ABCC1-Exported Sphingosine-1-phosphate, Produced by Sphingosine Kinase 1, Shortens Survival of Mice and Patients with Breast Cancer

Abstract

Sphingosine-1-phosphate (S1P), a bioactive sphingolipid mediator, has been implicated in regulation of many processes important for breast cancer progression. Previously, we observed 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 pathologic consequences of these events to breast cancer progression and metastasis have not been elucidated. Here, it is demonstrated that high expression of ABCC1, but not ABCB1, is associated with poor prognosis in breast cancer patients. Overexpression of ABCC1, but not ABCB1, in human MCF7 and murine 4T1 breast cancer cells enhanced S1P secretion, proliferation, and migration of breast cancer cells. Implantation of breast cancer cells overexpressing ABCC1, but not ABCB1, into the mammary fat pad markedly enhanced tumor growth, angiogenesis, and lymphangiogenesis with a concomitant increase 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 sphingosine kinase 1 (SPHK1), thus promoting more S1P formation. Finally, patients with breast cancers that express both activated SPHK1 and ABCC1 have significantly shorter disease-free survival. These findings suggest that export of S1P via ABCC1 functions in a malicious feed-forward manner to amplify the S1P axis involved in breast cancer progression and metastasis, which has important implications for prognosis of breast cancer patients and for potential therapeutic targets.Implication: Multidrug resistant transporter ABCC1 and activation of SPHK1 in breast cancer worsen patient's survival by export of S1P to the tumor microenvironment to enhance key processes involved in cancer progression. Mol Cancer Res; 16(6); 1059-70. ©2018 AACR.

Figure 1. Expression of ABCC1 but not ABCB1 correlates with poor prognosis in human breast cancer patients

Tissue microarrays containing 275 breast tumor tissues were stained with anti-ABCB1 or anti-ABCC1 antibodies. (A) Expression of ABCB1 and ABCC1 was scored as 0: negative, 1: weak, 2: moderate, or 3: strong. Score 0 and 1 are considered as low expression while score 2 and 3 are considered as high expression. Representative images are shown under high magnification for ABCB1 staining (upper), ABCC1 staining (lower). (B) Kaplan-Meier disease-free survival curves according to expression of ABCB1 and ABCC1. P values were calculated by the log-rank test. Scale bars, 50 μm.

Figure 2. Overexpression of ABCC1 but not ABCB1, enhances proliferation and cell migration of breast cancer cells and promotes S1P secretion mediated angiogenesis and lymphangiogenesis of endothelial cells. (A-E) MCF7 or 4T1 breast cancer cells were transfected with vector (V), ABCB1 (B1), or ABCC1 (C1) as indicated

(A) Immunohistochemistry reveals that expressed ABCB1 and ABCC are localized to the plasma membrane. (B, C) S1P secreted from the indicated breast cancer cells was determined by LC-ESI-MS/MS. (D) Proliferation of cells treated with vehicle or 20 μM MK571 for 48 hours was determined by WST8 assay. Data are expressed as fold increase compared to 0 time. (E, F) Monolayers of the indicated 4T1 cells treated with vehicle or 20 μM MK57 were wounded and migration of cells into the wounded area was measured 24 h later. (E) Representative photographs of wounded areas are shown. (F) Cell migration was determined as percent of initial wounded area and expressed as means ± SD of 6 determinations. (G, H) HUVECs and HLECs were cultured on reduced growth factor basement membrane matrix-coated 48-well plates and incubated for 6 h without or with S1P (1 μM) or conditioned medium from MCF cells overexpressing ABCC1 (C1) were pretreated with vehicle (NC, non-treated control) or 20 μM MK571 without or with 10 μM SK1-I for 12 h and conditioned medium prepared. +M, treated with MK571; +S, treated with SK1-I. Six random fields per condition were photographed (G) and total tube length determined (H). Scale bars, 20 μm (A), 200 μm (E), 100 μm (G). *, P < 0.05; **, P < 0.01 determined by Student t test. Data are means ± SD.

Figure 3. Overexpression of ABCC1, but not ABCB1, enhances MCF7 tumor growth in a mouse xenograft model

(A) BALB/c nude mice were ovariectomized and estrogen pellets implanted under anesthesia. Tumors were established by surgical implantation of MCF7 cells stably overexpressing vector, ABCB1, or ABCC1 into chest mammary fat pads. Tumor size was measured at the indicated times (n=5 mice per group). (B) Representative images of H&E and Ki67 staining and confocal immunofluorescent images of stained blood vessels with anti-CD31 (red) and nuclei co-stained with Hoechst (blue) in tumor sections 83 d after implantation. (C) Percentage of Ki67 positive cells and micro vessel density were determined. Scale bars, 100 μm. *, P < 0.05; **, P < 0.01 determined by Student t test. Data are means ± SEM.

