Systems Immunology Analysis Reveals an Immunomodulatory Effect of Snail-p53 Binding on Neutrophil- and T Cell-Mediated Immunity in KRAS Mutant Non-Small Cell Lung Cancer

Abstract

Immunomodulation and chronic inflammation are important mechanisms utilized by cancer cells to evade the immune defense and promote tumor progression. Therefore, various efforts were focused on the development of approaches to reprogram the immune response to increase the immune detection of cancer cells and enhance patient response to various types of therapy. A number of regulatory proteins were investigated and proposed as potential targets for immunomodulatory therapeutic approaches including p53 and Snail. In this study, we investigated the immunomodulatory effect of disrupting Snail-p53 binding induced by the oncogenic KRAS to suppress p53 signaling. We analyzed the transcriptomic profile mediated by Snail-p53 binding inhibitor GN25 in non-small cell lung cancer cells (A549) using Next generation whole RNA-sequencing. Notably, we observed a significant enrichment in transcripts involved in immune response pathways especially those contributing to neutrophil (IL8) and T-cell mediated immunity (BCL6, and CD81). Moreover, transcripts associated with NF-κB signaling were also enriched which may play an important role in the immunomodulatory effect of Snail-p53 binding. Further analysis revealed that the immune expression signature of GN25 overlaps with the signature of other therapeutic compounds known to exhibit immunomodulatory effects validating the immunomodulatory potential of targeting Snail-p53 binding. The effects of GN25 on the immune response pathways suggest that targeting Snail-p53 binding might be a potentially effective therapeutic strategy.

Keywords: T-cell mediated anticancer immunity; immunomodulation; neutrophil-mediated anticancer immunity; non-small cell lung cancer; snail-p53 binding; systems immunology; tumor sensitization.

Figure 1

Validation of Snail inhibition and p53 restoration in GN25 treated cells. The top 20 enriched pathways and functional clusters in (A) the upregulated genes and (B) downregulated genes in the GN25 treated cells analyzed using Metascape annotation tool (C). Top 20 pathways enriched in the differentially expressed genes in GN25 treated cell (both up- and down-regulated genes) using IPA. Green arrows indicate cell cycle related pathways; black arrows indicated cell morphogenesis related pathways; and black arrows indicate immune response related pathways.

Figure 2

Immunomodulatory effect of Snail-p53 binding inhibition. Enriched representation of the top 20 immune response pathways and functional clusters identified in analysis conducted using (A) Metascape and (B) IPA. The count of intersecting genes from the GN25 treated cells with the (C) Immunome set (D), immune cells activation gene ontology sets, and (E) immune cells chemotaxis gene ontology sets.

Figure 3

Most significantly enriched immune pathways within the immunomodulatory signature of GN25 (A). The differentially expressed genes (Fold changes cut-off >2 and <0.5) enriched in the gene ontology data sets for the activation and chemotaxis of each immune cell type (B). Pathway analysis of Differentially expressed genes enriched in the Immunome and activation/chemotaxis Gene ontology sets of each immune cell type. Pathway analysis done using Metascape (C). Representative FACS plots showing the percent of CFSE+ CD3+ cells co-culture with A549 cells treated with 5 and 10 µM GN25 as well as vehicle control (D). dotplot representation of mean percentage ± SEM of CD3+ cells proliferation in response to co-culture with A549 cells treated with 5 and 10 µM GN25 as well as vehicle control. Data shown are from four healthy individuals. * represents p-value <0.05.

Figure 4

String functional protein association networks analysis in all the differentially expressed genes (FC >2 or <0.5) enriched in the immune gene ontology sets. Red arrows indicate elements of our target molecules, p53 and Snail; black arrows indicate potential regulatory nodes induced by Snail-p53 binding to modulate immune response pathways.

Figure 5

Immune genes enrichment in the top differentially expressed genes. heatmap representation of the log2 Read counts of the (A) top 20 upregulated genes and the (B) top 20 downregulated genes in GN25 treated cells in comparison to the vehicle negative control. Green arrows indicate genes that intersect with the differentially expressed genes enriched in the immune gene ontology gene sets (C). Gene expression validation using qRT-PCR of immune response genes identified in the RNA-seq analysis including IL8, CXCL2, and DUSP1. *** represents p-value <0.005.