Sphingosine-1-phosphate produced by sphingosine kinase 1 promotes breast cancer progression by stimulating angiogenesis and lymphangiogenesis.

Abstract

Sphingosine-1-phosphate (S1P) is a pleiotropic bioactive lipid mediator that promotes breast cancer progression by diverse mechanisms that remain somewhat unclear. Here we report pharmacologic evidence of a critical role for sphingosine kinase 1 (SphK1) in producing S1P and mediating tumor-induced hemangiogenesis and lymphangiogenesis in a murine model of breast cancer metastasis. S1P levels increased both in the tumor and the circulation. In agreement, serum S1P levels were significantly elevated in stage IIIA human breast cancer patients, compared with age/ethnicity-matched healthy volunteers. However, treatment with the specific SphK1 inhibitor SK1-I suppressed S1P levels, reduced metastases to lymph nodes and lungs, and decreased overall tumor burden of our murine model. Both S1P and angiopoietin 2 (Ang2) stimulated hemangiogenesis and lymphangiogenesis in vitro, whereas SK1-I inhibited each process. We quantified both processes in vivo from the same specimen by combining directed in vivo angiogenesis assays with fluorescence-activated cell sorting, thereby confirming the results obtained in vitro. Notably, SK1-I decreased both processes not only at the primary tumor but also in lymph nodes, with peritumoral lymphatic vessel density reduced in SK1-I-treated animals. Taken together, our findings show that SphK1-produced S1P is a crucial mediator of breast cancer-induced hemangiogenesis and lymphangiogenesis. Our results implicate SphK1 along with S1P as therapeutic targets in breast cancer.

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Therapeutics, Targets, and Chemical Biology
Sphingosine-1-Phosphate Produced by Sphingosine
Kinase 1 Promotes Breast Cancer Progression by
Stimulating Angiogenesis and Lymphangiogenesis
Masayuki Nagahashi1,2, Subramaniam Ramachandran1,2, Eugene Y. Kim2, Jeremy C. Allegood2,
Omar M. Rashid1,2, Akimitsu Yamada1,2, Renping Zhao3, Sheldon Milstien2, Huiping Zhou3,
Sarah Spiegel2, and Kazuaki Takabe1,2
Abstract
Sphingosine-1-phosphate (S1P) is a pleiotropic bioactive lipid mediator that promotes breast cancer pro-
gression by diverse mechanisms that remain somewhat unclear. Here we report pharmacologic evidence of a
critical role for sphingosine kinase 1 (SphK1) in producing S1P and mediating tumor-induced hemangiogenesis
andlymphangiogenesisinamurinemodelofbreastcancermetastasis.S1Plevelsincreasedbothinthetumorand
the circulation. In agreement, serum S1P levels were significantly elevated in stage IIIA human breast cancer
patients, compared with age/ethnicity-matched healthy volunteers. However, treatment with the specific SphK1
inhibitorSK1-IsuppressedS1Plevels,reducedmetastasestolymphnodesandlungs,anddecreasedoveralltumor
burden of our murine model. Both S1P and angiopoietin 2 (Ang2) stimulated hemangiogenesis and lymphan-
giogenesis in vitro, whereas SK1-I inhibited each process. We quantified both processes in vivo from the same
specimen by combining directed in vivo angiogenesis assays with fluorescence-activated cell sorting, thereby
confirming the results obtained in vitro. Notably, SK1-I decreased both processes not only at the primary tumor
but also in lymph nodes, with peritumoral lymphatic vessel density reduced in SK1-I–treated animals. Taken
together, our findings show that SphK1-produced S1P is a crucial mediator of breast cancer–induced heman-
giogenesis and lymphangiogenesis. Our results implicate SphK1 along with S1P as therapeutic targets in breast
cancer. Cancer Res; 72(3); 726–35. ?2012 AACR.
Introduction
Breast cancer is the most commonly diagnosed cancer
among women accounting for 30% of all new cancer cases,
and close to 40,000 breast cancer deaths are expected to occur
amongU.S.womenin2011(1).Themajorityofdeathsofbreast
cancer patients occur after it metastasizes and becomes a
systemic disease (2). To date, most systemic therapies, includ-
ingcytotoxicchemotherapy,targetthetumoritselfandnotthe
host tumor microenvironment, which is known to play an
important role in the progression of cancer (3, 4).
It is well established that hemangiogenesis is one of the key
tumor microenvironmental factors in cancer progression.
Tumor growth beyond a diameter of a few millimetersrequires
hemangiogenesis which contributes to the development of
metastatic disease (5, 6). Although hemangiogenesis is known
to be an acquired capability of cancer (7), results of multiple
randomizedclinical trials that targeted hemangiogenesis using
bevacizumab,ananti-VEGFmonoclonalantibody(mAb),failed
to show an overall survival benefit in breast cancer (8–10).
