Loss of protein phosphatase 6 confers resistance to MAP kinase pathway inhibition in KRAS and BRAF mutant cells via NF-κB activation

To understand the global impact of PPP6C loss on gene expression, we carried out RNA-seq analysis in parental and PPP6C KO SW620 cells under 2D and 3D culture conditions. SW620 WT (sgNT) and PPP6C KO (sgPPP6C#2, sgPPP6C#3) were cultured in normal petri dish (2D culture) or U-bottom plates (3D culture) for 3 days with three independent replicates for each cell sample. A total of 4,337 genes were found to be differentially expressed in the KO cells under at least one condition (FDR < 0.05, ≥ 2-fold change). Among these, 1,123 genes were significant up- or down-regulated in PPP6C KO cells under both 2D and 3D conditions. Unsupervised clustering analysis identified three large clusters of gene expression patterns: cluster 1 were upregulated in the PPP6C KO cells primarily under 2D conditions, cluster 2 were upregulated in the PPP6C KO cells under both 2D and 3D conditions, and cluster 3 gene were downregulated genes in the PPP6C KO cells under both 2D and 2D conditions. We next carried out gene set enrichment analysis (GSEA) using the hallmark gene set database to identified pathways that were significantly altered in PPP6C KO cells. Among the pathways upregulated in the PPP6C KO cells under both 2D and 3D conditions, the “TNFA-signaling_via_NFKB” gene set scored as the top one. Both “KRAS_signaling_up” and “PI3K_AKT_mTOR_signaling” gene sets were also significantly upregulated. PPP6C KO has opposite effects on cell proliferation in 2D monolayer vs. 3D tumor spheroid culture. We noticed that 383 genes in the PPP6C KO cells showed opposite changes in 2D vs. 3D conditions. Taken together, gene expression analysis indicates that multiple signaling pathways including the KRAS, PI3K and NF-κB pathways were perturbed in the in PPP6C KO cells and they could contribute to the cell’s decreased sensitivity to MEK inhibitors.