Mutational activation of the epidermal growth factor receptor down-regulates major histocompatibility complex class I expression via the extracellular signal-regulated kinase in non-small cell lung cancer.
ABSTRACT: The efficacy of programmed cell death-1 (PD-1) blockade in patients with non-small cell lung cancer (NSCLC) positive for epidermal growth factor receptor (EGFR) gene mutations has been found to be limited, but the underlying mechanisms for this poor response have remained obscure. Given that the recognition by T cells of tumor antigens presented by major histocompatibility complex class I (MHC-I) molecules is essential for an antitumor immune response, we examined the effects of EGFR tyrosine kinase inhibitors (TKIs) on MHC-I expression in NSCLC cell lines. Appropriate EGFR-TKIs increased MHC-I expression at the mRNA and cell surface protein levels in NSCLC cells positive for EGFR mutations including those with the T790M secondary mutation. Trametinib, an inhibitor of the extracellular signal-regulated kinase (ERK) kinase MEK, also increased MHC-I expression, whereas the phosphatidylinositol 3-kinase (PI3K) inhibitor buparlisib did not, suggesting that the MEK-ERK pathway mediates the down-regulation of MHC-I expression in response to EGFR activation. Immunohistochemical analysis of EGFR-mutated NSCLC specimens obtained before and after EGFR-TKI treatment also revealed down-regulation of phosphorylated forms of EGFR and ERK in association with up-regulation of MHC-I, an increased number of infiltrating CD8+ T cells, and increased PD-1 ligand 1 expression after such treatment. Our results thus suggest that mutational activation of EGFR inhibits MHC-I expression through the MEK-ERK pathway in NSCLC and thereby contributes to the poor response of such tumors to immunotherapy. Further studies are warranted to evaluate the relation between EGFR-MEK-ERK signaling in and the immune response to EGFR-mutated NSCLC.?.
Project description:Lung adenocarcinomas harboring activating mutations in the epidermal growth factor receptor (EGFR) represent a common molecular subset of non-small cell lung cancer (NSCLC) cases. EGFR mutations predict sensitivity to EGFR tyrosine kinase inhibitors (TKIs) and thus represent a dependency in NSCLCs harboring these alterations, but the genetic basis of EGFR dependence is not fully understood. Here, we applied an unbiased, ORF-based screen to identify genetic modifiers of EGFR dependence in EGFR-mutant NSCLC cells. This approach identified 18 kinase and kinase-related genes whose overexpression can substitute for EGFR in EGFR-dependent PC9 cells, and these genes include seven of nine Src family kinase genes, FGFR1, FGFR2, ITK, NTRK1, NTRK2, MOS, MST1R, and RAF1. A subset of these genes can complement loss of EGFR activity across multiple EGFR-dependent models. Unbiased gene-expression profiling of cells overexpressing EGFR bypass genes, together with targeted validation studies, reveals EGFR-independent activation of the MEK-ERK and phosphoinositide 3-kinase (PI3K)-AKT pathways. Combined inhibition of PI3K-mTOR and MEK restores EGFR dependence in cells expressing each of the 18 EGFR bypass genes. Together, these data uncover a broad spectrum of kinases capable of overcoming dependence on EGFR and underscore their convergence on the PI3K-AKT and MEK-ERK signaling axes in sustaining EGFR-independent survival.
