Targeting endoplasmic reticulum stress and Akt with OSU-03012 and gefitinib or erlotinib to overcome resistance to epidermal growth factor receptor inhibitors

Abstract

Preexisting and acquired resistance to epidermal growth factor receptor (EGFR) inhibitors limits their clinical usefulness in patients with advanced non-small cell lung cancer (NSCLC). This study characterizes the efficacy and mechanisms of the combination of gefitinib or erlotinib with OSU-03012, a celecoxib-derived antitumor agent, to overcome EGFR inhibitor resistance in three NSCLC cell lines, H1155, H23, and A549. The OSU-03012/EGFR inhibitor combination induced pronounced apoptosis in H1155 and H23 cells, but not in A549 cells, suggesting a correlation between drug sensitivity and basal phospho-Akt levels independently of EGFR expression status. Evidence indicates that this combination facilitates apoptosis through both Akt signaling inhibition and up-regulation of endoplasmic reticulum (ER) stress-induced, GADD153-mediated pathways. For example, ectopic expression of constitutively active Akt significantly attenuated the inhibitory effect on cell survival, and small interfering RNA-mediated knockdown of GADD153 protected cells from undergoing apoptosis in response to drug cotreatments. Furthermore, the OSU-03012/EGFR inhibitor combination induced GADD153-mediated up-regulation of death receptor 5 expression and subsequent activation of the extrinsic apoptosis pathway. It is noteworthy that the ER stress response induced by this combination was atypical in that the cytoprotective pathway was not engaged. In addition, in vivo suppression of tumor growth and modulation of intratumoral biomarkers were observed in a H1155 tumor xenograft model in nude mice. These data suggest that the concomitant modulation of Akt and ER stress pathways with the OSU-03012/EGFR inhibitor combination represents a unique approach to overcoming EGFR inhibitor resistance in NSCLC and perhaps other types of cancer with elevated basal Akt activities.

Figure 1

OSU-03012 enhances EGFR inhibitor-induced apoptosis in gefitinib/erlotinib-resistant NSCLC cells that express high levels of phosphorylated Akt. A, effects of OSU-03012, gefitinib or erlotinib on the viability of H1155, H23 and A549 cells. Right panel. Cells were treated with drugs as indicated for 24 hours. Cell viability after drug treatment was determined by the MTT or MTS assay. Each data point represents the mean ± SD of three independent experiments. Left panel. Basal expression levels of EGFR, ⁴⁷³Ser-Akt and Akt in H1155, H23 and A549 cells were examined by immunoblotting as described in Materials and Methods. B, OSU-03012 dose-dependently decreased phosphorylation of Akt in H1155, H23 and A549 cells. Cells were treated at the indicated concentrations for 8 hours and immunoblotting was performed as described in Materials and Methods. C and D, induction of apoptosis in H1155, H23 and A549 cells treated with OSU-03012 in combination with gefitinib or erlotinib. Cells were treated as indicated for 24 hours and assessed for phosphatidylserine externalization by flow cytometry after staining with fluorescence-labeled Annexin V and propidium iodide. Representative quadrantal plots of data from dual-color flow cytometry of treated H1155 cells are shown (C). Flow cytometry data were used to determine the percentages of apoptotic H1155, H23 and A549 cells after the indicated treatments (D). Each bar represents the mean ± SD of three independent experiments.

