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. 2015 Jun 8;4(6):e156.
doi: 10.1038/oncsis.2015.16.

The Phosphorylated Prodrug FTY720 Is a Histone Deacetylase Inhibitor That Reactivates ERα Expression and Enhances Hormonal Therapy for Breast Cancer

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Free PMC article

The Phosphorylated Prodrug FTY720 Is a Histone Deacetylase Inhibitor That Reactivates ERα Expression and Enhances Hormonal Therapy for Breast Cancer

N C Hait et al. Oncogenesis. .
Free PMC article

Abstract

Estrogen receptor-α (ERα)-negative breast cancer is clinically aggressive and does not respond to conventional hormonal therapies. Strategies that lead to re-expression of ERα could sensitize ERα-negative breast cancers to selective ER modulators. FTY720 (fingolimod, Gilenya), a sphingosine analog, is the Food and Drug Administration (FDA)-approved prodrug for treatment of multiple sclerosis that also has anticancer actions that are not yet well understood. We found that FTY720 is phosphorylated in breast cancer cells by nuclear sphingosine kinase 2 and accumulates there. Nuclear FTY720-P is a potent inhibitor of class I histone deacetylases (HDACs) that enhances histone acetylations and regulates expression of a restricted set of genes independently of its known effects on canonical signaling through sphingosine-1-phosphate receptors. High-fat diet (HFD) and obesity, which is now endemic, increase breast cancer risk and have been associated with worse prognosis. HFD accelerated the onset of tumors with more advanced lesions and increased triple-negative spontaneous breast tumors and HDAC activity in MMTV-PyMT transgenic mice. Oral administration of clinically relevant doses of FTY720 suppressed development, progression and aggressiveness of spontaneous breast tumors in these mice, reduced HDAC activity and strikingly reversed HFD-induced loss of estrogen and progesterone receptors in advanced carcinoma. In ERα-negative human and murine breast cancer cells, FTY720 reactivated expression of silenced ERα and sensitized them to tamoxifen. Moreover, treatment with FTY720 also re-expressed ERα and increased therapeutic sensitivity of ERα-negative syngeneic breast tumors to tamoxifen in vivo more potently than a known HDAC inhibitor. Our work suggests that a multipronged attack with FTY720 is a novel combination approach for effective treatment of both conventional hormonal therapy-resistant breast cancer and triple-negative breast cancer.

