IKK is a therapeutic target in KRAS-Induced lung cancer with disrupted p53 activity

Abstract

Activating mutations in KRAS are prevalent in cancer, but therapies targeted to oncogenic RAS have been ineffective to date. These results argue that targeting downstream effectors of RAS will be an alternative route for blocking RAS-driven oncogenic pathways. We and others have shown that oncogenic RAS activates the NF-κB transcription factor pathway and that KRAS-induced lung tumorigenesis is suppressed by expression of a degradation-resistant form of the IκBα inhibitor or by genetic deletion of IKKβ or the RELA/p65 subunit of NF-κB. Here, genetic and pharmacological approaches were utilized to inactivate IKK in human primary lung epithelial cells transformed by KRAS, as well as KRAS mutant lung cancer cell lines. Administration of the highly specific IKKβ inhibitor Compound A (CmpdA) led to NF-κB inhibition in different KRAS mutant lung cells and siRNA-mediated knockdown of IKKα or IKKβ reduced activity of the NF-κB canonical pathway. Next, we determined that both IKKα and IKKβ contribute to oncogenic properties of KRAS mutant lung cells, particularly when p53 activity is disrupted. Based on these results, CmpdA was tested for potential therapeutic intervention in the Kras-induced lung cancer mouse model (LSL-Kras (G12D)) combined with loss of p53 (LSL-Kras (G12D)/p53 (fl/fl)). CmpdA treatment was well tolerated and mice treated with this IKKβ inhibitor presented smaller and lower grade tumors than mice treated with placebo. Additionally, IKKβ inhibition reduced inflammation and angiogenesis. These results support the concept of targeting IKK as a therapeutic approach for oncogenic RAS-driven tumors with altered p53 activity.

Keywords: IKK; KRAS; Lung cancer; NF-κB; p53.

Fig 1. Targeting IKKβ reduces NF-κB activity in KRAS positive lung cells

A) The indicated cell lines were transfected with 100ηg of an NF-κB-responsive firefly luciferase reporter vector (3x-κB-Luc) and 5ηg of Renilla luciferase vector (pRL-TK) and either treated with 0.1% DMSO or 5μM CmpdA for 16h.-LUC) negative control (cells transfected with pcDNA3 instead of 3x-κB-Luc and pRL-TK); RLUs) Relative luciferase units. B) The indicated cell lines were treated with 0.1% DMSO or 5μM CmpdA for 30 min and protein lysates were analyzed for NF-κB activity by Western Blotting. Antibodies used are indicated. C) H358 cells were transfected with the indicated siRNAs as described in methods and analyzed for NF-κB activity at 72h by Western Blotting. Antibodies used are indicated. Statistical significance was measured when appropriate by Student's t-test (*p<0.05) when compared to experimental control samples (DMSO). Error bars represent average ± 1s.d.

Fig 2. CmpdA reduces growth of lung cells in a KRAS dependent manner

A) Primary immortalized human airway cells (SALEB) and their KRAS-transformed counterpart (SAKRAS) were treated with 0.1% DMSO or 5μM CmpdA for the indicated timepoints. 2μg/mL Doxorubicin (Doxo) was used as a positive control. Cell growth was measured using a colorimetric MTS tetrazolium assay (CellTiter 96® AQ_ueous One Solution Cell Proliferation Assay from Promega, Madison, WI). B) H1703-TrexB and H1703-G12V lung cancer cells were treated with 2μg/mL doxycycline (Doxy) for 24h where indicated to induce KRAS expression. Subsequently cells were treated with 0.1% DMSO or 5μM CmpdA for 30 min and analyzed for NF-κB activity by Western Blotting. Antibodies used are indicated. C) H1703-TrexB and H1703-G12V cells were transfected with 100ηg of an NF-κB-responsive firefly luciferase reporter vector (3x-κB-Luc) and 5ηg of Renilla luciferase vector (pRL-TK). Cells were induced with 2μg/mL doxycycline (+DOX) or left untreated (-DOX) for 24h and subsequently treated for 16h with either 0.1% DMSO or 5μM CmpdA as indicated. Results are expressed as the luciferase activity ratio of induced/uninduced cells (+DOX/-DOX). RLUs) Relative luciferase units. D) H1703-TrexB and H1703-G12V cells were treated with 2μg/mL Doxycycline (+DOX) for 24h to induce KRAS expression. Control cells were left untreated (-DOX). Subsequently, they were treated either with 0.1% DMSO or 5μM CmpdA and cell growth was measured 48h later using a colorimetric MTS tetrazolium assay (CellTiter 96® AQ_ueous One Solution Cell Proliferation Assay from Promega, Madison, WI). Results are expressed as growth ratio of induced/uninduced cells (+DOX/-DOX). Statistical significance in all cases was measured by Student's t-test (*p<0.05) when compared to experimental control samples (DMSO). Error bars represent average ± 1s.d.

Fig 3. CmpdA reduced growth of KRAS positive lung cancer cells depends on loss or mutation of p53

