Targeting NFĸB mediated breast cancer chemoresistance through selective inhibition of sphingosine kinase-2

Abstract

Resistance to chemotherapy remains a significant obstacle in the treatment of hormone- independent breast cancer. Recent evidence suggests that altered sphingolipid signaling through increased sphingosine kinase activity may be an important mediator of breast cancer drug resistance. Sphingosine kinase-1 (Sphk1) is a proposed key regulator of breast cancer tumorigenesis, proliferation and resistance. There is, however, conflicting data on the role of sphingosine kinase-2 (Sphk2) in cancer biology and resistance, with some suggesting that Sphk2 has an opposing role to that of Sphk1. Here, we studied the effects of the novel selective Sphk2 inhibitor, ABC294640 (3-(4-chlorophenyl)-adamantane-1-carboxylic acid (pyridin-4-ylmethyl) amide), on human breast cancer. ABC294640 blocked both viability and survival at low micromolar IC(50) concentrations in the endocrine therapy-resistant MDA-MB-231 and chemoresistant MCF-7TN-R cell systems. Treatment with the inhibitor significantly reduced proliferation, as seen in immunofluorescence staining of Ki-67 in vitro. Interestingly, pharmacological inhibition of Sphk2 induced apoptosis through the intrinsic programmed cell death pathway. Furthermore, ABC294640 also diminished NF-ĸB survival signaling, through decreased activation of the Ser536 phosphorylation site on the p65 subunit. Xenografts of MCF-7TN-R cells growing in immunocompromised mice were utilized to validate the therapeutic efficacy of the sphingosine kinase-2 inhibitor. Treatment with 50 mg of ABC294640/kg completely blocked tumor volume in this model. These results indicate that pharmacological inhibition of Sphk2 with the orally bioavailable selective inhibitor, ABC294640, has therapeutic potential in the treatment of chemo- and endocrine therapy- resistant breast cancer.

Figure 1

Dysregulation of sphingolipid signaling in drug resistant breast cancer cells. (A) PCR analysis for endogenous expression of sphingosine kinase isoforms in drug resistant breast cancer cells. Data are expressed as fold-change relative to MCF10A cell control as normalized to internal β-actin ±SEM. Data points and error bars represent the mean ± SEM of three independent experiments. (B) MCF-7, MDA-MB-231 and MCF-7TN-R cells were measured for cellular levels of various sphingolipid species using ESI/MS/MS. Data points and error bars represent the mean ± SEM of three independent experiments. (C) MCF10A, MDA-MB-231 and MCF-7TN-R cells were plated at 7.5 × 10⁵ cells per 96 well plate. The following day cells were treated with indicated concentrations of ABC294640 for 24 h. Data are presented as percent of vehicle treated samples. Mean values of ±SEM of five different experiments in quadruplicate are reported. (D) MCF10A, MDA-MB-231 and MCF-7TN-R cells were plated at 500 cells per 60 mm². The following day, cells were treated with ABC294640 for 10–14 days. Colonies ≥50 cells were scored as positive for colony formation. Data are presented as percent of vehicle treated samples. Mean values of ±SEM of three different experiments in duplicate are reported.

Figure 2

Anti-proliferative effects of ABC294640 on drug resistant breast cancer cells. (A) MDA-MB-231 cells and (B) MCF-7TN-R cells were treated with vehicle or ABC294640 (10 µM) for 48 h. Following treatment, cells were fixed and stained with anti-Ki-67 (red) and nuclei counter stained with DAPI (blue). (A) Representative images of cells at ×250. (B) Quantification of cells positive for Ki-67 staining from ten fields of view per treatment. Data is represented as percent positive cells as compared to total cells normalized to vehicle control. Bars represent mean values ± SEM, (**p < 0.01, ***p < 0.001).

Figure 3

ABC294640 induces intrinsic apoptosis in drug resistant breast cancer cells. MCF-7TN-R cells were plated at 7.5 × 10⁵ cells per 96 well plate and treated with 50 µM of ABC294640 for 24 h. Following incubation, cells were (A) measured for fragmented DNA oligonucleotides using ELIZA assays, (B) analyzed for caspase-9 activation and (C) measured for viability using MTT analyses. Mean values of ±SEM of four different experiments in duplicate are reported.

Figure 4

ABC294640 increases doxorubicin and etoposide-induced intrinsic apoptosis. MCF-7TN-R cells were plated at 15,000 cells per well in a 96 well plate in phenol-free DMEM. The following day cells were treated with ABC294640 (10 µM), chemotherapeutic (etoposide 15 µM, doxorubicin, 0.1 µM) or ABC294640 + chemotherapeutic for 24 h. Following incubation, cells were (A) measured for fragmented DNA oligonucleotides using ELIZA assays, (B) analyzed for caspase-9 activation and (C) measured for viability using MTT analyses. Mean values of ±SEM of four different experiments in duplicate are reported.

Figure 5

ABC294640 blocks NFκB transcriptional activity in vitro. (A) MCF-7TN-R cells were transiently transfected with pFC-NFκB-luciferase plasmid. Following transfection, cells were treated with vehicle, TNFα, SKI or SKI + TNFα. Cells treated with TNFα were set to 1. Data points and error bars represent the mean ± SEM of three independent experiments. (B) MCF-7TN-R cells were treated with vehicle or ABC294640 for 18 h and analyzed for mRNA levels of ZEB1, BIRC2 and BIRC3 using qRT-PCR. Data are expressed as fold-change relative to vehicle control as normalized to internal β-actin ±SEM (*p < 0.05). Data points and error bars represent the mean ± SEM of three independent experiments. (C) MCF-7TN-R cells were treated with vehicle or ABC294640 (10 µM) for 6 h and analyzed by western blot for phosphorylated and total IκK, IκB and p65 proteins with densitometry analysis. Phosphorylated protein levels were normalized to corresponding tubulin and vehicle control set to 1. Data shown are representative of three independent experiments.

Figure 6

ABC294640 decreases chemoresistant breast cancer growth. 5 × 10⁶ MCF-7TN-R cells were injected in the mammary fat pads of female overiectomized mice with exogenous estrogen pellets. Tumors were allowed to form over 7 days. Mice were treated i.p with 50 mg/kg of ABC294640 for 14 days. Tumor volume was measured every 3 days. Diminished volume of treatment tumors at endpoint were statistically significant from vehicle (**p < 0.01). (B) Tumors from vehicle and ABC294640 treated mice were processed stained Ki-67. Representative images of Ki-67 staining in tumor sections are shown. (C) Quantitation of Ki-67 staining is expressed as percent positive of total number of cells per field of view (***p < 0.001, **p < 0.01, *p < 0.05).