Figure 4. Overexpression of ABCC1, but not ABCB1, enhances 4T1 tumor growth and decreases survival in a syngeneic mouse model

(A–F) 4T1-luc2 cells transfected with vector, ABCB1, or ABCC1 were implanted into mammary fat pads of BALB/c mice under direct vision (23). (A) Tumor burden was determined by in vivo bioluminescence (n=10 mice per group). (B) Representative images of Ki67 staining of tumor sections are shown and the percentage of Ki67 positive cells within tumors was enumerated. (n=5) (C) Confocal immunofluorescent images of tumors stained for blood vessels (anti-CD31, red), lymphatic vessels, (anti-lyve1, green), and nuclei (Hoechst, blue). Micro vessel density and lymphatic vessel density were determined. (D) Tumors were minced, digested with collagenase, and BECs and LECS were quantified by FACS. Representative panels of FACS analysis are shown. (E) Regional lymph node metastases and lung metastases were determined by ex vivo bioluminescence 12 d after implantation. (F) Confocal immune fluorescent images of lymph nodes stained for adenocarcinoma (anti-CK8, green, white arrows) and nuclei (Hoechst, blue) and H&E stained lung sections show metastases (black arrow). (G) Kaplan-Meier survival curves of mice bearing 4T1/V, 4T1/B1, and 4T1/C1 tumors. Days after cancer cell implantation. P values were calculated by log-rank test. Scale bars, 100 μm (B, C); 2 µm (F). *, P < 0.05; **, P < 0.01 based on Student t test. Data are means ± SEM.

Figure 5. S1P exported via ABCC1 upregulates expression of SphK1

(A) SphK1 expression in MCF7 and 4T1 cells transfected with vector or ABCC1 was determined by QPCR and normalized to GAPDH. Data are means ± SEM. (B) Activation of SphK1 in transfected MCF7 cells was determined by immunocytochemistry with pSphK1 antibody. Scale bars: 20 μm. (C) 4T1 cells transfected as indicated were treated with vehicle or 20 μM MK571 for 24 h in serum-free medium. Cells were then stimulated in medium containing 10% serum for 3 h. SphK1 mRNA levels were determined by QPCR and normalized to Gapdh. Data are means ± SD (D) Naïve 4T1 cells were starved for 24 h and treated with 100 nM or 1 μM S1P in 0.4% fatty acid free BSA for 3 or 8 hours as indicated. SphK1 and GAPDH mRNA was determined by QPCR.

Figure 6. Overexpression of ABCC1 in breast tumors enhances expression of SphK1 and increases S1P in tumor interstitial fluid

(A, B) MCF7 cells transfected with vector, ABCB1, or ABCC1 were implanted into chest mammary fat pads of ovariectomized BALB/c nude mice. pSphK1 in tumors determined by immunohistochemistry on day 32. (C-E) 4T1-luc2 cells transfected with vector, ABCB1, or ABCC1 were implanted into mammary fat pads of BALB/c mice and tumors analyzed on day 12. SphK1 expression in 4T1 tumors was examined by staining with anti-SphK1 antibody. (B, D) Relative intensity of immunostaining was quantified by NIH ImageJ. Scale bars: 200μm. (E) Sphk1 mRNA level in tumors were determined by QPCR and normalized to Gapdh. (F) S1P levels in tumors (n=10) and in tumor interstitial fluid (n=5) were determined by LC-ESI-MS/MS. Data are means ± SEM. *, P < 0.05; **, P < 0.01.

Figure 7. Patients with breast cancers that express both activated SphK1 and ABCC1 have shorter disease free survival

pSphK1 in 275 human breast tumors examined by immunohistochemistry. (A) Scoring of pSphK1 expression in human breast tumor samples. (B) Frequency of high pSphK1 expression in human breast tumors correlated with clinicopathological factors, tumor size, and lymph node metastasis status and TNM stage. *, P < 0.05; **, P < 0.01. (C) Kaplan-Meier disease free survival curves according to expression of pSphK1, co-expression of pSphK1 with ABCB1, and co-expression of pSphK1 with ABCC1. P values were calculated by log-rank test. (D) Kaplan-Meier survival analysis of breast cancer patients from the METABRIC database. Data was obtained from patients with clinical and expression information. Median survival is tabulated along with a Log Rank p-value representing the significance of high gene expression of SphK1 among all patients, among ABCB1 high patients, or among ABCC1 high patients on patient survival.