Project description:Several randomized trials have demonstrated non-small cell lung cancer (NSCLC) patients with activating epidermal growth factor receptor (EGFR) mutations can achieve favorable clinical outcomes on treatment with EGFR tyrosine kinase inhibitors (TKIs). EGFR mutation is considered as a predictive marker for efficacy of EGFR-TKIs in NSCLC. Here we show miR-200c overexpression was correlated with the epithelial phenotype and sensitivity to gefitinib in EGFR wild-type NSCLC cell lines. Up-regulated miR-200c could regain the sensitivity to gefitinib in the EGFR wild-type cell lines and miR-200c could regulate epithelial to mesenchymal transition through PI3K/AKT and MEK/ERK pathways. NSCLC patients at advanced stage (N=150) who received EGFR-TKIs (gefitinib or erlotinib) as second- or third-line therapy from September 2008 to December 2012 were included in the study. In 66 NSCLC patients with wild-type EGFR, high levels of miR-200c expression was associated with higher disease control rate (DCR), longer progression-free survival (PFS) and longer overall survival (OS) compared with low miR-200c expression subgroup. In the subgroup with EGFR mutation, the trend remained the same but not statistically significant. Overall, these findings indicated that miR-200c might be a predictive biomarker for sensitivity to EGFR-TKIs in advanced NSCLC patients with wild-type EGFR.
Project description:Most non-small cell lung cancers (NSCLC) display elevated expression of epidermal growth factor receptor (EGFR), but response to EGFR kinase inhibitors is predominantly limited to NSCLC harboring EGFR-activating mutations. These mutations are associated with increased activity of survival pathways, including phosphatidylinositol 3-kinase/AKT and signal transducer and activator of transcription 3/5. We report that EGFR-activating mutations also surprisingly lead to decreased ability to activate extracellular signal-regulated kinase (ERK) compared with wild-type EGFR. In NSCLC cells and mouse embryonic fibroblasts expressing mutant EGFR, this effect on ERK correlates with decreased EGFR internalization and reduced phosphorylation of SHP2, a tyrosine phosphatase required for the full activation of ERK. We further show that ERK activation levels affect cellular response to gefitinib. NSCLC cells with EGFR mutation display reduced gefitinib sensitivity when ERK activation is augmented by expression of constitutively active mutants of mitogen-activated protein kinase/ERK kinase (MEK). Conversely, in a NSCLC cell line expressing wild-type EGFR, gefitinib treatment along with or following MEK inhibition increases death response compared with treatment with gefitinib alone. Our results show that EGFR-activating mutations may promote some survival pathways but simultaneously impair others. This multivariate alteration of the network governing cellular response to gefitinib, which we term "oncogene imbalance," portends a potentially broader ability to treat gefitinib-resistant NSCLC.
Project description:Our previous study demonstrated that long non-coding RNA (lncRNA) BC087858 could stimulate acquired resistance to EGFR-TKIs in non-small cell lung (NSCLC) but the specific regulatory mechanism remained unknown. We aimed to explore the role and mechanism of lncRNA BC087858 on EGFR-TKIs acquired resistance. LncRNA BC087858 mRNA expression was detected by reverse transcription polymerase chain reaction in different NSCLC cell lines and tissues. The relationship between BC087858 expression and clinicopathological factors was performed by Cox multivariate regression analysis. Small-interfering RNA, flow cytometry and trans-well assay were conducted to explore the biological functions of BC087858. Western blotting was used to analyze the target proteins expression. Over-expression was observed in NSCLC cells and patients with acquired resistance to EGFR-TKIs and significantly associated with a shorter progression-free survival (PFS) (12.0 vs. 17.0 months, P = 0.0217) in tumors with respond to EGFR-TKIs. The significant relationship was not observed in patients with T790M mutation (median PFS 17.6 vs. 12.5 months, P = 0.522) but in patients with non-T790M (median PFS 8.0 vs. 18.25 months,P = 0.0427). Down-regulation of BC087858 could significantly promote PC9/R and PC9/G2 cells invasion (P < 0.05; respectively). BC087858 knockdown restored gefitinib sensitivity in acquired resistant cells with non-T790M and inhibited the activation of the PI3K/AKT and MEK/ERK pathways and epithelial-mesenchymal transition (EMT) via up- regulating ZEB1 and Snail. In conclusion, LncRNA BC087858 could promote cells invasion and induce non-T790M mutation acquired resistance to EGFR-TKIs by activating PI3K/AKT and MEK/ERK pathways and EMT via up- regulating ZEB1 and Snail in NSCLC.