Breast cancer first metastasizes to its sentinel lymph node,
and the level of lymph node metastasis is a major determinant
of staging and prognosis of breast cancer (2, 11). Recently,
lymphangiogenesis, a process that generates new lymphatic
vessels from preexisting ones, was found to be mediated by
several factors including VEGF-C, D, and angiopoietins (Ang1
and Ang2), which provided new insights into how the lym-
phatic vessels grow and affect metastasis (12, 13). Although
clinical and experimental evidence suggest a roleforlymphan-
giogenesis in lymph node metastasis, this process is far less
understood than hemangiogenesis (13, 14).
The pleiotropic bioactive lipid mediator sphingosine-1-
phosphate (S1P) is now emerging as a key regulatory molecule
in cancer through its ability to promote cell proliferation,
migration,invasion,andhemangiogenesis(15,16).S1Pisgene-
rated intracellularly by 2 sphingosine kinases, sphingosine
kinase 1 (SphK1) and SphK2, and is exported out of the cells
Authors' Affiliations:
Biochemistry and Molecular Biology, and the Massey Cancer Center, and
3Department of Microbiology and Immunology, Virginia Commonwealth
University School of Medicine, Richmond, Virginia
1Division of Surgical Oncology,
2Department of
Note: Supplementary data for this article are available at Cancer Research
Online (http://cancerres.aacrjournals.org/).
Corresponding Author: Kazuaki Takabe, Division of Surgical Oncology,
VirginiaCommonwealthUniversitySchoolofMedicineandMasseyCancer
Center, West Hospital 7-402, 1200 East Broad Street, PO Box 980011,
Richmond, VA 23298. Phone: 804-828-9322; Fax: 804-828-4808; E-mail:
ktakabe@vcu.edu
doi: 10.1158/0008-5472.CAN-11-2167
?2012 American Association for Cancer Research.
Cancer
Research
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where it regulates many functions by binding to and signaling
through a family of 5 G protein-coupled receptors (S1P1–5).
This process known as "inside-out" signaling explains the
autocrine and paracrine actions of S1P (15). We previously
showed that SphK1, but not SphK2, is involved in S1P export
frombreastcancercellsmediatedbytheATP-bindingcassette
transporters, ABCC1 and ABCG2 (17). Expression of SphK1 is
upregulated in breast cancer (18, 19), correlates with poor
prognosis (20), and is associated with resistance to chemo-
therapy (18). Furthermore, abundant evidence implicates the
SphK1/S1P/S1P1axis in hemangiogenesis and vasculogenesis
(21). In contrast, only a few studies so far have examined the
involvement of SphK1 and S1P in lymphangiogenesis. S1P has
been shown to induce in vitro lymphangiogenesis via the
S1P1receptor (22), and it was recently reported that SphK1
and S1P in lymphatic endothelial cells (LEC) are required for
the proper development of lymphatic vessels (23). However,
nothing is yet known of the role of SphK1 and S1P in tumor-
induced lymphangiogenesis in vivo.
In this study, we utilized an improved syngeneic breast
cancer cell implantation method, which models human breast
cancer biology better than conventional xenograft subcutane-
ous implantation, to explore the role of SphK1 and S1P in
hemangiogenesis and lymphangiogenesis. We found not only
that circulating S1P levels correlated with tumor burden but
also that targeting SphK1 with a specific inhibitor reduced
tumorgrowth,lymphnodeandlungmetastasis,anddecreased
hemangiogenesis and lymphangiogenesis around the primary
tumor and in the lymph nodes. Our results suggest that
targeting SphK1 and S1P may be a useful additional modality
for the treatment of metastatic breast cancer.
Materials and Methods
Cell culture
4T1-luc2 cells, a mouse mammary adenocarcinoma cell
line that has been engineered to express luciferase (PerkinEl-
mer), were cultured in RPMI Medium 1640 with 10% FBS.
Green fluorescent protein (GFP)-expressing human umbilical
vein endothelial cells (HUVEC) and GFP-expressing human
lymphatic endothelial cells (HLEC) purchased from Angio-
Proteomie were maintained in endothelial cell medium sup-
plemented with 5% FBS and endothelial cell growth supple-
ment (ScienCell Research Laboratories).
Construction of lentiviral shRNAs for mouse SphK1
Three sense sequences of siRNA cassettes specifically tar-
geting the nucleotides of mouse SphK1 (accession number:
NM_011451.1) were designed using siRNA Target Finder
(Ambion). The specificity of the selected sequences was con-
firmed by BLAST search. The lentiviral short hairpin RNAs
(shRNA) were constructed using a pLL3.7 expression vector as
described previously (24). The sequences of the shRNAs for
mouse SphK1 are as follows: shRNA 1, 50- GCACCCAAAC-
TACCTTTGGAT-30; shRNA 2, 50- GCACCTTCTTTCGCCTAG-
CAA-30; shRNA 3,50-GAGGCAGAGATAACCTTTAAA-30.
