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

Abstract

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 GluIIβ 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.

Keywords: Cell adhesion molecules (CAMs); Glucosidase II beta subunit; Non-small cell lung cancers (NSCLCs); PRKCSH; T cell; Transcriptomic analysis.

Fig. 1

Differential expression, Gene Ontology (GO), KEGG and Reactome analysis compared between GluIIß knockout cells (KO) and non-target transfected cells (control). Western blot analysis showing GluIIß expression levels in GluIIß KO and non-target transfected cells (A). Volcano plots (B) showing the overall distribution of differentially expressed genes. GO Enrichment analysis histogram showing top 30 significantly (padj <0.05) affected pathways according to major categories of biological processes (BP), cell components (CC), molecular functions (MF) (C) and according to categories of up and down expressed genes (D) in response to GluIIß knockout. KEGG enrichment analysis histogram (E), scatter plot (F) and Reactome enrichment analysis histogram (G) and scatter plot (H) showing pathways significantly (padj <0.05) affected by GluIIß knockout

Fig. 2

Quantitative RT-PCR validation of significant changes demonstrated by NGS data. 9 genes encoded for CAMs were chosen for verification and 8 genes were confirmed to be significantly decreased in all 3 GluIIß knockout clones (PD-L1, VCAN, CDH2, CDH4, ITGA4, ESAM, NECTIN3, CADM1) while PTPRF was significantly decreased only in 1 out of 3 clones compared to non-target transfected cells. Further investigation on the effect of GluIIß knockout on PD-L2 also demonstrated the significant impact on the down regulation of PD-L2

Fig. 3

Phase-contrast microscopy (A) and crystal violet staining (B) images at 40x magnification of GluIIß KO cells (KO1, KO2, KO6) compared to parental (A549) and non-target transfected cells (NT)

Fig. 4

Fluorescence images showing the green fluorescence (GFP) expressing cells, GluIIß KO A549 cells and non-target transfected cells, surrounded by Jurkat E6.1 T cells in the co-culture experiment (A). Viability and proliferation of Jurkat E6.1 T cells after being exposed to GluIIß KO A549 cells or non-target control cells for 24 hours as measured at 0, 12, 24, 48 and 72 hours using alamarBlue^® (B). Cancer cell lysing activity of Jurkat E6.1 T cells and PBMCs co-cultured with GluIIß KO A549 cells compared to those co-cultured with non-target transfected cells (C). Bar graphs represent means and standard deviations (SDs) from three independent experiments. *Significant different from those co-cultured with non-target transfected cells (p<0.05 by Mann Whitney U test)

Fig. 5

Analysis of secreted cytokines in media of Jurkat E6.1 cells co-cultured with GluIIß knockout cells compared to those co-cultured with non-target transfected cells. Image of cytokine array signals containing 42 different anti-cytokine antibodies spotted in duplicate, including 3 positive and 2 negative controls, hybridized with co-culture media (A). Array signals were quantified using “ImageJ software with the Protein Array Analyzer plugin16”. Values of quantified cytokine signals from duplicate spots were averaged and plotted as scatter plot (B) with mean values from Jurkat E6.1 cells co-cultured control cells as X-axis and those from Jurkat E6.1 cells co-cultured with KO cells as Y-axis. Mean and standard deviation of quantified signals of each cytokine were plotted as bar graph (C) Verification of angiogenin and ENA-7expression level by real time RT-PCR (D) *Significant different from those co-cultured with non-target transfected cells (p<0.05 by Mann Whitney U test)