Downregulation of death receptor 4 is tightly associated with positive response of EGFR mutant lung cancer to EGFR-targeted therapy and improved prognosis

Abstract

Death receptor 4 (DR4), a cell surface receptor, mediates apoptosis or induces inflammatory cytokine secretion upon binding to its ligand depending on cell contexts. Its prognostic impact in lung cancer and connection between EGFR-targeted therapy and DR4 modulation has not been reported and thus was the focus of this study. Methods: Intracellular protein alterations were measured by Western blotting. Cell surface protein was detected with antibody staining and flow cytometry. mRNA expression was monitored with qRT-PCR. Gene transactivation was analyzed with promoter reporter assay. Drug dynamic effects in vivo were evaluated using xenografts. Gene modulations were achieved with gene overexpression and knockdown. Proteins in human archived tissues were stained with immunohistochemistry. Results: EGFR inhibitors (e.g., osimertinib) decreased DR4 levels only in EGFR mutant NSCLC cells and tumors, being tightly associated with induction of apoptosis. This modulation was lost once cells became resistant to these inhibitors. Increased levels of DR4 were detected in cell lines with acquired osimertinib resistance and in NSCLC tissues relapsed from EGFR-targeted therapy. DR4 knockdown induced apoptosis and augmented apoptosis when combined with osimertinib in both sensitive and resistant cell lines, whereas enforced DR4 expression significantly attenuated osimertinib-induced apoptosis. Mechanistically, osimertinib induced MARCH8-mediated DR4 proteasomal degradation and suppressed MEK/ERK/AP-1-dependent DR4 transcription, resulting in DR4 downregulation. Moreover, we found that DR4 positive expression in human lung adenocarcinoma was significantly associated with poor patient survival. Conclusions: Collectively, we suggest that DR4 downregulation is coupled to therapeutic efficacy of EGFR-targeted therapy and predicts improved prognosis, revealing a previously undiscovered connection between EGFR-targeted therapy and DR4 modulation.

Keywords: EGFR inhibitors; acquired resistance; apoptosis; death receptor 4; osimertinib.

Figure 1

Osimertinib and other EGFR-TKIs decrease DR4 levels in EGFRm NSCLC cell lines accompanied with induction of apoptosis. A-D, Both PC-9 and HCC827 cells were exposed to the indicated concentrations of EGFR-TKIs for 8 h (A and C) or to 100 nM osimertinib (Osim) for different times (B) or 12 h (D). E-G, The indicated cell lines were exposed to 100 nM of a given EGFR-TKI (E and F) or to different concentrations of osimertinib for 8 h (G). H, The tested cell lines were exposed to DMSO or 100 nM osimertinib for 12 h. Total cellular DR4 and cell surface DR4 were detected with Western blotting (A-C and E-G) and flow cytometry (D and H), respectively. The data in D and H are means ± SDs of duplicate determinations. CF, cleaved form.

Figure 2

Osimertinib decreases DR4 levels in an EGFRm NSCLC xenografts in vivo accompanied with induction of apoptosis. PC-9 xenografts at ~ 300 mm³ were treated with vehicle control (Ctrl) or osimertinib (Osim; 10 mg/kg; og; once/daily) and collected at the indicated times (N = 3). The given proteins were detected with Western blotting (A). The results or band intensities were quantified with NIH Image J software (B and D) in comparison with tumor weight alteration (C). The data presented in B-D are means ± SEs (N = 3). Control and tumor tissues on day 9 were also stained for cPARP with IHC (E). The scale bar is 50 μm. CF, cleaved form.

Figure 3

Osimertinib decreases DR4 mRNA levels, inhibits AP-1-dependent DR4 transactivation, and facilitates proteasomal degradation of DR4. A, PC-9 and HCC827 cells were exposed to the indicated concentrations of osimertinib (Osim) for 6 h. DR4 mRNA levels were detected by RT-PCR. B, PC9 cells were transfected with the given DR4 reporter plasmids. Approximately 18 h after transfection, the cells were exposed to 100 nM osimertinib or erlotinib for another 10 h and lysed for luciferase activity assay. The data are mean ± SDs of triplicate determinations. C, PC-9 and HCC827 cells were exposed to DMSO or 100 nM osimertinib for 8 h, followed by the addition of 10 µg/ml CHX. Cells were then harvested at the indicated times post CHX for Western blotting. DR4 levels were plotted relative to those at time 0 of CHX treatment after being quantified by NIH Image J software and normalized to Actin or GAPDH. D and F, The indicated cells were pre-treated with 10 µM MG132, or 50 nM Baf A1 for 1 h and then co-treated with 100 nM osimertinib, CO1686 or erlotinib for an additional 6 h. Total cellular and cell surface DR4 were detected with Western blotting (D and E) and flow cytometry (F), respectively. D, DMSO; MG, MG132; A1, Baf A1.

