IKKβ Kinase Promotes Stemness, Migration, and Invasion in KRAS-Driven Lung Adenocarcinoma Cells

Abstract

KRAS oncogenic mutations are widespread in lung cancer and, because direct targeting of KRAS has proven to be challenging, KRAS-driven cancers lack effective therapies. One alternative strategy for developing KRAS targeted therapies is to identify downstream targets involved in promoting important malignant features, such as the acquisition of a cancer stem-like and metastatic phenotype. Based on previous studies showing that KRAS activates nuclear factor kappa-B (NF-κB) through inhibitor of nuclear factor kappa-B kinase β (IKKβ) to promote lung tumourigenesis, we hypothesized that inhibition of IKKβ would reduce stemness, migration and invasion of KRAS-mutant human lung cancer cells. We show that KRAS-driven lung tumoursphere-derived cells exhibit stemness features and increased IKKβ kinase activity. IKKβ targeting by different approaches reduces the expression of stemness-associated genes, tumoursphere formation, and self-renewal, and preferentially impairs the proliferation of KRAS-driven lung tumoursphere-derived cells. Moreover, we show that IKKβ targeting reduces tumour cell migration and invasion, potentially by regulating both expression and activity of matrix metalloproteinase 2 (MMP2). In conclusion, our results indicate that IKKβ is an important mediator of KRAS-induced stemness and invasive features in lung cancer, and, therefore, might constitute a promising strategy to lower recurrence rates, reduce metastatic dissemination, and improve survival of lung cancer patients with KRAS-driven disease.

Keywords: IKKβ kinase; KRAS; NF-κB signalling; cancer stem cells; invasion; lung cancer; migration; stemness.

Figure 1

KRAS-mutant tumoursphere-derived cells exhibit stemness features and increased IKKβ kinase activity. (A) Clonogenic assays of adherent (AD) and tumoursphere-derived (TS) A549 and H358 cells. Cells were plated and colonies formed were stained with crystal violet and colony area was analysed using Image J software. Images shown are representative of three independent experiments. (B) Relative expression of SOX2, OCT4, NANOG, CXCR4, BMI1, and CD24 was analysed by real-time quantitative PCR in adherent (AD) and tumoursphere-derived (TS) A549 and H358 cells using β-ACTIN as endogenous control. (C) Western blotting of adherent (AD) and tumoursphere-derived (TS) A549 and H358 cells. Antibodies used are indicated. Protein bands were quantitated and normalized to the reference sample using ImageJ software. Nitrocellulose membrane was cut before probing with the respective primary antibody and full membrane blots are presented in Figure S1. Images shown are representative of three independent experiments. In all cases, bar graphs represent average ±1 SD of three independent experiments (n = 3). Statistical significance was determined by Student’s t-test (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001). Groups being compared are indicated by horizontal bars.

Figure 2

IKKβ targeting reduces the expression of stemness-associated genes in KRAS-mutant lung cancer cells. (A) Western blotting of A549 and H358 cells treated with 0.1% DMSO or 5 µM Compound A (CmpdA) for 30 min. Antibodies were used as indicated. Nitrocellulose membrane was cut before probing with the respective primary antibody and full membrane blots are presented in Figure S2. Representative western blots are shown. Protein bands were quantitated and normalized to the reference samples (0.1% DMSO-treated samples) using Image J software. (B) A549 and H358 cells were treated with 0.1% DMSO or the indicated concentrations of CmpdA for 48 h and expression of SOX2, OCT4, NANOG, BMI1, and CXCR4 was evaluated by qRT-PCR using β-ACTIN as endogenous control. Bar graphs represent average ±1 SD of three independent experiments (n = 3). Statistical significance was determined by one-way ANOVA with a post hoc Turkey test. (* p < 0.5, ** p < 0.01, *** p < 0.001, **** p < 0.0001) by comparing CmpdA-treated groups with the DMSO-treated group.

Figure 3

IKKβ targeting with CmpdA reduces tumoursphere formation and preferentially impairs proliferation of KRAS-mutant tumoursphere-derived cells. (A) A549 and H358 cells were treated with the indicated concentrations of CmpdA or with vehicle control (0.1% DMSO) and plated for tumoursphere cultures. Tumoursphere number was determined by manual counting. Images shown are representative of three independent experiments (n = 3). White scale bars represent 50 µm. (B) Clonogenic assays of A549 adherent cells treated with CmpdA or vehicle control (0.1% DMSO) were compared to clonogenic assays of A549 cells derived from CmpdA-treated or control-treated (0.1% DMSO) tumourspheres. Adherent or tumoursphere-derived cells were plated and colonies formed were stained with crystal violet. Colony number and colony area were quantified using Image J software. Images shown are representative of three independent experiments (n = 3). (C) Growth curves measured by IncuCyte time-lapse video microscopy of adherent-derived and tumoursphere-derived A549 cells upon treatment with 0.1% DMSO and 5 µM CmpdA (left) or 10 µM CmpdA (right). A representative growth curve of two independent experiments (n = 2) is shown for each condition. Error bars show ±1 SD for technical triplicates. In all cases, bar graphs represent average ± 1 SD of three independent experiments (n = 3). Statistical significance was determined by one-way ANOVA with a post hoc Turkey test (* p < 0.5, *** p < 0.001) (A) or by Student’s t-test (* p < 0.05, ** p < 0.01, *** p < 0.001, ns = not significant) (B), by comparing CmpdA-treated groups with the DMSO-treated group.

