Targeting the IκB Kinase Enhancer and Its Feedback Circuit in Pancreatic Cancer

Abstract

Pancreatic ductal adenocarcinoma (PDAC) is a deadly disease with an overall median 5-year survival rate of 8%. This poor prognosis is because of the development of resistance to chemotherapy and radiation therapy and lack of effective targeted therapies. IκB kinase enhancer (IKBKE) overexpression was previously implicated in chemoresistance. Because IKBKE is frequently elevated in PDAC and IKBKE inhibitors are currently in clinical trials, we evaluated IKBKE as a therapeutic target in this disease. Depletion of IKBKE was found to significantly reduce PDAC cell survival, growth, cancer stem cell renewal, and cell migration and invasion. Notably, IKBKE inhibitor CYT387 and IKBKE knockdown dramatically activated the MAPK pathway. Phospho-RTK array analyses showed that IKBKE inhibition leads to rapid upregulation of ErbB3 and IGF-1R expression, which results in MAPK-ERK pathway activation-thereby limiting the efficacy of IKBKE inhibitors. Furthermore, IKBKE inhibition leads to stabilization of FOXO3a, which is required for RTK upregulation on IKBKE inhibition. Finally, we demonstrated that the IKBKE inhibitors synergize with the MEK inhibitor trametinib to significantly induce cell death and inhibit tumor growth and liver metastasis in an orthotopic PDAC mouse model.

Figure 1

IKBKE is an important oncogene in pancreatic ductal adenocarcinoma. A, Lysates from human pancreatic tumor (T) and normal pancreas (N) and B, mutant KRAS or wild type KRAS PDAC cells were subjected to Western blot analyses with IKBKE and actin. C and D, PDAC cell lines were transfected with 2 siRNAs for IKBKE. MTT assay was performed 72 h after transfection. Knockdown efficiency of the siRNAs is shown. E and F, Soft agar colony formation assays in MiaPaCa-2 and metastatic L3.6 pl cells transfected with 2 siRNAs for IKBKE. Colonies were counted after 10 days. IKBKE knockdown significantly inhibited the colony formation. G–H, Aldefluor assays were performed on L3.6 pl cells with IKBKE knockdown or control cells as described in Materials and Methods. Diethylaminobenzaldehyde (DEAB) was used as a control. IKBKE knockdown significantly reduced the number of cancer stem cells. I, CSC spheres from control or IKBKE siRNA-transfected L3.6 pl cells. IKBKE knockdown significantly inhibited the sphere formation. J–K, L3.6 pl cells transfected with control siRNA or IKBKE siRNAs were plated in Boyden chambers for migration (J) or invasion (K) assays. *P < .05 and **P < .002.

Figure 2

IKBKE/TBK1 inhibitor CYT387 reduced cell viability, cell motility, and cancer stem cell populations in PDAC cells. A, PDAC cell lines were treated with increasing concentrations of CYT387 and MTT assay was performed after 72 h. B, CYT387 inhibited L3.6 pl cell sphere formation compared with vehicle. C and D, L3.6 pl cells were plated with indicated amounts of CYT387 or vehicle for cell invasion (C) or migration (D) assays. E and F, L3.6 pl cells were treated with 2 μM of CYT387 for 96 h and ALDH-positive CSC population was assessed using Aldefluor kit. G, Western blot analyses of L3.6 pl cells treated with different doses of CYT387 for 24 h showed decreased phosphorylation of STAT3 and AKT and increased phosphorylation of ERK1/2. *P < .05 and **P < .002.

Figure 3

Inhibition of IKBKE leads to transcriptional upregulation of several RTKs. A, 50 μg of lysates from L3.6 pl cell treated with 2 μM of CYT387 for 24 h were incubated with phospho-RTK arrays. Western blot with pan-tyrosine antibody revealed activation of ErbB3. B, Phospho-RTK array analysis with 250 μg of lysates from L3.6 pl cells treated with 2 μM of CYT387 for 36 h showed significant activation of IGF-1R. C, Western blot analysis of lysates from L3.6 pl cells treated with 2 μM of CYT387 for indicated times showed increased activation and protein expression of IGF-1R and ErbB3. D, Quantitative PCR with ErbB3 and IGF-1R primers using RNA from L3.6 pl cells treated with 2 μM of CYT387 for indicated amount of time. Actin was used as a control. *P < .05.