Project description:Compensatory activation of the signal transduction pathways is one of the major obstacles for the targeted therapy of non-small cell lung cancer (NSCLC). Herein, we present the therapeutic strategy of combined targeted therapy against the MEK and phosphoinositide-3 kinase (PI3K) pathways for acquired resistance to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) in NSCLC. We investigated the efficacy of combined trametinib plus taselisib therapy using experimentally established EGFR-TKI-resistant NSCLC cell lines. The results showed that the feedback loop between MEK/ERK and PI3K/AKT pathways had developed in several resistant cell lines, which caused the resistance to single-agent treatment with either inhibitor alone. Meanwhile, the combined therapy successfully regulated the compensatory activation of the key intracellular signals and synergistically inhibited the cell growth of those cells in vitro and in vivo. The resistance mechanisms for which the dual kinase inhibitor therapy proved effective included (MET) mesenchymal-epithelial transition factor amplification, induction of epithelial-to-mesenchymal transition (EMT) and EGFR T790M mutation. In further analysis, the combination therapy induced the phosphorylation of p38 MAPK signaling, leading to the activation of apoptosis cascade. Additionally, long-term treatment with the combination therapy induced the conversion from EMT to mesenchymal-to-epithelial transition in the resistant cell line harboring EMT features, restoring the sensitivity to EGFR-TKI. In conclusion, our results indicate that the combined therapy using MEK and PI3K inhibitors is a potent therapeutic strategy for NSCLC with the acquired resistance to EGFR-TKIs.
Project description:Resistance to tyrosine kinase inhibitors (TKIs) results in tumor relapse and poor prognosis in patients with lung adenocarcinoma. TKI resistance caused by epidermal growth factor receptor (EGFR) mutations at T790M and c-Met amplification occurs through persistent activation of the MEK/ERK and PI3K/AKT signaling pathways. We therefore expected that dual inhibitors of both signaling pathways could overcome TKI resistance in lung adenocarcinoma. Here, dioscin was selected from a product library of Chinese naturally occurring compounds and overcame TKI resistance in EGFR-mutated lung adenocarcinoma cells. Mechanistically, dioscin may down-regulate the expression of SH2 domain-containing phosphatase-2 (SHP2) at the transcription level by increasing p53 binding to the SHP2 promoter due to reactive oxygen species (ROS). Simultaneous inhibition of MEK/ERK and PI3K/AKT activation via decreased SHP2 expression and its interaction with GAB1 may be responsible for dioscin-mediated TKI sensitivity. A higher unfavorable response to TKI therapy occurred more commonly in patients with high SHP2 mRNA expression than in patients with low SHP2 mRNA expression. Therefore, we suggest that dioscin may act as a dual inhibitor of the MEK/ERK and PI3K/AKT signaling pathways to overcome TKI resistance via dysregulation of SHP2 expression in lung adenocarcinoma.
Project description:Drug treatment of 3D cancer spheroids more accurately reflects in vivo therapeutic responses compared to adherent culture studies. In EGFR-mutated lung adenocarcinoma, EGFR-TKIs show enhanced efficacy in spheroid cultures. Simultaneous inhibition of multiple parallel RTKs further enhances EGFR-TKI effectiveness. We show that the common RTK signaling intermediate SOS1 was required for 3D spheroid growth of EGFR-mutated NSCLC cells. Using two distinct measures of pharmacologic synergy, we demonstrated that SOS1 inhibition strongly synergized with EGFR-TKI treatment only in 3D spheroid cultures. Combined EGFR- and SOS1-inhibition markedly inhibited Raf/MEK/ERK and PI3K/AKT signaling. Finally, broad assessment of the pharmacologic landscape of drug-drug interactions downstream of mutated EGFR revealed synergy when combining an EGFR-TKI with inhibitors of proximal signaling intermediates SOS1 and SHP2, but not inhibitors of downstream RAS effector pathways. These data indicate that vertical inhibition of proximal EGFR signaling should be pursued as a potential therapy to treat EGFR-mutated tumors.