Figures

Figure 1
Figure 1
FTY720-P is produced in the nucleus of breast cancer cells by SphK2. Breast cancer cell lines, murine 4T1 (a, b), human MDA-MB-231 (c, d) and human MCF7 (f) were treated with 5 μM FTY720. (a, c) Nuclear levels of FTY720-P and S1P were determined by liquid chromatography, electrospray ionization/tandem mass spectrometry (LC-ESI-MS/MS) at the indicated times. Equal amounts of protein from nuclear and cytosolic fractions were analyzed by immunoblotting with SphK2 antibody. Antibodies against histone H3 or laminA/C and tubulin were used as nuclear and cytosol markers. (b, d) Total intracellular and secreted FTY720-P were determined in 4T1 cells after 8 h and MDA-MB-231 cells after 6 h of FTY720 treatment, respectively. MDA-MB-231 cells (e) and MCF7 cells (g) transfected with vector, SphK2 or catalytically inactive SphK2G212E (ciSphK2) were treated with vehicle or 5 μM FTY720 for 6 and 24 h, respectively. Nuclear levels of FTY720-P and S1P were determined by LC-ESI-MS/MS. Data are mean±s.d. *P<0.005 compared with vector; #P<0.005 compared with vehicle. Equal expression of nuclear SphK2 was confirmed by immunoblotting.
Figure 2
Figure 2
Nuclear FTY720-P enhances specific histone acetylations in breast cancer cells. MCF7 cells (a) and 4T1 cells (b) were treated with FTY720 (5 μM) for the indicated times. Histone acetylations in nuclear extracts were detected by immunoblotting with antibodies to specific histone acetylation sites. (c) Purified nuclei from naive MCF7 cells were incubated for the indicated times with vehicle, S1P (1 μM) or FTY720-P (1 μM) and histone acetylations determined. (d, e) Purified nuclei were isolated from MCF7 cells transfected with vector, SphK2 or ciSphK2 and treated with FTY720 (1 μM) for 15 min. (f, g) Purified nuclei were isolated from MCF7 cells transfected with siControl or siSphK2 and incubated with the indicated concentrations of FTY720 for 15 min. Histone acetylations were determined by immunoblotting (d, f) and levels of FTY720-P by liquid chromatography, electrospray ionization/tandem mass spectrometry (LC-ESI-MS/MS) (e, g). *P<0.05. (h, i) Naive MCF7 cells were treated with vehicle, FTY720-P (100 nM), FTY720 (1 μM) or SAHA (2 μM) for 2 h, nuclear extracts were analyzed by western blotting with the indicated antibodies (h) and HDAC activity measured and expressed as arbitrary fluorescence units (AFU) (i). *P<0.001.
Figure 3
Figure 3
Microarray analysis of genes regulated by FTY720 and SAHA. Gene expression in naive MCF7 cells or MCF7 cells treated with vehicle, S1P (100 nM), FTY720 (1 μM) or SAHA (1 μM) for 24 h was determined by microarray analyses. (a) Heatmap showing supervised hierarchical clustering of 713 genes differentially expressed in FTY720-treated cells compared with naive. Expression level of a given gene is indicated by red (high) and green (low). Note that not all of the genes differentially regulated by SAHA are shown. (b) Venn diagram of genes differentially regulated by FTY720 and SAHA. (c) The gene ontology (GO) Biological Process analyses of 276 common probe sets regulated by SAHA and FTY720 treatment ranked for biological processes.
Figure 4
Figure 4
FTY720 treatment suppresses advanced tumorigenic mammary lesions in HFD-fed PyMT transgenic mice. Female PyMT transgenic mice were fed with a normal diet (ND) or a Western HFD, and were treated daily with saline or FTY720 (1 mg/kg) by gavage starting after weaning. (a) Tumor volumes were determined at the indicated times. (b) Representative images of 10-week-old female PyMTTg mice fed with ND or HFD without or with FTY720. Note the difference in the size of the tumors in the mammary pads. (c) Tumor volumes and weights were determined at 11 weeks. (d, e) Tumor sections were stained with hematoxylin and eosin (H&E), proliferation determined by Ki67 staining, apoptosis by TUNEL and cyclin D1 expression determined by immunohistochemistry. Scale bars: 200 μm and 100 μm, as indicated. (e) Quantification of Ki67- and TUNEL-positive cells. Data are mean±s.e.m. #P<0.05 compared with ND; *P<0.05 compared with HFD.
Figure 5
Figure 5
FTY720 treatment reverses HFD-induced loss of estrogen and progesterone receptors in PyMT transgenic mice. Female PyMT transgenic mice were fed with a normal diet (ND) or a Western HFD, and were treated daily with saline or FTY720 (1 mg/kg) by gavage starting after weaning (n=5 each), as indicated. (a, b) Nuclear extracts from tumors were analyzed by western blotting with the indicated antibodies. (c) Representative images of tumor sections immunostained with anti-ERα or anti-H3-K9ac antibodies. Scale bars: 20 μm. (d) HDAC activity in nuclear extracts of tumors was determined and expressed as arbitrary fluorescence units. (e) FTY720 and FTY720-P levels in nuclear extracts of tumors from mice on HFD treated with FTY720 were measured by liquid chromatography, electrospray ionization/tandem mass spectrometry (LC-ESI-MS/MS). (f) ERα, PR and ErbB2 mRNA levels in tumors were quantified by quantitative real-time PCR (QPCR) and normalized to Gapdh. Data are mean±s.e.m. *P<0.05 compared with ND; #P<0.05 compared with HFD.
Figure 6
Figure 6
FTY720 induces ERα expression in ERα-negative human and murine breast cancer cells and sensitizes them to tamoxifen. 4T1 (a) and MDA-MB-231 (b) cells were treated with FTY720 (5 μM) or SAHA (1 μM) for 24 h. ERα, PR and ERβ mRNA levels were determined by quantitative real-time PCR (QPCR) and normalized to GAPDH. (c) Proteins in MDA-MB-231 nuclear extracts were analyzed by immunoblotting with the indicated antibodies. LaminA/C was used as a loading control. (d) MDA-MB-231 cells were subjected to chromatin immunoprecipitation (ChIP) analyses with antibodies to H3-ac, H3 or normal rabbit IgG, as indicated. The precipitated DNA was analyzed by real-time PCR with primers amplifying the core promoter sequence of the ERα gene. Relative binding to the promoter is expressed as fold enrichment compared with input. Data are mean±s.d. *P<0.003 compared with vehicle. (e) MDA-MB-231 cells were treated with FTY720 (1 nM) without or with 10 nM E2 for the indicated days and cell proliferation was determined by WST assay. (f, g) MDA-MB-231 cells (f) or 4T1 cells (g) were treated with the indicated concentrations of TAM or FTY720, or with 2.5 μM FTY720 with increasing concentrations of TAM for 48 h and cell proliferation determined. Data are expressed as % of untreated control.
Figure 7
Figure 7
FTY720 reduces breast tumor growth and enhances anticancer effectiveness of TAM in ERα-negative 4T1 syngeneic xenografts. 4T1 cells were surgically implanted into the second mammary fat pads under direct vision. Tumor-bearing mice were randomized into five groups 2 days after implantation and then treated with vehicle, FTY720 (1 mg/kg), TAM (25 mg/kg), FTY720 plus TAM or SAHA (intraperitoneal (i.p.) 20 mg/kg) plus TAM by gavage daily till day 15 (n=8). (a) Tumor volumes were measured daily. (Insert) Tumor volumes on day 15. (b) Representative tumors. *P<0.01, #P<0.05 compared with vehicle. (c, d) Immunohistochemical staining of tumor sections for TUNEL (c) and ERα (d). Scale bar: 20 μm. Quantifications of TUNEL-positive cells and ERα intensity are shown. *P<0.05 compared with vehicle or TAM. (e) HDAC activity in nuclear extracts of tumors was determined and expressed as arbitrary fluorescence units. (f) Expression of ERα in the tumors was analyzed by quantitative real-time PCR (QPCR) and normalized to Gapdh. (g) Nuclear extract proteins were analyzed by western blotting with the indicated antibodies. Histone H3 was used as loading control. Data are mean±s.e.m. *P<0.01 compared with vehicle or TAM.

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