Figure 2

The combination of OSU-03012 with gefitinib or erlotinib induces concomitant inhibition of Akt signaling and upregulation of ER stress-inducible apoptotic, but not cytoprotective, proteins in H1155 and H23 cells. A, effects of combined treatment with OSU-03012 (03012) and gefitinib (Gef) or erlotinib (Erl) on the phosphorylation status of ⁴⁷³Ser-Akt (p-Akt) and the expression levels of GADD153 were examined in H1155, H23 and A549 cells. Cells were treated as indicated for 8 or 24 hours followed by immunoblotting of cell lysates for the indicated markers as described in Materials and Methods. B, effects of PI3 kinase inhibition by LY294002 or shRNA-mediated knockdown of PDK-1 in combination with EGFR inhibitors on apoptosis was assessed in H1155 cells by flow cytometric analysis of phosphatidylserine externalization as described in the Materials and Methods. Left panel, Cells were treated with OSU-03012, LY294002 and/or erlotinib at the indicated concentrations for 24 hours, and stained with fluorescence-labeled Annexin V and propidium iodide. Effects of treatments on Akt phosphorylation status were examined by immunoblotting (upper panel). Percentages of apoptotic cells after the indicated treatments were calculated from flow cytometry data. Each bar represents the mean ± SD of three independent experiments. Right panel, cells were stably transfected with shRNA constructs against human PDK-1 or GFP as controls, treated with OSU-03012, gefitinib or erlotinib at the indicated concentrations for 24 hours, and stained with fluorescence-labeled Annexin V and propidium iodide. Efficiency of PDK-1 knockdown and suppression of phospho-Akt (p-Akt) was verified by immunoblotting (upper panel). Percentages of apoptotic cells after the indicated treatments were calculated from flow cytometry data. Each bar represents the mean ± SD of three independent experiments (*, P<0.05, Student’s t-test). C, the combination of OSU-03012 and erlotinib enhances the expression of proteins in the apoptotic arm, but not the cytoprotective arm, of the ER stress response. Cells were co-treated with 3 µmol/L OSU-03012 and 6 µmol/L erlotinib for 8 hours. Immunoblotting was performed as described in Materials and Methods. Right panel, Immunoblot of ER stress-inducible proteins, GADD153, CREB2, GRP78 and GRP94, in treated H1155, H23 and A549 cells. Left panel, Diagram of apoptotic and cytoprotective signaling pathways of the ER stress response.

Figure 3

Enforced expression of constitutively active Akt or siRNA-mediated knockdown of GADD153 protects H1155 cells from the apoptotic effects of OSU-03012/EGFR inhibitor combinations. A, survival and apoptosis of H1155 cells overexpressing constitutively active Akt were assessed after treatment with the combination of OSU-03012 and gefitinib or erlotinib. Cells were transfected with the expression vector for the constitutively active, hemagglutinin (HA)-tagged, mutant (T308D/S473D) mouse Akt-1 (HA-CA Akt). Two stable clones with the highest expression of HA-Akt (upper left panel) were treated for 24 hours, after which cell viability was determined by the MTS assay (lower left panel) and, for the higher HA Akt-expressing clone, apoptosis was assessed by flow cytometry (lower right panel) as described in Materials and Methods. Each bar represents the mean ± SD of three independent experiments (*, P<0.05, Student’s t-test). Upper right panel, Constitutively active Akt protects H1155 cells against OSU-03012/EGFR inhibitor-induced dephosphorylation of the Akt substrate, GSK3β. A stable HA-CA Akt-expressing clone was treated with the combination of OSU-03012 and gefitinib or erlotinib as indicated for 24 hours and the phosphorylation status of GSK3β was assessed by immunoblotting. B, effect of siRNA-mediated knockdown of GADD153 on induction of apoptosis in H1155 cells co-treated with OSU-03012 and gefitinib was assessed by flow cytometric analysis of phosphatidylserine externalization. Cells were transfected with siRNA for the human DDIT3 gene or scrambled siRNA as controls, treated with OSU-03012 and gefitinib at the indicated concentrations for 24 hours, and stained with fluorescence-labeled Annexin V and propidium iodide. Efficiency of GADD153 knockdown was verified by immunoblotting (upper panel). Percentages of apoptotic cells after the indicated treatments were calculated from flow cytometry data. Each bar represents the mean ± SD of three independent experiments. O, 3 µmol/L OSU-03012; G, 6 µmol/L gefitinib.