Figure 4

Inhibition of ErbB3 or IGF-1R inhibits CYT387-mediated ERK1/2 activation. A, 24 h after L3.6 pl cells were transfected with control siRNA or siRNAs targeting ErBB3 or IGF-1R, cells were treated with 2 μM of CYT387 for an additional 24 h and Western blot was performed using indicated antibodies. B, 36 h, h after L3.6 pl cells were transfected with control or FOXO3a-specific siRNAs, cells were treated with CYT387 for an additional 24 h and Western blot was performed using indicated antibodies. Band intensities were quantified using ImageJ software. C, L3.6 pl cells were treated as described in B and RNA was isolated. Quantitative PCR with ErbB3 and IGF-1R primers were performed. *P < .05 and **P < .002. Knockdown of FOXO3 reduced CYT387-induced expression of ERB3 and IGFR-1R miRNA levels.

Figure 5

CYT387 synergizes with trametinib in pancreatic cells in vitro and in vivo. A, An MTT assay was performed on L3.6 pl cells that were treated with indicated amounts of CYT387 and trametinib for 72 h. CYT387 and trametinib show synergy in reducing cell viability. Combination indices are shown. B, L3.6 pl cells were cultured in 1 μM of CYT387 and 15 nM of trametinib or the combination for 10 days, and colonies were visualized by crystal violet staining. C, L3.6 pl cells were cultured under CSC culture conditions with 2 μM of CYT387 and/or 30 nM of trametinib. After 10 days, the number of spheres was counted. Two passages of cells were performed, with representative colony sizes shown. D, Tumor volumes of the L3.6 pl orthotopic tumors were measured once per week for 4 weeks. E, Tumor weights from mice bearing L3.6 pl orthotopic tumors and representative images of tumors at endpoint are shown. F, Liver metastasis scores at the endpoint. *P < .05 and **P < .002.

Supplementary Figure 1

IKBKE inhibitor Amlexanox reduced cell viability, cell motility, invasion and cancer stem cell sphere formation of PDAC cells. A, Five PDAC cell lines were treated with increasing concentrations of amlexanox and MTT assay was performed after 72 hours. B, Amlexanox significantly inhibited L3.6pl cell sphere formation compared to vehicle. C and D, L3.6pl cells were plated with indicated amounts of Amlexanox 0or vehicle for cell invasion (C) or migration (D) assays. *P < .05 and **P < .002.

Supplementary Figure 2

Magnified images of the reduced colonies in Figure 1. A, magnified colonies from panel 1D. B, magnified colonies from panel 1E.

Supplementary Figure 3

IKBKE inhibitor Amlexanox decreased PDAC tumor growth and metastasis in mice. A, B, C, Tumor volumes of the L3.6pl orthotopic tumors were measured once per week for 4 weeks. Amlexanox significantly decreased tumor volume and tumor weight compared to vehicle. D and F, Amlexanox significantly decreased liver metastasis 4 weeks after treatment compared to vehicle. E, Tumor immunohistology shows that amlexanox decreased tumor cell proliferation index (Ki-67) and angiogenesis marker (CD31) and increased pERK expression compared to vehicle. *P < .05.

Supplementary Figure 4

Inhibition of IKBKE leads to feedback activation of the RTKs/MAPK pathway. A, Amlexanox treatment at a concentration of 50 and 100 μM to L3.6pl cells for 24 h increased pERK1/2 expression and decreased pSTAT3 and pAKT expression compared to vehicle. B, IKBKE inhibitor (CYT387) treatment at a concentration of 1 and 4 μM for 24 h led to increased pERK1/2 expression in multiple PDAC cell lines compared to vehicle. C, L3.6pl cells treated with CYT387 (2 μM) showed prolonged activation of the BRaf-MEK-ERK pathway at a time-dependent manner. D, L3.6pl cells transfected with IKBKE siRNAs also demonstrated the increased pERK1/2 expression compared to control siRNA.

Supplementary Figure 5

CYT387 synergizes with trametinib in a panel of PDAC cells in vitro. The combination of CYT387 and trametinib at different concentrations on PDAC cells viability. The combination index were calculated to show the synergistic effects of the combination.

Supplementary Figure 6

The combined treatment of CYT387 and trametinib synergistically inhibits the tumor cell signaling and induces apoptosis. A, L3.6pl cells were treated with CYT387 and trametinib as shown and Western blot was performed using indicated antibodies. B, Western blot analyses of mouse tissues were performed with indicated antibodies. C, Immunohistochemistry staining with pERK1/2, CD31, Ki67, and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay of the orthotopic tissues. D, Quantitative PCR with ErbB3 and IGF-1R primers was performed with RNA isolated from mouse tissues. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a reference.