Recombinant lentivirus was produced by cotransfection of
293FT cells with lentiviral vector and the packaging vectors
using calcium phosphate. The viruses were collected from the
culture medium and purified by polyethylene glycol precipi-
tation.Thetransductionefficiencyoflentivirusin4T1cellswas
determined by fluorescence microscopy. The silencing effi-
ciency was determined using quantitative PCR (qPCR) and
Western blot analysis.
Syngeneic tumor model
All animal studies were conducted in the Animal Research
CoreFacilityatVCUSchoolofMedicineinaccordancewiththe
institutional guidelines. All procedures were approved by the
VCU Institutional Animal Care and Use Committee (IACUC)
that is accredited by the Association for Assessment and
Accreditation of Laboratory Animal Care (AAALAC). Female
BALB/cmice,8to12weeksofage,weighingapproximately20g
were obtained from Harlan. 4T1-luc2 cells (1 ? 105cells in 10
mLRPMI)weresurgicallyimplantedintheupperfatpadunder
direct vision. One day after implantation, mice were random-
ized by tumor size determined by bioluminescence with the
IVIS Imaging System (Xenogen). SK1-I in PBS was injected
intraperitoneally, asindicated,everydayatadoseof20mg/kg.
At the indicated times, the animals were sacrificed by exsan-
guination, blood was collected, tumors excised, weighed, fixed
in formalin, and embedded in paraffin or frozen in liquid
nitrogen.
Bioluminescent quantification of tumor burden
D-Luciferin (0.2 mL of 15 mg/mL stock; PerkinElmer) was
injected intraperitoneally into mice previously implanted with
4T1-luc2 cells. Living Image Software (Xenogen) was used to
quantify the photons/sec emitted by the cells. Biolumines-
cence was measured and quantified at 5-minute intervals over
30 minutes using a subject height of 1.5 cm, medium binning
and an exposure time of 0.5 seconds to 1 minutes. Biolumi-
nescence was then determined by the peak number of
photons/sec calculated over this time frame. Axillary lymph
node metastasis was quantified in vivo after intraperitoneal
injection of luciferin and primary tumor resection. Lung
metastasiswasquantifiedexvivoafterthelungswereremoved.
Quantification of sphingolipids by mass spectrometry
Lipids were extracted from serum and tissues and sphingo-
lipids quantified by liquid chromatography–electrospray ion-
ization–tandem mass spectrometry (LC-ESI-MS/MS; 4000
QTRAP, AB Sciex) as described (25).
Directed in vivo angiogenesis assays and cell isolation
Angioreactors[directedinvivoangiogenesisassays(DIVAA)
kit; Trevigen] were implanted into the subcutaneous layer of
the back of 8-week-old nude mice under anesthesia according
to the manufacturer's instructions. Micewere euthanizedwith
CO2at day 11 after implantation and the angioreactors were
removed under stereomicroscopy (SZ51, Olympus).
Fluorescence-activated cell sorting
Single-cell suspensions were obtained from lymph nodes as
described (26). For DIVAA/FACS (fluorescence-activated cell
sorting) analyses, Matrigel and fibrotic reactive tissue in the
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open end of the angioreactors were removed with Dulbecco's
modified Eagle's medium and digested with a mixture of 9 mL
of Cell Sparse (Trevigen) containing 0.1% collagenase type 2
(Gibco), 1 mL of 0.25 U/mL dispase (Gibco), and 75 mL of 0.1%
DNase(Invitrogen)for30minutestoobtainsingle-cellsuspen-
sions. Tumors were minced and digested similarly. Cell sus-
pensions were blocked with anti-CD16/CD32 (Mouse BD
Fc Block; BD Biosciences) and then stained with the follow-
ing antibodies as indicated: Alexa 488–conjugated LYVE-1
(eBioscience); PE-conjugated podoplanin, PerCP-Cy5.5–con-
jugated CD45, APC-conjugated CD31, Alexa 700–conjugated
TER-119 (Biolegend); or appropriate matched, fluorochrome-
labeled isotype control mAb. The LIVE/DEAD Viability Assay
kit (Invitrogen) was used to eliminate dead cells. Cells were
analyzed by FACS using BD FACSCanto II and BD FACSAria II
(BD Biosciences) and corollary data assessed with BD FACS-
Diva Software version 6.1.3 (BD Biosciences).
Histopathologic analysis and vessel density
determination
Immediately after the sacrifice of the animals, tumor sam-
ples were fixed in 10% neutral buffered formalin for immuno-
histochemical analyses of cell proliferation and apoptosis.
4T1-luc2 cell proliferation in the tumor was determined by
staining histologic sections with mAbs against Ki67 (Dako), a
nuclear protein expressed in proliferating cells. Apoptosis was
determined by terminal uridine deoxynucleotidyl transferase
dUTP nick end labeling (TUNEL) assay using the ApopTag
Peroxidase In Situ Apoptosis Detection Kit S7100 (Millipore).
The tumors were also embedded in optimal cutting medi-
um (OCT 4583; Sakura Finetek) and frozen for immunofluo-
rescent analysis for hemangiogenesis and lymphangiogenesis.
