Table 1 Hallmarks of erlotinib resistance in EGFR-mutant cell lines.
Cholesterol metabolism | • Enzymes of almost the whole pathway, from acetoacetyl coenzyme A transferase (ACAT2) and CYP51A1 all of the way down to cholesterol, were expressed at gene level relatively lower in DT cells, together with the low-density lipoprotein receptor (LDLR) responsible for cholesterol uptake, STARD4, involved in intracellular cholesterol transport, the sterol-sensing protein INSIG1, and sterol response element-binding proteins (SREBPs) SREBF-1 and 2, while the ATP-binding cassette A1 (ABCA1) involved in lipid efflux from cells, was upregulated. • The SREBP targets FASN, FDFT1, FDPS, HMGCS1, HSD17B7, IDI1, INSIG1, LDLR, PCSK9, RDH11, SQLE, STARD4, which are directly involved in cholesterol biosynthesis128, were downregulated in all DT clusters. • Biosynthesis of unsaturated fatty acids that involves regulation through PPAR-γ signaling pathway (ACSL1, ACSL3, ACSL4, FADS1, FADS2, ME1, SCD) is likely to be impaired in DT clusters (P = 5.4 × 10−12). |
EMT | • Among the transcription factors that regulate EMT56, SOX4, SLUG/SNAI2, and GATA6 were highly increased in DT cells. Analysis for enrichment of TF-binding sites identified lymphoid enhancer-binding factor 1 (LEF1/TCF7L1) that favors EMT and is downstream of WNT/β‐catenin. • The TGFB2 marker expression correlated with genes involved in protein secretion and increased activity of promoters occupied by SMAD2 or SMAD3129 (73 promoters in Cluster 4 and 68 promoters in Cluster 5, Supplementary Fig. 7c), indicating SMAD pathway activation130,131. There was an increase in the transactivator for TGF-β-dependent transcription CITED2. • IGFBP3, the main carrier protein for insulin-like growth factors (IGF), along with the homologous protein IGFBP5, were increased. • Features of senescence associated secretory phenotype (SASP)132 included growth arrest and secretion of extracellular matrix proteins such as fibronectin FN1 and the CCN family of matricellular ligand CYR61/CCN1, which binds to integrins and has been linked to chemotherapy resistance, and proteases such as serpin E1 SERPINE1/PAI125. SASP initiated stimulator of interferon-γ (IFN-γ) genes (STING) response, enhancing the production of chemokines and type I interferons. • Many markers functionally belonged to vesicle-mediated transport. • Genes encoding cytokines and associated proteins such as SPARC were highly increased. SPARC regulates cell growth through interactions with the extracellular matrix oxidized low density lipoprotein (oxLDL) receptor 1 OLR1. Its association with oxLDL induces the activation of NF-κB. • Focal adhesion pathway (FN1, COL5A1, MYL12A, PDGFC, TNC, IGF1R, COL4A4, SHC3, PXN, CAV1, CAV2, LAMC1, ITGA4, MET, FYN) or apical junction complex (ACTB, ACTN1, ACTN4, ACTG2, MYL9, RSU1, NEXN, LIMA1, ZYX, VCL, CNN2, MYH9) were characteristics of METhigh and TPMhigh modules. Figs. 2d, 3b, 4c, 5d, e, 6c, f, and 7b, d and Supplementary Fig. 7b–f, 9c, d, h, and 14f |
Epithelium development | • Enrichment of GO terms related to tissue development, multicellular organismal development and CGP gene sets related to lung epithelium differentiation133. • Upregulation of TFs such as THBS1, FOXJ1/HFH4, JUN, KLF9 and NFIB that influence epithelial cell growth and differentiation134. • A shift towards the GATA6-high program and repression of putative markers of distal airway epithelium STEAP1, GPR87, vascular and ECM remodeling genes VEGFA and PLAU, and cytokeratins KRT6A and KRT6B135. Figs. 2d, 3b, 4c, 6c, d, and 7b, d and Supplementary Fig. 7b, c. |
Drug metabolism | • TF-binding site analysis predicted activation of nuclear factor of activated T cells (NFAT), which is indicative of the imbalance in superoxide/hydrogen peroxide. • Upregulation of the ROS genes GPX4 and PDLIM1 across all DT clusters. • Upregulation of targets downstream of PI3K/AKT/mTOR, SQSTM1, and AP2M1, which may trigger synthesis of detoxifying enzymes that prevent oxidative stress. SLC3A2 in complex with SLC7A5 imports essential amino acids and promotes mTORC1 activity136. SLC3A2 and SLC7A5 were both upregulated, which may be important in managing erlotinib-induced ROS, as it was accompanied by an increase in the high-affinity glutamine transporter SLC1A5 and SLC7A11, whose products form the cystine transporter complex with SLC3A2, and are required for glutathione synthesis. • Enrichment for drug metabolism through cytochrome P450, represented by GSTK1, GSTM3, GSTM4, MGST3, ALDH1A3, ALDH3A1, ALDH3B1, and CYP1B1. Figs. 2d, 4c, 6c, e, and 7b, d, and Supplementary Fig. 7b, d, f |
Epigenetic regulation | • Enrichment of upregulated EZH2 targets (e.g., P < 10−16, 124 genes137). • Enrichment of upregulated targets of class I and II HDACs (e.g., P < 10−16, 71 genes138). • Enrichment of gene sets associated with inhibition of DNA methylation (e.g., P < 10−16, 37 genes139). The P values are provided from data in Fig. 4c. See also Figs. 3c, and 6d, and Supplementary Fig. 14e, l. |