Figure 4

Knockdown of MARCH8 elevates basal levels of DR4 levels and rescues DR4 reduction induced by osimertinib through preventing its degradation. A and B, The indicated cell lines were infected with pLKO.1 or MARCH8 shRNA lentiviruses for 48 h. Western blotting and flow cytometry were used to detect total cellular proteins (A) and cell surface DR4 (B), respectively. C, PC-9 and HCC827 cells expressing shMARCH8 were treated with 100 nM osimertinib or erlotinib for 6 h. MARCH8 knockdown and DR4 levels were detected by Western blotting. D, Cell surface DR4 levels of PC-9/shMARCH8 cells exposed to 100 nM osimertinib for 6 h were detected by flow cytometry. The data are means ± SDs of duplicate determinations. E, The indicated cell lines were treated with DMSO or 100 nM osimertinib for 6 h followed by addition of 10 µg/ml CHX. Cells were then harvested at the indicated times post CHX for Western blotting. DR4 levels were plotted relative to those at time 0 of CHX treatment (right panels) after being quantified by NIH Image J software and normalized to tubulin.

Figure 5

EGFR-TKI-resistant cell lines and tissues possess elevated DR4 expression. A, The basal levels of DR4 in the indicated cell lines were detected with Western blotting. B-E, DR4 in human NSCLC tissues with EGFR mutations was detected with IHC. Representative cases in which DR4 expression was increased post relapse from EGFR-TKI treatment are presented in B. Quantitation of DR4 expression for all relapsed cases is presented in C-E. Pt, patient.

Figure 6

DR4 knockdown enhances osimertinib-induced apoptosis, whereas induction of ectopic DR4 overexpression transiently compromises osimertinib-induced apoptosis in the sensitive EGFRm NSCLC cell cells. A, Cells were infected with DR4 shRNA lentiviruses for 24 h followed by treatment with 100 nM osimertinib (Osim) for another 24 h. B, PC-9/DR4i cells were exposed to the indicated concentration of DOX for 8 h (DOX 8 h), followed by washing and culture with fresh medium for another 16 h (DOC 8 h/W/16 h) or continuously for 24 h (DOX 24 h). C, PC-9/DR4i cells were treated with 100 nM osimertinib for 48 h, followed by 20 ng/ml DOX for another 8 h. D, PC-9/DR4i cells were treated with 10 ng/ml DOX. After 8 h, the cells were washed with fresh medium and exposed to 100 nM osimertinib for another 24 h. DR4 expression and PARP cleavage were detected by Western blotting and apoptosis was measured by annexin V/flow cytometry. The data are means ± SDs of duplicate determinations (A, C and D). CF, cleaved form.

Figure 7

DR4 knockdown in osimertinib-resistant cell lines sensitizes the cells to osimertinib-induced apoptosis, whereas MEK inhibition combined with osimertinib potentiates DR4 reduction with augmented induction of apoptosis in osimertinib-resistant cells. A-C, The indicated osimertinib-resistant cell lines were infected with DR4 shRNA lentiviruses for 24 h followed by treatment with 100 nM osimertinib (Osim) for another 48 h. DR4 knockdown and PARP cleavage were detected by Western blotting and apoptosis was measured by annexin V/flow cytometry. D and E, PC-9/AR cells were treated with 100 nM osimertinib alone, 25 nM trametinib (Tram) or 50 nM selumetinib (Sel) or PD0325901 (PD901) alone, and the combination of osimertinib with a given MEK inhibitor for 24 h (D) or 8 h (E). The cells were then harvested for Western blotting to detect the given proteins (D and E) and for annexin V/flow cytometric analysis to detect apoptosis (D). **, P < 0.01; *** P < 0.001.CF, cleaved form.

Figure 8

DR4 expression, like pathological grade and clinical stage, is significantly associated with poor survival of lung adenocarcinoma patients. A, Analysis of the association between DR4 expression and clinico-pathological features of lung adenocarcinomas (n = 242). Chi-square test was used to calculate statistical significance. LNM, lymph node metastasis. B, Representative images of DR4 staining. C, Kaplan-Meier survival analyses of the impact of DR4 expression, pathological grade and clinical stage on the survival of lung adenocarcinoma patients.