The sections were stained with the following antibodies as
indicated: CD31 (BD), LYVE-1 (Abcam) and F4/80 (kindly
providedbyBin-ZhiQianinJeffreyPollardlaboratoryatAlbert
Figure 1. The SphK1/S1P axis in breast cancer progression. A, 4T1 tumors were established by surgical implantation of 4T1-luc2 cells into the chest
mammary fat pad under direct vision. The tumor burden was determined by bioluminescence technology. Representative IVIS images (left) and
bioluminescentquantification(right)ontheindicateddaysareshown.B,axillarylymphnodemetastasesweremeasuredbybioluminescenceattheindicated
times when mammary tumor was removed for accurate measurement (left). Lung metastases were measured separately ex vivo (middle). The incidence of
metastasis to the axillary lymph nodes and the lung was determined on the indicated days (right). C, mRNA was isolated from mammary fat pads of
naive BALB/c mice (light gray filled bars), cultured 4T1-luc2 cells (black cross hatched bars), and primary tumors in the chest mammary fat pads formed by
4T1-luc2 cells (dark gray filled bars) 10 days after implantation. Expression of SphK1 and SphK2 was determined by qPCR and normalized to levels of
glyceraldehyde-3-phosphatedehydrogenase(GAPDH)mRNA.Dataareexpressedasfoldincreases?SD.D,S1PlevelsweredeterminedbyLC-ESI-MS/MS
intheserumofshamsurgerymiceandinmicewith4T1-luc2xenografttumors15daysafterimplantation.Dataareexpressedasfoldincreases?SEM.E,S1P
was measured in serum from stage IIIA breast cancer patients and age/ethnicity-matched healthy volunteers (n ¼ 5).?, P < 0.001;??, P < 0.05.
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Einstein College of Medicine; ref. 27). The stained sections
were examined with LSM510 laser scanning confocal micro-
scopes (Zeiss) and microvessel density was determined as
previously described (28).
Patient samples
All studies using patient samples were conducted in accor-
dance with the institutional guidelines after approval by VCU
Institutional Review Board. Human sera were collected by the
TissueandDataAcquisitionandAnalysisCoreofVCUandS1P
levels determined in serum from stage IIIA breast cancer
patients and age/ethnicity-matched healthy volunteers.
In vitro assays
Cell number of 4T1-luc2 cells was determined by mea-
surement of luciferase activity with the luciferin substrate
(PerkinElmer) using a VICTOR X4 Multilabel Plate Reader
(PerkinElmer). qPCR (15) and tube formation assay were
conducted as described previously (29).
Statistical analysis
Results were analyzed for statistical significance with a 2-
tailed Student's t test, with P < 0.05 considered significant.
Experiments were repeated at least 3 times in triplicate with
consistent results. In vivo experiments were repeated 3 times
and each experimental group consisted of at least 6 mice.
Results
Upregulation of SphK1 and increased S1P levels in 4T1-
luc2 tumor progression
Previous studies showed that S1P and SphK1, the enzyme
that producesit,regulatemanyprocessesimportantforbreast
cancer (15). To examine their involvement in breast cancer
progression in vivo, we utilized an enhanced syngeneic mouse
metastatic breast cancer model in which 4T1-luc2 murine
mammary cancer cells were orthotopically implanted under
direct vision into the chest mammary fat pad of immune-
competent mice. We have found that this chest orthotopic
model more accurately mimics human breast cancer progres-
sion than subcutaneous models. Indeed, 4T1-luc2 cells pro-
duced large tumors in the chest mammary fat pad that rapidly
metastasized and increased total tumor burden (Fig. 1A).
Metastatic spread to the regional axillary lymph nodes and
the lungs was quantified by bioluminescence after removal of
the 4T1-luc2 tumors from the implantation sites (primary
tumors). As can be seen in Fig. 1B, lymph node metastases
emerged earlier than lung metastases, with a rapid increase in
Figure 2. Inhibition of SphK1 decreases growth of 4T1-luc2 mammary tumors. A, 4T1-luc2 cells were treated with the indicated concentration of
SK1-I and total cell number was determined by measurement of luciferase activity. 4T1-luc2 cells were surgically implanted in mammary fat
pads under direct vision. Tumor-bearing mice were randomized into 2 groups and injected intraperitoneally with PBS or SK1-I (20 mg/kg) daily. Tumor
volumes were measured on the indicated days (B) and tumor weights were determined after excision on day 18 (C) (n ¼ 5 per group). D, tumor histology.
Paraffin-embedded tumor sections were immunostained with Ki67 and counterstained with hematoxylin. Apoptotic cells were visualized by
TUNEL staining. Scale bar, 50 mm. E, S1P levels were determined by LC-ESI-MS/MS in mammary fat pads of naive (open bar) and sham operated mice
(dotted bars) and in mammary tumors from mice that were treated with PBS (filled bars) or SK1-I (hatched bars) on the indicated days. F, SK1-I
levels were determined on the indicated days by LC-ESI-MS/MS in mammary tumors from mice that were treated with SK1-I (filled bars), and
means ? SEM are shown.?, P < 0.01;??, P < 0.05.