Figure 4

Small interfering RNA (siRNA)-mediated IKKβ targeting reduces tumoursphere formation and self-renewal of KRAS-mutant lung cancer cells. A549 and H358 cells were transfected with a non-targeting control siRNA (siCtrl) or with siRNA SMARTpools targeting KRAS (siKRAS) or IKKβ (siIKKβ) as described in methods. (A) Protein lysates were collected 96 h post-transfection and evaluated by Western Blotting with the indicated antibodies. Protein bands were quantitated and normalized to the reference samples (siCtrl samples). Nitrocellulose membrane was cut before probing with the respective primary antibody. Lanes from blots cropped from different membranes are separated by a black line and full membrane blots are presented in Figure S4. Images shown are representative of three independent experiments (n = 3). (B) Expression of KRAS (left panel) or IKKβ (right panel) was analysed 72 h post-transfection by RT-qPCR in each cell line as indicated using GAPDH as endogenous control. (C) Serial tumoursphere formation assay of siKRAS- or siIKKβ-transfected A549 cells compared to siCtrl-transfected A549 cells. Representative images of primary, secondary and tertiary A549 tumourspheres for each siRNA transfection condition are shown. White scale bar represents 100 µm. (D) Primary tumoursphere formation assay of siKRAS- or siIKKβ-transfected H358 cells compared to siCtrl-transfected H358 cells. Representative images of H358 tumourspheres for each siRNA transfection condition are shown. White scale bar represents 100 µm. In all cases, bar graphs represent average ±1 SD of three independent experiments (n = 3). Statistical significance was determined by the Student’s t-test (**** p < 0.0001) by comparing siCtrl-transfected groups with siKRAS- or with siIKKβ-transfected groups (B) or by one-way ANOVA with a post hoc Turkey test (* p < 0.5, ** p < 0.01, *** p < 0.001) (C and D). Groups being compared are indicated by horizontal bars.

Figure 5

IKKβ kinase targeting with Compound A reduces the expression of matrix metalloproteinase genes, tumour cell migration and invasion. A549 and H358 cells were treated with 0.1% DMSO or the indicated concentrations of CmpdA. (A) After 24 h, expression of MMP2 and MMP9 was evaluated by RT-qPCR using β-ACTIN as endogenous control. (B) After 24 h, transwell migration assays were performed as described in methods. Images shown are representative of three independent experiments (n = 3). White scale bar represents 100 µm. (C) Real-time wound healing assays of A549 cells were performed by IncuCyte time-lapse video microscopy over 72 h of treatment with 0.1% DMSO or the indicated concentrations of CmpdA. Results are expressed as percentage of confluence in the wound. A representative wound confluence curve of two independent experiments is shown for each condition. Error bars show ±1 SD for technical triplicates. Representative images of A549 wound-healing assays at 0, 24 and 48 h are shown. White scale bar represents 300 µm. (D) After 24 h of treatment of A549 and (E) H358 cells with 0.1% DMSO or the indicated concentrations of CmpdA, transwell invasion assays were performed as described in methods. Images shown are representative of three independent experiments (n = 3). White scale bars represent 100 µm. In all cases, bar graphs represent average ±1 SD of three independent experiments (n = 3). Statistical significance was determined by one-way ANOVA with a post hoc Turkey test. (* p < 0.5, ** p < 0.01, *** p < 0.001, ns = not significant). Groups being compared are indicated by horizontal bars.

Figure 6

siRNA-mediated targeting of IKKβ or KRAS reduces the expression and activity of matrix metalloproteinases, tumour cell migration and invasion. A549 and H358 cells were transfected with a non-targeting control siRNA (siCtrl) or with siRNA SMARTpools targeting KRAS (siKRAS) or IKKβ (siIKKβ) as described in methods. (A) After 72 h, expression of MMP2 and MMP9 was evaluated by RT-qPCR using GAPDH as endogenous control. (B) Conditioned culture medium was collected 96 h post-transfection and matrix metalloproteinase 2 (MMP2) and matrix metalloproteinase 9 (MMP9) activity was determined using ELISA-based Biotrack Activity Assay Systems (GE Healthcare). (C) Transwell cell migration assays were performed as described in methods 96 h post-transfection. Images shown are representative of three independent experiments (n = 3). White scale bar represents 50 µm. (D) Transwell cell invasion assays for A549 and (E) H358 were performed as described in methods 72 h post-transfection. Images shown are representative of three independent experiments (n = 3). White scale bars represent 50 µm. In all cases, bar graphs represent average ±1 SD of three independent experiments (n = 3). Statistical significance was determined by one-way ANOVA with a post hoc Turkey test (* p < 0.05, *** p < 0.001, **** p < 0.0001). Groups being compared are indicated by horizontal bars.