A) Human and mouse KRAS positive lung cancer cell lines harboring wildtype (WT) p53 (A549, H460, KE67), mutant p53 (H1792) or lacking (null) p53 (H358, KPF54) were treated with either 0.1% DMSO or 5μM CmpdA as indicated. 2μg/mL Doxorubicin (Doxo) was used as a positive control. Cell growth was measured at the indicated timepoints using a colorimetric MTS tetrazolium assay (CellTiter 96® AQ_ueous One Solution Cell Proliferation Assay from Promega, Madison, WI). B) p53 WT KE67 and p53 null KPF54 cell lines were transfected with 100ηg of an NF-κB-responsive firefly luciferase reporter vector (3x-κB-Luc) and 5ηg of Renilla luciferase vector (pRL-TK) and NF-κB activity was analyzed by dual luciferase reporter assays. –LUC) negative control (cells transfected with pcDNA3 instead of 3x-κB-Luc and pRL-TK); RLUs) Relative luciferase units. C) H358 and A549 cells were analyzed for NF-κB activity by Western Blotting. Antibodies used are indicated. D) p53 null H358 cells were transfected with 100ηg of an NF-κB-responsive firefly luciferase reporter vector (3x-κB-Luc), 5ηg of Renilla luciferase vector (pRL-TK) and 200ηg of either empty pcDNA3 or pcDNA3-p53. After transfection, cells were treated with either 0.1% DMSO or 5μM CmpdA for 16h. NF-κB activity was analyzed by dual luciferase reporter assays. Expression of recombinant p53 was evaluated by Western Blotting. Antibodies used are indicated. RLUs) Relative luciferase units. E) p53 WT murine KE67 cells were transfected as described in methods with a siRNA targeting murine p53 (sip53) or non-targeting siRNA (NTctrl). At 48h these cells were transfected with 100ηg of an NF-κB-responsive firefly luciferase reporter vector (3x-κB-Luc) and 5ηg of Renilla luciferase vector (pRL-TK) and analyzed at 72h after siRNA transfection for knockdown efficiency by Western Blotting. Antibodies used are indicated. F) KE67 cells were transfected as described in (E) and NF-κB activity was analyzed by dual luciferase reporter assays. –LUC) negative control (cells transfected with pcDNA3 instead of 3x-κB-Luc and pRL-TK); RLUs) Relative luciferase units. G) KE67 cells were transfected as described in methods with a siRNA targeting murine p53 (sip53) or non-targeting siRNA (NTctrl). Subsequently cell growth was measured at 72h post-transfection using a colorimetric MTS tetrazolium assay (CellTiter 96® AQ_ueous One Solution Cell Proliferation Assay from Promega, Madison, WI). H) A549 cells were transfected as described in methods with a siRNA smartpool targeting human p53 (sip53) or a non-targeting siRNA (NTctrl). Subsequently, cells transfected with sip53 were transfected either with pcDNA3 (empty vector control) or with pcDNA3-p53 vector. Analysis of p53 knockdown and p53 re-expression was performed by Western Blotting. Antibodies used are indicated. I) A549 cells were transfected as described in H and cell growth was evaluated at 72h post-transfection using a colorimetric MTS tetrazolium assay (CellTiter 96® AQ_ueous One Solution Cell Proliferation Assay from Promega, Madison, WI). Statistical significance in all cases was measured by Student's t-test (*p<0.05) when compared to experimental control samples (siCtrl). Error bars represent average ± 1s.d.

Fig 4. IKK targeting reduces lung cell growth by inhibiting cell proliferation

A) Proliferation was evaluated by BrdU incorporation to measure DNA synthesis in a colorimetric assay (BrdU Cell Proliferation Assay from EMD Millipore, Billerica, MA) in all indicated cell lines after 0.1% DMSO or 5μM CmpdA treatment for 48h. 2μg/mL Doxorubicin (Doxo) was used as a positive control. B) The indicated cell lines were transfected as described in methods with a non-targeting control siRNA (siCtrl) or with siRNA smartpools targeting KRAS (siKRAS), IKKβ (siIKKβ) or IKKα (siIKKα) and efficacy of siRNA targeting was evaluated at 72h by Western Blotting with the indicated antibodies. C) Cell growth of siRNA-transfected cells (as in B) was measured using a colorimetric MTS tetrazolium assay (CellTiter 96® AQ_ueous One Solution Cell Proliferation Assay from Promega, Madison, WI); D) Proliferation of siRNA transfected cells (as in B) was evaluated at 72h by BrdU incorporation to measure DNA synthesis (BrdU Cell Proliferation Assay from EMD Millipore, Billerica, MA). Statistical significance in all cases was measured by Student's t-test (*p<0.05) when compared to experimental control samples (DMSO). Error bars represent average ± 1s.d.

Fig 5. Targeting IKKβ in the KRAS^G12D/p53Δ conditional mouse lung tumor model reduces tumor burden in vivo

KRAS^G12D/p53Δ mice were analyzed at 12 weeks post-infection and after 4 weeks of CmpdA or placebo (DMSO) treatment (as described in methods). A) Immunohistochemistry for phospho-IκBα (positive cells are brown). A representative picture of stained lung tumor slides of CmpdA-treated and placebo-treated lungs is displayed on the left panel. The graph on the right panel is a quantitative representation of the data. B) Western Blotting analysis of IκBα phosphorylation/degradation of CmpdA-treated and placebo-treated lung tumors. Antibodies used are indicated. C) A representative picture of Hematoxylin/Eosin (H/E) stained lung slides of CmpdA-treated and placebo-treated lungs. D) Number of KRAS-induced neoplastic lesions was determined by counting lesions in H/E stained lung sections as described in methods. E) Average tumor area was determined by measuring tumor area using ImageJ software. F) TUNEL positive cells (brown) were stained according to the Apoptag Plus In Situ Apoptosis Detection Kit (EMD Millipore, Billerica, MA). A representative picture of stained lung slides of CmpdA-treated and placebo-treated lungs is displayed (rare positive cells are indicated by arrows). A DNase treated slide was used as a positive control (+ control). G) Immunohistochemistry for KI67 (positive cells are brown). A representative picture of stained lung slides of CmpdA-treated and placebo-treated lungs is displayed on the top panel. The graph on the bottom panel is a quantitative representation of the data. H) Analysis of lung tumor grade was performed on H/E stained lung sections as described in methods. Statistical significance in all cases was measured by Student's t-test (*p<0.05) when compared to experimental control samples (DMSO). Error bars represent average ± 1s.d.