IKK inhibition by BMS-345541 suppresses breast tumorigenesis and metastases by targeting GD2+ cancer stem cells

Abstract

We have identified that the ganglioside GD2 is a marker for breast cancer stem cells (BCSCs), and that targeting the enzyme GD3 synthase (GD3S, which regulates GD2 biosynthesis) reduces breast tumorigenesis. The pathways regulating GD2 expression, and their anomalous functions in BCSC, are unclear. Proteomic analysis of GD2+ and GD2- cells from breast cancer cell lines revealed the activation of NFκB signaling in GD2+ cells. Dose- and time-dependent suppression of NFκB signaling by the small molecule inhibitor BMS-345541 reduced GD2+ cells by > 90%. Likewise, BMS-345541 inhibited BCSC GD3S expression, mammosphere formation, and cell migration/invasion in vitro. Breast tumor-bearing mice treated with BMS-345541 showed a statistically significant decrease in tumor volume and exhibited prolonged survival compared to control mice, with a median survival of 78 d for the BMS-345541-treated group vs. 58 d for the controls. Moreover, in an experimental metastases model, treatment with BMS-345541 reduced the lung metastases by > 5-fold. These data suggest that GD2 expression and function,and NFκB signaling, are related, and they control BCSCs tumorigenic characteristics. Thus, the suppression of NFκB signaling by BMS-345541 is a potentially important advance in controlling breast cancer growth and metastases.

Keywords: GD2; GD3 synthase; NFκB; breast cancer; cancer stem cells.

Figure 1. Activation of NFκB signaling in GD2⁺ cells

A. MDA-MB-231 cells were stained with anti-GD2 conjugated with phycoerythrin (fluorochrome) or Sm152 (metal) and analyzed on an LSR II flow cytometer or by CyTOF, respectively. The data were analyzed on FlowJo software. B. MDA-MB-231 cells were incubated with anti-GD2 antibody conjugated with Sm152, anti-pNFκB(p65) conjugated with Sm149, anti-pPI3K(Y607) antibody conjugated with Gd158, and anti-pmTOR(S248) antibody conjugated with Dy164. All the samples were co-stained with and iridium (DNA-intercalator) conjugated with Rh103 for gating. The cells were analyzed on by CyTOF. The data were analyzed using the SPADE complex data analysis tool. A gradient from green (low) to red (high) indicates expression of each marker in different populations in the SPADE tree structure.. C. qRT-PCR analysis of IKKα in control and IKKα knockdown MDA-MB-231 cells. D. Western blot analysis to determine IKKα and GD3S protein expression in control and IKKα knockdown MDA-MB-231 cells. E. Bar graph represents quantification of IKKα and GD3S protein expression in control and IKKα knockdown MDA-MB-231 cells. GAPDH served as loading control F. Flow cytometry analysis was performed to measure GD2 expression in control and IKKα knockdown MDA-MB-231 cells. G. Bar graph represents the percentage of GD2⁺ cells in control and IKKα knockdown MDA-MB-231 cells.

Figure 2. BMS-345541 inhibits GD2 and GD3S expression by inhibiting NFκB signaling in breast cancer cells

A) MDA-MB-231 cells were treated with or without varying concentrations of BMS-345541 for 24 h. For the western blot, the cell lysates were run on a gel, and the transfer membranes were incubated with anti-pNFκB or anti-RelB antibodies. GAPDH served as a loading control. B. MDA-MB-231 cells were treated with BMS-345541 at concentrations of 1, 2.5, and 5 μM for 72 h. The cells were then stained with anti-GD2 antibody conjugated with allophycocyanin and analyzed on an LSR II flow cytometer. The cells were also stained with DAPI to exclude dead cells. C. The bar graph represents the absolute number of GD2⁺ cells from the experiment in Figure 2A. The assay was performed using TruCount absolute counting beads. D. GD3S mRNA expression analysis by quantitative RT-PCR. MDA-MB-231 cells were treated with BMS-345541 at concentrations of 0.1, 1, or 5 µM for 24 h. Relative GD3S mRNA expression levels were analyzed by TaqMan quantitative RT-PCR using the 7900HT fast real-time polymerase chain reaction system. 18S RNA served as the equal loading control.

Figure 3. BMS-345541 inhibits function of BCSCs

A. The bar graphs represent the number of mammospheres formed per 5000 MDA-MB-231 (A) or SUM159 B. cells seeded in low-adherent conditions with varying concentrations of BMS-345541 for 2 wk. C. and D. MDA-MB-231 (C) or SUM159 (D) cells were plated into soft agar with varying concentrations of BMS-345541. After 3 weeks of incubation, colonies were fixed with MTT and then counted using an automated colony counter. E. SUM159 cells were plated into trans-well migration plates with varying concentrations of BMS-345541. After 6 h, the membrane was fixed and stained, and then cells were counted. F. SUM159 cells were plated into trans-well invasion plates with matrigel; the media contained varying concentrations of BMS-345541. After 24 h, the membrane and Matrigel were removed, and the cells were counted.

Figure 4. BMS-345541 reduces the rate of tumor growth and increases survival in tumor-bearing mice

A. GFP/luciferase-expressing MDA-MB-231 cells were implanted into the mammary fat pads of mice (n = 14), who were then split into two treatment groups (Seven mice per group); one group was treated with BMS-345541 (25 mg/kg for three treatments per wk for 4 wk), while the other group was treated with control. The images show the luciferase activity in the mice over time. B. The bar graph represents the luminescence levels in the two groups of tumor-bearing mice. The y-axis represents the number of photons emitted. C. Survival analysis: Ten mice received MDA-MB-231 cells (1 × 10⁶ cells per mouse) in the mammary fat pads. One group was treated with BMS-345541, while the other group was treated with PBS as a control (three injections per week for 1 week). The survival curves were generated using Prism software (GraphPad Software, San Diego, CA).

Figure 5. BMS-345541 inhibits cancer metastasis in in vivo settings

A. GFP/luciferase-expressing MDA-MB-231 cells were injected into the tail veins of NSG mice (n = 10) in an experimental metastatic model. The mice were then split into two treatment groups; one group (5 mice per group) was treated with BMS-345541, while the other group was treated with PBS. The bar graph represents the luciferase activity of the groups. B. H and E staining of lung tissues derived from mice in experiment described in Figure 5A. The sections are derived from mice on d 34 after tumor implantation. C. and D. To quantitate the amount of metastasis in each group, lungs derived from treated and untreated groups on d 34 were stained with hematoxylin and eosin, and the sections were scanned using EVOS-FL auto microscope and the metastasis was quantitated using inForm software (PerkinElmer). E. The image illustrates the mechanism of action for BMS-345541, an IKK inhibitor. Through inhibition of IKK activity, IκB fails to get phosphorylated, leading to the inhibition of NFκB translocation across the nuclear membrane and inhibition of GD3S and GD2 expression.