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the incidence of metastasis to the lymph nodes, similar to
human breast cancer progression.
ExpressionsofbothSphK1andSphK2wereverylowinnaive
mammary fat pads, whereas 4T1-luc2 cells express much
higher levels of SphK1 mRNA than SphK2 (Fig. 1C). However,
after implantation in mammary fat pads, SphK1 mRNA levels
in breast tumors were significantly increased (Fig. 1C), with
concomitantly increased S1P levels in the tumors (Fig. 2E).
Interestingly, levels of S1P in serum from these mice were also
significantly elevated (Fig. 1D), suggesting that overexpression
of SphK1 in the tumors may be responsible for the increased
circulating S1P.
Because it has been shown that overexpression of SphK1
correlates with poor prognosis of breast cancer patients (20),
it was of interest to measure serum levels of S1P in breast
cancer patients. We found that the serum S1P levels were
significantly elevated in stage IIIA breast cancer patients who
have lymph node metastases, compared with age/ethnicity-
matched healthy volunteers (Fig. 1E).
Growth of primary mammary tumors, tumor burden,
and lymph node and lung metastases are reduced by
inhibition of SphK1
We next examined the effect of inhibition of SphK1 with
SK1-I [(2R,3S,4E)-N-methyl-5-(4-pentylphenyl)-2-aminopent-
4-ene-1,3-diol (BML-258); ref. 30]. SKI-I is a potent, water-
soluble, isoenzyme-specific inhibitor of SphK1 that in contrast
to pan-SphK inhibitors does not inhibit SphK2, protein kinase
C, or numerous other protein kinases (30). Consistent with
previous studies in other types of cancer cells (30, 31), SK1-I
inhibitedgrowthof4T1-luc2cellsinadose-dependentmanner
(Fig.2A).Asignificanteffectwasobservedataconcentrationof
3mmol/Landcompleteinhibition ofgrowthat10mmol/L(Fig.
2A). Weconfirmed thatSK1-I decreasedthe enzymeactivity of
SphK1 and that downregulation of SphK1 with specific siRNA,
similartoSK1-I,alsosuppressedthegrowthofthesecells(data
not shown). We first examined circulating levels of SK1-I
followingasingleintraperitonealinjectionof20mg/kg.Plasma
SK1-I levels reached a maximum concentration of 0.6 mmol/L
within 2 hours but still could be detected up to 12 hours.
Concomitantly,plasmalevelsofS1Pweresignificantlyreduced
up to 12 hours (Supplementary Fig. S1). We then investigated
the effect of SK1-I on the growth of 4T1-luc2 tumors in mouse
mammary fat pads. Intraperitoneal injections of SK1-I signif-
icantlyreducedbothtumorvolumeandweight(Fig.2BandC).
The tumors from animals treated with SK1-I also showed
reduced mitotic activity compared with those from vehicle-
treatedanimals,asshownbyKi67immunohistochemistry(Fig.
2D). Conversely, TUNEL staining revealed a large increase in
apoptotic cells in tumors from SK1-I–treated mice (Fig. 2D).
Consistent with the upregulation of SphK1 intumors (Fig. 1C),
S1P levels in tumors gradually increased compared with naive
Figure 3. SK1-I decreases tumor burden, lymph node, and lung metastases, and circulating levels of S1P. 4T1-luc2 cells were surgically implanted in
mammary fat pads under direct vision. Tumor-bearing mice were randomized into 2 groups and injected intraperitoneally daily with PBS or SK1-I (20 mg/kg;
n ¼ 5). A, tumor burden was quantified by in vivo bioluminescence on the indicated days. Right panels show representative IVIS bioluminescent images.
B, S1P levels were determined by LC-ESI-MS/MS in serum of naive (open bar) and sham operated mice (dotted bars), mice bearing 4T1-luc2 tumors
thatweretreatedwithPBS(filledbars)orSK1-I(hatchedbars)ontheindicateddays.C,SK1-IlevelsweredeterminedontheindicateddaysbyLC-ESI-MS/MS
in serum from the mice that were treated with SK1-I (filled bars). Representative bioluminescent images (right) and quantification of regional lymph
node (D) and lung (E) metastasis was determined by bioluminescence 7 or 10 days after treatment with PBS or SK1-I, respectively. Data are expressed as
mean ? SEM.?, P < 0.01;??, P < 0.05.
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or sham operated mammary fat pads as measured by LC-ESI-
MS/MS. This increase of S1P in the tumors was prevented by
treatment with SK1-I (Fig. 2E). Notably, SK1-I was taken up by
the mammary tumors and its levels gradually increased with
time (Fig. 2F).