Figure 4

The combination of OSU-03012 with EGFR inhibitors induces GADD153-mediated upregulation of death receptor DR5 expression and activation of extrinsic apoptosis signaling. A, effect of OSU-03012 in combination with gefitinib or erlotinib on mRNA expression levels of the DR5 gene in H1155 cells. Total RNA was isolated from H1155 cells after treatment with OSU-03012 alone and in combination with erlotinib or gefitinib for 12 hours, and then subjected to semi-quantitative RT-PCR as described in Materials and Methods. B, effect of OSU-03012 in combination with gefitinib or erlotinib on cell surface expression of DR5 protein in H1155 cells. Cells were treated with OSU-03012, gefitinib or erlotinib alone and in the indicated combinations for 24 hours and then stained with phycoerythrin-labeled antibody against the extracellular domain of DR5. Numbers of cells with enhanced fluorescence intensity were determined by flow cytometry (left panel), and expressed as percentages of cells with elevated cell surface expression of DR5 protein (right panel). Each bar represents the mean ± SD of three independent experiments (*, P<0.05, Student’s t-test). C, involvement of GADD153 in drug-induced transcriptional upregulation of DR5 gene expression was determined by analysis of DR5 gene promoter activity. H1155 cells were transfected with reporter vectors encoding modified firefly luciferase driven by the wild-type DR5 promoter (pDR5Pro) or the DR5 promoter containing a mutated GADD153-binding sequence (pDR5Pro-GADD153mt). Transfectants were treated with drugs as indicated for 12 hours and then assayed for luciferase activity as an indicator of reporter gene expression. Firefly luciferase activities were normalized to that of co-transfected Renilla luciferase. Each bar represents the mean ± SD of three independent experiments (*, P<0.05, Student’s t-test). D, effect of OSU-03012 in combination with gefitinib or erlotinib on caspase 8 activity and Bid cleavage in H1155 cells. Cells were treated as indicated for 24 hours and then, for assessment of caspase 8 activity, incubated with a fluorogenic substrate of caspase 8. Numbers of cells with enhanced fluorescence intensity were determined by flow cytometry (top panel), and expressed as percentages of cells with enhanced caspase 8 activity (middle panel). Each bar represents the mean ± SD of three independent experiments (*, P<0.05, Student’s t-test). For evaluation of Bid cleavage (bottom panel), lysates from treated cells were immunoblotted for intact and truncated Bid (tBid) as described in Materials and Methods. G, 6 µmol/L gefitinib; E, 6 µmol/L erlotinib; O, 3 µmol/L OSU-03012. E, effect of siRNA-mediated knockdown of GADD153 on induction of apoptosis in H1155 cells co-treated with OSU-03012 and gefitinib or erlotinib was assessed by flow cytometric analysis of phosphatidylserine externalization. Cells transfected with siRNA for the human DR5 gene or scrambled siRNA as controls were treated as indicated for 24 hours, and stained with fluorescence-labeled Annexin V and propidium iodide. The effects of treatments on DR5 mRNA levels (upper panel) and apoptosis (lower panel) in transfected cells were determined by RT-PCR and flow cytometry, respectively. Each bar represents the mean ± SD of three independent experiments (*, P<0.05, Student’s t-test).

Figure 5

Ultrastructural evidence of ER stress in EGFR inhibitor-resistant NSCLC cells treated with the combination of OSU-03012 and gefitinib. H1155 cells were treated with DMSO (A, D), the combination of OSU-03012 and gefitinib (B, E) and thapsigargin as a positive control (C, F) for 8 hours at the indicated concentrations and then visualized by transmission electron microscopy as described in Materials and Methods. Arrows indicate stacked vacuole-like cytoplasmic structures indicative of expanded and dilated endoplasmic reticulum. N, nucleus; M, mitochondria; G, golgi apparatus.

Figure 6

OSU-03012 in combination with erlotinib suppresses the growth of EGFR inhibitor-resistant tumor xenografts in vivo. Athymic nude mice bearing established subcutaneous H1155 xenograft tumors were randomized to four groups (n = 10) that received the following treatments by gavage for the duration of the study: Vehicle, OSU-03012 at 100 mg/kg body weight q.d., erlotinib at 100 mg/kg q.d., or OSU-03012 combined with erlotinib both at 100 mg/kg q.d. (Combination). A, Kaplan-Meier survival curves are shown for each treatment group. Survival in the Combination group was significantly extended (P<0.05) in comparison to all other treatment groups. Tumors were measured once per week and survival analysis was performed with survival time defined as the time for tumors to reach 1,200 mm³. The log-rank test was used to calculate P values. B, effects of treatments on intratumoral biomarkers of drug activity in H1155 xenograft tumors. Athymic nude mice bearing established subcutaneous H1155 xenograft tumors were treated as described above for two weeks. Tumors were harvested at terminal sacrifice and intratumoral expression of phosphorylated ⁴⁷³Ser-Akt (p-Akt) and GADD153 were examined by immunohistochemistry in formalin-fixed, paraffin-embedded tissues (upper panel) and by immunoblotting of the homogenates of two representative H1155 tumors from each treatment group (lower panel). C, immunohistochemical evaluation of intratumoral proliferation and apoptosis in H1155 xenograft tumors. Tumors were harvested from mice treated as described above in B. Immunostaining for PCNA and cleaved caspase 3 in formalin-fixed, paraffin-embedded tumor tissues was performed, and apoptotic and proliferation indices were calculated as described in Materials and Methods. Upper panel, Immunohistochemistry showing PCNA and cleaved caspase 3 in H1155 tumors from each treatment group. Normal skin from vehicle-treated mice was included as control. Lower panel, Apoptotic and proliferation indices in H1155 tumors from each treatment group. Each bar represents the mean of five 400x fields ± SD.