Abstract P5-03-05: Sphingosine-1-phosphate produced by sphingosine kinase 1 and exported via ABCC1 shortens survival of mice and humans with breast cancer
Authors: Yamada A , Nagahashi M , Aoyagi T , Huang W-C , Lima S , Miyazaki H , Narui K , Ishikawa T , Endo I , Waters MR , Milstien S , Spiegel S , Takabe K 
Published: February 2018

Abstract
Background: Sphingosine-1-phosphate (S1P), a bioactive sphingolipid mediator that is generated by sphingosine kinase 1 (SphK1) when it is phosphorylated (pSphK1) inside cells, has been implicated in regulation of many process important for breast cancer progression. Previously we have shown that S1P is exported out of human breast cancer cells by ATP-binding cassette (ABC) transporter ABCC1, but not by ABCB1, both known multidrug resistance proteins that efflux chemotherapeutic agents. However, the pathological consequences of these events to breast cancer progression and metastasis have not been elucidated. Here, we report that high expression of ABCC1, but not ABCB1, is associated with poor prognosis in breast cancer patients via exporting S1P.
Materials and methods: Microarray based gene expression data of 2509 patients associated with their survival were obtained from METABRIC database. Single gene survival analysis based on expressin of SphK1, and dual ABCC1 or ABCB1 and SphK1 survival analyses were perfomerd. For protein analyses, tissues were obrained from 275 patients with stage 1-3 breast cancers treated in Yokohama City University Medical Center in Japan between 2006 and 2008. The expression of pSphK1 was analyzed by immunohistochemistry and investigate the relationship with clinicopathological findings. For in vitro and in vivo experiments, breast cancer cell lines were transfected by ABCB1, ABCC1 or vector transiently or stably. BALB/c nu/nu mice and BALB/c mice were used for in vivo experiments. S1P was measured by LC-ESI-MS/MS.
Results: SphK1 expression significantly associate with worse overall survival (median survival of 124 months with high SphK1 expression compared to 163 months for patients with low SphK1 expression, p=0.0014). Although patients with high ABCC1 expression had only a slightly worse overall survival of 150 months, those with high levels of both SphK1 and ABCC1 had much worse prognosis with median overall survival of 114 months (p &lt; 0.0068). Such association was not observed with ABCB1 expression. The frequency of strong pSphK1 protein expression was higher in HER2 enrhiched or TNBC than in Luminal. pSphK1 was more prevalent and increased in a larger tumors and in tumors from patients with lymph node metastases. Patients with breast cancers that express both pSphK1 and ABCC1 proteins have significantly shorter disease free survival. Overexpression of ABCC1, but not ABCB1, in human MCF7 and murine 4T1 cells enhanced S1P secretion, proliferation and migration of breast cancer cells. Implantation of breast cancer cells overexpressing ABCC1, but not ABCB1, into the mammary pad markedly enhanced tumor growth, angiogenesis and lymphangiogenesis with concomitant increases in lymph node and lung metastases as well as shorter survival of mice. Interestingly, S1P exported via ABCC1 from breast cancer cells upregulated transcription of SphK1 and its own formation.
Conclusions: Our findings suggest that production and export of S1P via ABCC1, but not ABCB1, is associated with worse overall and disease free survival of breast cancer patients and that S1P axis play a role in aggressive biology of breast cancer progression and metastasis.
Citation Format: Yamada A, Nagahashi M, Aoyagi T, Huang W-C, Lima S, Miyazaki H, Narui K, Ishikawa T, Endo I, Waters MR, Milstien S, Spiegel S, Takabe K. Sphingosine-1-phosphate produced by sphingosine kinase 1 and exported via ABCC1 shortens survival of mice and humans with breast cancer [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P5-03-05.