The total tumor burden determined by the IVIS Imaging
System was also significantly suppressed by SK1-I treat-
ment (Fig. 3A). Serum S1P levels, which are elevated in the
tumor-bearing mice, were also reduced by SK1-I treatment
(Fig. 3B). Serum levels of SK1-I in these mice increased with
time (Fig. 3C). Importantly, SK1-I significantly reduced
lymph node and lung metastasis (Fig. 3D and E). Taken
together, these results suggested that S1P produced by
SphK1 in the mammary tumors is involved in lymph node
and lung metastasis.
SK1-I suppresses in vitro hemangiogenesis and
lymphangiogenesis
Because hemangiogenesis and lymphangiogenesis are con-
sideredtoplaycriticalrolesinlymphnodeandlungmetastasis
(28),itwasofinteresttonextexaminetheroleofS1Pgenerated
by SphK1 in these processes. We first determined the effect of
S1P on in vitro hemangiogenesis and lymphangiogenesis. In
agreementwithpreviousstudies(22,32),wefoundthatS1Pisa
potent hemangiogenic and lymphangiogenic factor for human
blood and lymphatic endothelial cells, respectively, as shown
by enhanced tube formation (Fig. 4A and B). This reflects cell
migration and organization of a network architecture, key
aspects of both hemangiogenesis and lymphangiogenesis.
Ang2, whose levels in breast cancer correlate with metastasis
tolymphnodesandpoorprognosis(33),wasusedasapositive
control. SK1-I inhibited in vitro hemangiogenesis and lym-
phangiogenesis (Fig. 4A and B), suggesting that the effects of
Ang2 are mediated at least in part by SphK1 and formation of
S1P.
We next sought to determine whether high expression of
SphK1 and S1P production in 4T1-luc2 cells contribute to
enhanced lymphangiogenesis and hemangiogenesis. Whereas
supernatants from 4T1-luc2 cells significantly stimulated tube
formation of HUVECs and HLECs, shRNA knockdown of
SphK1, which decreased SphK1 expression (Supplementary
Fig. S2A and S2B) and the release of S1P (Supplementary
Figure 4. Inhibition of SphK1 suppresses in vitro hemangiogenesis and lymphangiogenesis. GFP expressing HUVECs and HLECs were cultured on
reduced growth factor basement membrane matrix–coated 48-well plates and incubated for 6 hours with or without S1P (1 mmol/L), Ang2 (500 ng/mL),
or SK1-I (10 mmol/L), as indicated. A, representative images. B, two random fields per well were photographed. Total tube length determined and
means ? SD are shown.?, P < 0.01;??, P < 0.05.
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Fig.S2C),markedlyreducedthem(SupplementaryFig.S2Dand
S2E).Moreover,treatmentwithS1Pdidnotaffectexpressionof
either VEGF-A or VEGF-C mRNA in 4T1-luc2 cells (Supple-
mentary Fig. S3). These results indicated that S1P producedby
SphK1 in 4T1-luc2 mammary cancer cells is an important
contributor to hemangiogenesis and lymphangiogenesis.
Involvement of SphK1 and S1P in in vivo
hemangiogenesis and lymphangiogenesis
DIVAA provides a simple and quantitative method to mea-
sure hemangiogenesis in vivo (34). In contrast, one of the
challenges of studying lymphangiogenesis in vivo is the lack
of assays for objective quantification (12). To this end, we
developed a similar approach to simultaneously quantify both
hemangiogenesis and lymphangiogenesis by combining
DIVAA with FACS (DIVAA/FACS).
WeinitiallymodifiedtheFACSgatingschemeestablishedby
Pham and colleagues (23) to separate lymph node blood
endothelial cells (BEC) and lymphatic endothelial cells (LEC)
from other cells. TER-119 and CD45 were used to gate out red
blood cells (RBC) and leukocytes, respectively (Fig. 5A). LECs
and BECs were quantified using gp38 (podoplanin) and lym-
phatic vessel endothelial hyaluronan receptor (LYVE-1), spe-
cific markers for LECs, and CD31, a specific marker for both
BECs and LECs. LECs were identified as CD45?CD31þgp38þ,
BECs as CD45?CD31þgp38?, and fibroblast reticular cells
(FRC) as CD45?CD31?gp38þ. As expected, the selected popu-
lation of LECs showed a higher expression of LYVE-1 than
BECs or FRCs (Fig. 5B).
In the DIVAA approach, mice were implanted subcutane-
ously with silicone cylinders (angioreactors) that were closed
atoneendandfilledwith20mLofMatrigel(34).Theadditionof
stimuli enables directional migration of BECs and LECs into
the angioreactors, which proliferate and form appropriate
vessels. In agreement with previous studies (34), hemangio-
genesis was readily observable by the appearance of blood
vessels (Fig. 5C). Both S1P and Ang2 significantly induced
hemangiogenesis 11 days after implantation of the angioreac-
tors (Fig. 5C). To simultaneously quantify hemangiogenesis
and lymphangiogenesis, cells were isolated from the angior-
eactors by collagenase digestion, followed by FACS analysis to
separate the BECs from LECs and other cells as described
above. Interestingly, both S1P and Ang2 enhanced the migra-
tion of BECs and LECs into the angioreactors, although
hemangiogenesis was morepronounced than lymphangiogen-
esis (Fig. 5D and E). Similar to its effect on in vitro hemangio-
genesisandlymphangiogenesis(Fig.4B),theinclusionofSK1-I
in the angioreactors together with Ang2 completely abolished
its ability to stimulate both of these processes (Fig. 5E).
