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. 2014 Aug 15;5(15):5920-33.
doi: 10.18632/oncotarget.1874.

SPHK1 Regulates Proliferation and Survival Responses in Triple-Negative Breast Cancer

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Free PMC article

SPHK1 Regulates Proliferation and Survival Responses in Triple-Negative Breast Cancer

Arpita Datta et al. Oncotarget. .
Free PMC article

Abstract

Triple-negative breast cancer (TNBC) is characterized by unique aggressive behavior and lack of targeted therapies. Among the various molecular subtypes of breast cancer, it was observed that TNBCs express elevated levels of sphingosine kinase 1 (SPHK1) compared to other breast tumor subtypes. High levels of SPHK1 gene expression correlated with poor overall and progression- free survival, as well as poor response to Doxorubicin-based treatment. Inhibition of SPHK1 was found to attenuate ERK1/2 and AKT signaling and reduce growth of TNBC cells in vitro and in a xenograft SCID mouse model. Moreover, SPHK1 inhibition by siRNA knockdown or treatment with SKI-5C sensitizes TNBCs to chemotherapeutic drugs. Our findings suggest that SPHK1 inhibition, which effectively counteracts oncogenic signaling through ERK1/2 and AKT pathways, is a potentially important anti-tumor strategy in TNBC. A combination of SPHK1 inhibitors with chemotherapeutic agents may be effective against this aggressive subtype of breast cancer.