Project description:First-generation epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs), including gefitinib and erlotinib, have proven to be highly effective agents for advanced non-small cell lung cancer (NSCLC) in patients harboring an activating EGFR mutation such as the exon 19 deletion mutation and L858R. Although those reversible small molecular targeted agents provide a significant response and survival benefit, all responders eventually acquire resistance. Second-generation EGFR-targeting agents, such as irreversible EGFR/HER2 tyrosine kinase inhibitors and pan-HER TKIs, may improve survival further and be useful for patients who acquired resistance to ?rst-generation EGFR-TKIs. This review discusses novel therapeutic strategies for EGFR-mutated advanced NSCLC using first- and second-generation EGFR-TKIs.
Project description:Lung cancer leads cancer-related mortality worldwide. Non-small-cell lung cancer (NSCLC), the most prevalent subtype of this recalcitrant cancer, is usually diagnosed at advanced stages, and available systemic therapies are mostly palliative. The probing of the NSCLC kinome has identified numerous nonoverlapping driver genomic events, including epidermal growth factor receptor (EGFR) gene mutations. This review provides a synopsis of preclinical and clinical data on EGFR mutated NSCLC and EGFR tyrosine kinase inhibitors (TKIs). Classic somatic EGFR kinase domain mutations (such as L858R and exon 19 deletions) make tumors addicted to their signaling cascades and generate a therapeutic window for the use of ATP-mimetic EGFR TKIs. The latter inhibit these kinases and their downstream effectors, and induce apoptosis in preclinical models. The aforementioned EGFR mutations are stout predictors of response and augmentation of progression-free survival when gefitinib, erlotinib, and afatinib are used for patients with advanced NSCLC. The benefits associated with these EGFR TKIs are limited by the mechanisms of tumor resistance, such as the gatekeeper EGFR-T790M mutation, and bypass activation of signaling cascades. Ongoing preclinical efforts for treating resistance have started to translate into patient care (including clinical trials of the covalent EGFR-T790M TKIs AZD9291 and CO-1686) and hold promise to further boost the median survival of patients with EGFR mutated NSCLC.
Project description:Despite initial and sometimes dramatic responses of specific NSCLC tumors to EGFR TKIs, nearly all will develop resistance and relapse. Gene expression analysis of NSCLC cell lines treated with the EGFR TKI, gefitinib, revealed increased levels of FGFR2 and FGFR3 mRNA. Analysis of gefitinib action on a larger panel of NSCLC cell lines verified that FGFR2 and FGFR3 expression is increased at the mRNA and protein level in NSCLC cell lines in which the EGFR is dominant for growth signaling, but not in cell lines where EGFR signaling is absent. A luciferase reporter containing 2.5 kilobases of fgfr2 5' flanking sequence was activated after gefitinib treatment, indicating transcriptional regulation as a contributing mechanism controlling increased FGFR2 expression. Induction of FGFR2 and FGFR3 protein as well as fgfr2-luc activity was also observed with Erbitux, an EGFR-specific monoclonal antibody. Moreover, inhibitors of c-Src and MEK stimulated fgfr2-luc activity to a similar degree as gefitinib, suggesting that these pathways may mediate EGFR-dependent repression of FGFR2 and FGFR3. Importantly, our studies demonstrate that EGFR TKI-induced FGFR2 and FGFR3 are capable of mediating FGF2 and FGF7 stimulated ERK activation as well as FGF-stimulated transformed growth in the setting of EGFR TKIs. In conclusion, this study highlights EGFR TKI-induced FGFR2 and FGFR3 signaling as a novel and rapid mechanism of acquired resistance to EGFR TKIs and suggests that treatment of NSCLC patients with combinations of EGFR and FGFR specific TKIs may be a strategy to enhance efficacy of single EGFR inhibitors.