Inhibition of SphK1 suppresses hemangiogenesis and
lymphangiogenesisintumorsandinlymphnodesinvivo
ToestablishthatS1PgeneratedbySphK1playsacriticalrole
in tumor-induced hemangiogenesis and lymphangiogenesis in
vivo, we next examined the effect of SK1-I on hemangiogenesis
and lymphangiogenesis in the tumors themselves and in the
draining lymph nodes. FACS analysis of mammary pad site
tumorsrevealedthatbothBECsandLECsweregreatlyreduced
by treatment of mice with SK1-I (Fig. 6A and B). Interestingly,
the number of BECs and LECs in lymph nodes, the host tumor
microenvironment, was significantly increased in tumor-bear-
ing animals compared with sham animals, and treatment with
SK1-I inhibited the increase of both BECs and LECs in lymph
nodes (Fig. 6C and D). Morphometric analysis confirmed that
SK1-I reduced both tumor-induced hemangiogenesis and
Figure 5. Inhibition of SphK1 suppresses hemangiogenesis and
lymphangiogenesis quantified by DIVAA/FACS. A and B, FACS gating
schemetoquantifyBECsandLECsfromthesamelymphnodesingle-cell
suspension. A, TER-119 and CD45 were used to gate out the RBCs and
leukocytes, respectively. CD31 and gp38 (podoplanin) expression
allowedseparationof LECs and BECsfrom other TER-119?CD45?cells:
FRCs and double-negative stromal cells (Others). B, LYVE-1 expression
ontheindicatedpopulationsorisotypecontrol(IC)onthetotalpopulation
was quantified by FACS and mean values ? SEM are shown in the right
panel.?, P < 0.001. C–E, quantification of BECs and LECs by DIVAA/
FACS.C,angioreactorsareclosedatthe bottomendandfilledwith20mL
of Matrigel. The addition of stimuli in the Matrigel enables directional
migrationofBECsandLECsintotheangioreactors,whichproliferateand
form appropriate vessels(left). Hemangiogenesiswas readily observable
by the appearance of blood vessels in the angioreactors containing S1P
(1 mmol/L), or Ang2 (1 mg/mL) compared with PBS. D, cells were isolated
from theangioreactors andBECsandLECs wereanalyzedbyFACS with
antibodiestoTER-119,CD45,CD31,andgp38asin(A).E,totalnumberof
BECs and LECs were calculated and means ? SEM are shown.
?, P < 0.01;??, P < 0.05.
Nagahashi et al.
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lymphangiogenesis as determined by densities of CD31þ
LYVE-1?blood vessels (BVD) and CD31þLYVE-1þlymphatic
vessels (LVD), respectively (Fig. 6E and F).
Discussion
Previousstudies haveshown that SphK1 isoverexpressed in
breast cancer, and its level of expression correlates with
resistance to treatments and poor patient outcomes
(16, 18, 20). However, several factors have hindered progress
in determining the role of S1P, the product of SphK1, in
tumorigenesis and tumor-induced hemangiogenesis and lym-
phangiogenesis in vivo. First, because S1P has such a profound
role in immune function (35), studies of traditional in vivo
metastatic breast cancer models with xenografts in immune-
compromised mice that ignore the host immune response to
cancer may be difficult to interpret. Second, it has not been
possible to accurately quantify S1P in tumors until recently
with the development of high sensitivity mass spectrometric
assays (25). To this end, we utilized our newly established
syngeneicbreastcancermodel,whichnotonlyretainsthehost
immuneresponsesbutalsomorecloselymodelsprogressionof
thehumandiseasebyforminglymphnodeandlungmetastasis
leading to metastatic spread and death. Our data indicate that
SphK1 is the predominant isoenzyme expressed in 4T1-luc2
cells in culture dish, and is further upregulated in tumors, in
Figure 6. Inhibition of SphK1 suppresses hemangiogenesis and lymphangiogenesis in tumors and lymph nodes in vivo. 4T1-luc2 cells implanted in
chest mammary fat pads were allowed to form tumors for 2 days, which were quantified by IVIS. Mice with similar amounts of tumor were randomized
and treated with either PBS or SK1-I (20 mg/kg/d i.p.). Tumors (A, B) and regional axillary lymph nodes (C, D) were harvested after 4 days of treatment,
minced, digested with collagenase, and BECs and LECs quantified by FACS. Total numbers of BECs or LECs in 4T1 tumors (B) and lymph nodes (D) are
shown as means ? SEM.?, P < 0.05. E, immunofluorescent staining for CD31 and LYVE-1. CD31þLYVE-1?cells were considered as blood vessels,
CD31þLYVE-1þcells as lymphatic vessels (arrows). F, morphometric analysis of blood and lymphatic vessel densities were determined in the
peritumoral regions after 7 days of treatment and are shown as means ? SEM.?, P < 0.05. Scale bar, 50 mm.
