Sphingosine-Kinase-1 Signaling Promotes Metastasis of Triple-Negative Breast Cancer

Author Manuscript Published OnlineFirst on June 25, 2019; DOI: 10.1158/0008-5472.CAN-18-3803 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Sphingosine-kinase-1 signaling promotes metastasis of triple-negative breast cancer

Sunil Acharya1,2, Jun Yao1, Ping Li1, Chenyu Zhang1, Frank J. Lowery1,2, Qingling Zhang1, Hua

Guo3, Jingkun Qu1, Fei Yang4, Ignacio I. Wistuba4, Helen Piwnica-Worms5, Aysegul A. Sahin3,

& Dihua Yu1,2*

1 Department of Molecular and Cellular Oncology, The University of Texas MD Anderson

Cancer Center, Houston, TX 77030, USA; 2 Cancer Biology Program, The University of Texas

MD Anderson Cancer Center UTHealth Graduate School of Biomedical Sciences, Houston, TX

77030, USA; 3 Department of Pathology, The University of Texas MD Anderson Cancer Center,

Houston, TX 77030, USA; 4 Department of Translational Molecular Pathology, The University

of Texas MD Anderson Cancer Center, Houston, TX 77030, USA; 5 Department of Experimental

Radiation Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX 77030,

USA

*Address correspondence to: Dihua Yu, M.D., Ph.D., Department of Molecular and Cellular

Oncology, Unit 108, Rm Z11.5034, The University of Texas MD Anderson Cancer Center, 6565

MD Anderson Blvd., Houston, Texas 77030. Phone: 713-792-3636; Fax: 713-792-4544; E-mail:

[email protected]

Running title: SPHK1 enhances TNBC metastasis

Key words: SPHK1, TNBC, metastasis, FSCN1, NFĸB

Declaration of Conflict of Interest: The authors declare no competing financial interests.

Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 2019 American Association for Cancer Research. Author Manuscript Published OnlineFirst on June 25, 2019; DOI: 10.1158/0008-5472.CAN-18-3803 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Abstract

Triple negative breast cancer (TNBC) is the most aggressive breast cancer subtype. To identify TNBC therapeutic targets, we performed integrative bioinformatics analysis of multiple breast cancer patient-derived gene expression datasets and focused on kinases with

FDA-approved or in-pipeline inhibitors. Sphingosine kinase 1 (SPHK1) was identified as a top candidate. SPHK1 overexpression or downregulation in human TNBC cell lines increased or decreased spontaneous metastasis to lungs in nude mice, respectively. SPHK1 promoted metastasis by transcriptionally upregulating the expression of the metastasis-promoting gene

FSCN1 via NFκB activation. Activation of the SPHK1/NFκB/FSCN1 signaling pathway was associated with distance metastasis and poor clinical outcome in TNBC patients. Targeting

SPHK1 and NFκB using clinically-applicable inhibitors (safingol and bortezomib, respectively) significantly inhibited aggressive mammary tumor growth and spontaneous lung metastasis in orthotopic syngeneic TNBC mouse models. These findings highlight SPHK1 and its downstream target NFκB as promising therapeutic targets in TNBC.

Significance: Sphingosine kinase 1 (SPHK1) is overexpressed in TNBC and promotes metastasis, targeting SPHK1 or its downstream target NFκB with clinically available inhibitors could be effective for inhibiting TNBC metastasis.

Introduction

Breast cancer, which arises mainly from mammary ducts or lobules, is the leading cause

of cancer-related death and most commonly diagnosed cancer in women worldwide (1).

Approximately 10%-20% of breast cancers are triple-negative, i.e., they do not express estrogen

receptor (ER), progesterone receptor (PR), or human epidermal growth factor receptor 2 (HER2)

(2, 3). Triple-negative breast cancer (TNBC) tends to occur at higher frequency in young women

and is particularly aggressive, with high recurrence and metastasis rates (4). Compared with

patients having other subtypes of breast cancer, TNBC patients have a poor overall prognosis,

e.g., the 5-year survival rate for patients with stage IV TNBC is about 22%, mainly due to early-

onset of metastasis (4). It has been reported that TNBC tumors are about 2.5 times more likely to

metastasize within 5 years than are breast tumors of other subtypes (5). Since TNBC tumors lack

expression of hormone and HER2 receptors, i.e., negative for therapeutic targets, TNBCs do not

respond to, and patients cannot benefit from, currently available hormonal and HER2-targeted

therapies.

In contrast to the successful development of therapies for hormone receptors positive,

and/or HER2 positive breast cancers, little progress has been made in identifying positively

expressed molecular targets in TNBC that are druggable (6). