Doxorubicin effect is enhanced by sphingosine-1-phosphate signaling antagonist in breast cancer

Abstract

Background: Doxorubicin is one of the most commonly used chemotherapeutic drugs for breast cancer; however, its use is limited by drug resistance and side effects. We hypothesized that adding FTY720, a sphingosine-1-phosphate (S1P) receptor functional antagonist, to doxorubicin would potentiate its effects by suppression of drug-induced inflammation.

Materials and methods: The Cancer Genome Atlas, Gene Expression Omnibus data sets, and National Cancer Institute-60 panel were used for gene expressions and gene set enrichment analysis. E0771 syngeneic mammary tumor cells were used. OB/OB mice fed with western high-fat diet were used as an obesity model.

Results: STAT3 expression was significantly increased after doxorubicin treatment in human breast cancer that implicates that doxorubicin evokes inflammation. Expression of sphingosine kinase 1, the enzyme that produces S1P and links inflammation and cancer, tended to be higher in doxorubicin-resistant human cancer and cell lines. In a murine breast cancer model, sphingosine kinase 1, S1P receptor 1, interleukin 6, and STAT3 were overexpressed in the doxorubicin-treated group, whereas all of them were significantly suppressed with addition of FTY720. Combination therapy synergistically suppressed cancer growth both in vitro and in vivo. Furthermore, combination therapy showed higher efficacy in an obesity breast cancer model, where high body mass index demonstrated trends toward worse disease-free and overall survival, and high-serum S1P levels in human patients and volunteers.

Conclusions: We found that FTY720 enhanced the efficacy of doxorubicin by suppression of drug-induced inflammation, and combination therapy showed stronger effect in obesity-related breast cancer.

Keywords: Breast cancer; Doxorubicin; FTY720; Mouse model; Obesity; Sphingosine-1-phosphate.

Figure 1

Differences in S1P signaling and inflammation related gene expression with doxorubicin treatment: (A) gene expression in patient tumors pre- and post- doxorubicin treatment, (B) gene expression in good and bad responders pre- and post- doxorubicin treatment, (C) gene expression in doxorubicin sensitive and resistant cell lines. **: p<0.05. *: p<0.1

Figure 2

FTY720 enhanced doxorubicin efficacy in vitro. (A) Drug sensitivity assay of FTY720 for E0771. (B) Gene expression of E0771 after doxorubicin with and without FTY720 treatment. *: p<0.05.

Figure 3

Doxorubicin induced S1P and inflammation related gene overexpression in E0771 syngeneic breast cancer model. (A) Tumor growth with doxorubicin, FTY720 and combination therapy. Tumor weight in the combination therapy group was significantly less than the single drug treatment groups. (B) mRNA expression of SphK1, S1PR1, IL6 and STAT3 in the tumor were determined by qPCR and normalized to levels of GAPDH. (C) Immunohistochemistry of SphK1. SphK1 expression was upregulated in doxorubicin treated tumors. Data are expressed as means ± SEM. *, p < 0.05.

Figure 4

Breast cancer prognosis and serum S1P levels according to BMI. (A, B) Higher BMI trended toward significantly worse OS and DFS. (C) Higher BMI individuals showed significantly higher plasma S1P levels in healthy individuals. Data are expressed as means ± SEM.

Figure 5

Doxorubicin and FTY720 suppressed tumor growth in an obesity syngeneic breast cancer model. (A) Tumor growth in doxorubicin, FTY720 and combination therapy groups in OB/OB mice. Tumor weight in each single drug treatment and combination therapy group was significantly less than the vehicle treated group. (B) FTY720 significantly suppressed tumor growth in an obesity model compared to a normal weight model. (C, D) GSEA of TCGA contributor from RPCI whole cohort and ER positive only. Lower BMI was significantly correlated with doxorubicin resistant genes, and higher BMI was significantly correlated with the S1P signaling pathway in the ER positive cohort. Data are expressed as means ± SEM. *, p < 0.05.