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contrast to SphK2. This is in agreement with previous reports
correlating expression of SphK1, but not of SphK2, with poor
prognosis in breast cancer (20). We found that levels of S1P
gradually increased both in tumors and in serum determined
by LC-ESI-MS/MS and correlated with tumor burden. More-
over, treatment of tumor-bearing animals with the specific
SphK1 inhibitor SK1-I reduced S1P levels in the tumor and in
circulation, and greatly reduced the size of the primary tumor,
lymph node, and lung metastasis. This is in agreement with a
previousreportthatbloodandtumorS1Plevelswereincreased
in mice with colon cancer (36).
Quantificationoftumor-inducedhemangiogenesisandlym-
phangiogenesis has remained a challenge (12). This is usually
examined by histologic determinations of microvessel density
or LVD, which rely on selective morphometric analysis (e.g.,
vesselcounts,vascularmorphology,etc.;ref.12).Thestrengths
of the morphologic approach are that it can evaluate the
location of the vessels in relationship to the tumor and
identify/quantify morphologic changes that lymphatic vessels
undergo during tumor progression. The limitations include
variable sites of tissue sectioning, variable immunostaining
techniques, different vessel density quantification methods,
and the lack of standardization in the estimation of heman-
giogenesis and lymphangiogenesis (12). To compliment this
approach and overcome some of these limitations, we devel-
opedaflowcytometrymethodtoquantifybothBECsandLECs
in the same sample, to simultaneously evaluate both heman-
giogenesis and lymphangiogenesis. Flow cytometric analysis
can provide supportive data to quantify the changes in BECs
and LECs.
ConsistentwithboththeinvitrotubeformationandDIVAA/
FACSassays,weshowedthatexogenousS1Penhancedheman-
giogenesis and lymphangiogenesis. In this regard, Anelli and
colleagues showed that downregulation of SphK1 did not
change the levels of VEGF-A or VEGF-C secretion in glioma
cells, and downregulation of SphK1, but not VEGF-A or VEGF-
C, suppressed glioma-induced hemangiogenesis (37). Utilizing
shSphK1, we confirmed that tumor-derived S1P mediates
tumor-induced hemangiogenesis and lymphangiogenesis
without altering VEGF-A or VEGF-C (Supplementary Fig.
S3), which is in agreement with Anelli and colleagues (37).
Taken together, S1P is an important stimulator of hemangio-
genesis and lymphangiogenesis in vitro.
SK1-I decreased hemangiogenesis and lymphangiogenesis
not only around the primary tumor but also in lymph nodes
that are distant from the tumor. Furthermore, we have
observed by F4/80 immunofluorescent staining that SK1-I
also reduced macrophage recruitment surrounding the tumor
(Supplementary Fig. S4). Macrophages are cellular compo-
nents of the tumor microenvironment that promote heman-
giogenesisandlymphangiogenesis(38,39).Thus,inadditionto
directeffectsonhemangiogenesisandlymphangiogenesis,S1P
can indirectly affect the tumor microenvironment to further
enhance these processes.
In sum, S1P generated by SphK1 is important not only for
tumor progression but also for tumor-induced hemangiogen-
esis and lymphangiogenesis and, therefore, targeting SphK1
and its product S1P would be a multipronged attack against
breast cancer.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
Acknowledgments
The authors thank the Tissue and Data Acquisition and Analysis Core
(TDAAC) of VCU for collection of human samples, and the Anatomic
Pathology Research Services (APRS) Director, Dr. Jorge A. Almenara, and
the histotechnologists for technical assistance with tissue processing, sec-
tioning, and staining. The authors also thank Dr. Bin-Zhi Qian in Jeffrey
Pollard laboratory at Albert Einstein College of Medicine, Bronx, NY, for anti-
F4/80 antibody.
Grant Support
This work was supported by NCI grant R01CA61774 to S. Spiegel and NIH
grant K12HD055881 and Susan G. Komen for the Cure Research Foundation
Career Catalyst Research Grant KG090510 to K. Takabe. M. Nagahashi was
supported by the SUMITOMO Life Social Welfare Services Foundation.
The Flow Cytometry and Confocal microscopy Shared Resource Cores were
supported in part by NIH Grant P30CA16059 to the Massey Cancer Center and
NINDS Center core Grant 5P30NS047463, respectively.
The costs of publication of this article were defrayed in part by the
payment of page charges. This article must therefore be hereby marked
advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate
this fact.
Received July 5, 2011; revised November 30, 2011; accepted December 1, 2011;
published online February 1, 2012.
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