Figures

Figure 1
Figure 1. SPHK1 expression in breast tumors
(A) SPHK1mRNA expression in human breast tumors and paired adjacent normal breast tissues by real-time PCR. Expression levels were normalized with GAPDH. A two-tailed Student t-test was used to calculate statistical significance. (B) SPHK1 mRNA expression in ER-positive, ER (+), and ER-negative, ER (-) cases. Each dot corresponds to an individual patient's fold change in relative SPHK1 mRNA levels between tumor and adjacent normal tissue. ER (-) patients showed significantly higher expression of SPHK1 than ER (+) patients (p = 0.007). (C) SPHK1 gene expression levels in breast cancer subtypes. Basal subtype has the highest SPHK1 gene expression (Mann Whitney Test, p = 4.16e−81), whereas Luminal-A and –B subtypes have the lowest SPHK1 gene expression (Mann Whitney Test, p = 1.1e−21, and p = 6.74e−37, respectively). (D) SPHK1 gene expression correlates with poor overall (left) and progression-free survival (right). Kaplan-Meier plots of overall and progression-free survival in all samples. Median expression was used to define SPHK1-Low and SPHK1-High. The p-value shown was computed by log-rank test. HR indicates the hazard ratio, and n in parentheses indicates number of samples.
Figure 2
Figure 2. SPHK1 expression in breast cancer cells
(A) SPHK1 mRNA levels by real-time PCR in breast cancer cell lines and the breast epithelial cell line MCF10A (normalized to GAPDH). The data is expressed as the fold change in SPHK1 expression compared to MCF-10A. Data represents the mean ± SD of at least three independent experiments; *p < 0.05. (B) The protein expression of SPHK1 in breast cancer cell lines was determined by Western blot. (C) Endogenous SPHK activity in cell lines. Data represents the mean ± SD of at least three independent experiments; *p < 0.05.
Figure 3
Figure 3. Inhibition of SPHK1 activity induces apoptosis and inhibits proliferation of breast cancer cells
(A) Flow cytometric analysis of PI staining in MDA-MB-231, MCF-7 and MCF-10A cell lines fixed after treatment with 10-50μM SKI-5C for 24 hours. The graph shows percentages of cells in the Sub-G1 fraction for MDA-MB-231, MCF-7 and MCF-10A. Data represents the mean ± SD of at least three independent experiments; *p < 0.05. (B) MDA-MB-231 and MCF-7 cells were treated with 10-25μM SKI-5C for 24 hours and apoptotic cells were detected by flow-cytometric analysis of Annexin V staining. Data represents the mean ± SD of at least three independent experiments; *p < 0.05. (C) The effect of SKI-5C on cell proliferation was determined by culturing MCF-7, MDA-MB-231 and MCF10A cells under standard culture conditions in the presence of 10μM SKI-5C for 6 to 48 hours by BrdU assay. The relative fold changes in O.D. at 450nm compared to cells at the start of the assay (‘0’ hours) are shown, indicated as ‘Relative Proliferation’ on the y-axis. Data represents the mean ± SD of at least three independent experiments; *p < 0.05.
Figure 4
Figure 4. Inhibition of SPHK1 activity impairs colony formation and breast tumor formation by MDA-MB-231 cells in immunodeficient mice
(A) Top: Representative western blot of SPHK1 and GAPDH in MDA-MB-231 after transfection with SPHK1-specific siRNAs (SPHK1 siRNA1 and siRNA2) or control siRNA for 48 hours. Bottom: Representative images of colony formation assay in MDA-MB-231 cells transfected with either control siRNA or SPHK1 siRNAs. 48 hours after transfection, equivalent numbers of live cells were replated, cultured for 7 days, then stained with crystal violet. (B) Number of colonies from (A) were counted. Data are expressed as mean ± S.D. of triplicate measurements and of three independent experiments; *p < 0.01. (C) The effect of SKI-5C on colony formation was determined by treating MDA-MB-231 breast cancer cells grown at low cell density (1000 cells/well in a 6-well plate) with 5 - 25μM SKI-5C for 7 days. Colonies were stained with crystal violet and quantified. Data are expressed as mean ± S.D. of triplicate measurements and of three independent experiments; *p < 0.01. (D) SCID mice (6 mice per group) with palpable MDA-MB-231 tumors (2 tumors per mouse) were injected intraperitoneally with saline (control) or SKI-5C 1 or 10mg/kg body weight for 14 consecutive days. Tumor volumes were recorded daily. Animals were euthanised and tumors excised. (E) Representative picture shows MDA-MB-231 tumors treated with saline control or with SKI-5C at 10mg/kg body weight. Animals were euthanised and tumors excised and weighed. SKI-5C inhibits tumor growth and reduces tumor weight in a dose-dependent manner. Data are expressed as mean ± S.D. (F) Tumor histology. Paraffin-embedded tumor sections were stained with either H&E or PCNA. Apoptotic cells were visualized by TUNEL staining and counterstained with methyl green. Slides were analyzed by fluorescence microscopy. Representative section from n=3 individual treated tumors. Scale bar represents 50 μm.
Figure 5
Figure 5. SKI-5C inhibits the serum-dependent activation of ERK1/2 and AKT signalling and S1P export
Western blots showing the effect of SKI-5C on serum-dependent activation of ERK1/2 and AKT in MDA-MB-231 cells stimulated with 10% FBS for 5 - 120 minutes after treatment with 10μM SKI-5C. Control cells were stimulated with 10% FBS after pretreatment with DMSO. (B) Secreted S1P levels determined by ELISA in MDA-MB-231 cells pretreated with 10μM SKI-5C, followed by stimulation with 10% FBS for up to 240 minutes. Control cells were pretreated with DMSO. Data are expressed as mean ± S.D. of triplicate measurements and of three independent experiments; *p < 0.01. (C) Representative Western blot (of three independent experiments) showing the effect of PTX on serum induced ERK1/2 and AKT phosphorylation.
Figure 6
Figure 6. SPHK1 affects breast cancer sensitivity to Doxorubicin and 5-FU
(A) Comparison of baseline SPHK1 levels between clinical non-responders and responders to Doxorubicin-based treatment (8212±14901 vs 2948±3411, p=0.021). (B) Percentages of sub-G1 population by PI analysis in cells transfected with either control siRNA or SPHK1 siRNA1 for 48 hours (p = 0.00024), followed by addition of 20μM 5-FU or 10μM Doxorubicin for 24 hours. Data are expressed as mean ± S.D. of triplicate measurements and of three independent experiments; *p < 0.05 compared to SPHK1 siRNA 1, # p < 0.05 compared to 5-FU or Doxorubicin (Student's t- test). (C-E) Sub-G1 populations by PI analysis in combination treatment of cells using 10μM SKI-5C with low doses (below IC50) of 1-10μM Doxorubicin or 2 - 20μM 5-FU or 10-50nM Docetaxel for 24 hours compared to treatment with either SKI-5C, Doxorubicin, 5-FU or Docetaxel alone. Data represents the mean ± SD of at least three independent experiments; *p < 0.05.

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