, 1068 genes were significantly up-regulated and 807 genes greatly down-regulated. The KEGG enrichment analysis showed significant down-regulation of genes related extracellular matrix (ECM), ECM-receptor interaction, cytokine-cytokine receptor interaction and cell adhesion molecules (CAMs) in GluIIß knockout cells. Of 9 CAMs encoded DEG identified by KEGG enrichment analysis, real time RT-PCR confirmed 8 genes to be significantly down-regulated in all 3 different GluIIß knockout clones, which includes cadherin 4 (CDH4), cadherin 2 (CDH2), versican (VCAN), integrin subunit alpha 4 (ITGA4), endothelial cell-selective adhesion molecule (ESAM), CD274 (program death ligand-1 (PD-L1)), Cell Adhesion Molecule 1 (CADM1), and Nectin Cell Adhesion Molecule 3 (NECTIN3). Whereas PTPRF (Protein Tyrosine Phosphatase Receptor Type F) was significantly decreased only in 1 out of 3 knockout clones. Microscopic analysis revealed distinctively different cell morphology of GluIIß knockout cells with lesser cytoplasmic and cell surface area compared to parental A549 cells and non-targeted transfected cells.Further investigations revealed that Jurkat E6.1 T cells or human peripheral blood mononuclear cells (PBMCs) co-cultured with GluIIß knockout A549 exhibited significantly increased viability and tumor cell killing activity compared to those co-cultured with non-target transfected cells. Analysis of cytokine released from Jurkat E6.1 T cells co-cultured with GluIIß knockout A549 cells showed significant increased level of angiogenin and significant decreased level of ENA-78. In conclusion, knockout of GluIIß from cancer cells induced altered gene expression profile that improved anti-tumor activities of co-cultured T lymphocytes and PBMCs thus suppression of GluIIß may represent a novel approach of boosting anti-tumor immunity.

Navigating PRKCSH's impact on cancer: from N-linked glycosylation to death pathway and anti-tumor immunity.

PRKCSH, also known as Glucosidase II beta subunit (GluIIß), is a crucial component of the endoplasmic reticulum (ER) quality control system for N-linked glycosylation, essential for identifying and eliminating misfolded proteins. Glucosidase II consists of the catalytic alpha subunit (GluIIα) and the regulatory beta subunit (GluIIß), ensuring proper protein folding and release from the ER. The induction of PRKCSH in cancer and its interaction with various cellular components suggest broader roles beyond its previously known functions. Mutations in the PRKCSH gene are linked to autosomal dominant polycystic liver disease (ADPLD). Alternative splicing generates distinct PRKCSH isoforms, which can influence processes like epithelial-mesenchymal transition (EMT) and the proliferation of lung cancer cells. PRKCSH's involvement in cancer is multifaceted, impacting cell growth, metastasis, and response to growth factors. Additionally, PRKCSH orchestrates cell death programs, affecting both autophagy and apoptosis. Its role in facilitating N-linked glycoprotein release from the ER is hypothesized to assist cancer cells in managing increased demand and ER stress. Moreover, PRKCSH modulates anti-tumor immunity, with its suppression augmenting NK cell and T cell activity, promising enhanced cancer therapy. PRKCSH's diverse functions, including regulation of IGF1R and IRE1α, implicate it as a therapeutic target and biomarker in cancer immunotherapy. However, targeting its glucosidase II activity alone may not fully counteract its effects, suggesting broader mechanisms in cancer development. Further investigations are needed to elucidate PRKCSH's precise role and validate its therapeutic potential in cancer treatment.

Knockout of glucosidase II beta subunit inhibits growth and metastatic potential of lung cancer cells by inhibiting receptor tyrosine kinase activities.

Glucosidase II (GluII) plays a major role in regulating post-translation modification of N-linked glycoproteins. We have previously reported that the expression of glucosidase II beta subunit (GluIIß) was significantly increased in lung tumor tissues and its suppression triggers autophagy and/or apoptosis. Here, we investigated the role of GluIIß in cell growth, metastatic potential, and receptor tyrosine kinases (RTKs) signaling activity in lung carcinoma cell lines. CRISPR-CAS9 technology was used to knockout the GluIIß encoding gene (PRKSH) in lung carcinoma cells. GluIIß knockout cells exhibited drastically slower growth rates in comparison to non-target transfected cells, particularly with lower concentrations of fetal bovine serum, indicating impairment of their ability to survive under nutritional deprivation. Cell migration and anchorage-independent growth, the fundamental components of cancer cell metastasis, were significantly decreased in GluIIß knockout cells. Knockout of GluIIß increased the sensitivity of lung cancer cells to cisplatin but reduced their sensitivity to gefitinib. Interestingly, knocking out of GluIIß lowered overall RTK signaling activities to less than half of those in non-target transfected cells, which could represent a novel strategy for blocking multiple RTKs in tumor cells in an effort to improve lung cancer treatment.

Glucosidase II beta subunit (GluIIß) plays a role in autophagy and apoptosis regulation in lung carcinoma cells in a p53-dependent manner.

PURPOSE: Glucosidase II plays a major role in regulating the post-translational modification of N-linked glycoproteins. Previously, we found that the beta subunit of glucosidase II (GluIIß) levels are significantly increased in lung carcinoma tissues, indicating a potential role in lung tumorigenesis. Here, we investigated the role of GluIIß in the regulation of autophagy and apoptosis in lung carcinoma- and immortalized human bronchial epithelial-derived cells. METHODS: A selective glucosidase II inhibitor, bromoconduritol, was used to inhibit GluII enzyme activity and a siRNA-based technology was used to suppress the expression of the GluIIß encoding gene PRKCSH in lung carcinoma cells differing in p53 status. Cell viability was assessed using a MTT assay, cell cycle progression was assessed using flow cytometry, autophagy was assessed using Western blotting and apoptosis was assessed using an annexin V-FITC/PI double labeling method. RESULTS: We found that GluIIß inhibition resulted in the induction of autophagy in all cell lines tested, but apoptosis in only wild-type p53 cells. We also found that GluIIß inhibition dose-dependently decreased activation of the EGFR/RTK and PI3K/AKT signaling pathways. Although the apoptosis inducing effect of GluIIß inhibition appeared to be p53-dependent, we found that a combined treatment with lysosomal inhibitors to block autophagy enhanced the apoptotic effect of GluIIß inhibition in both wild-type p53 and p53-null cells. CONCLUSIONS: Our data indicate that GluIIß inhibition results in autophagy and apoptosis in lung carcinoma-derived cells, supporting the hypothesis that this enzyme may play a role in blocking these two tumor suppressive processes. Since blocking autophagy by lysosomal inhibitors enhanced the apoptosis-inducing effect of bromoconduritol, independent of p53 status, their combined use may hold promise for the treatment of cancer, particularly lung cancer.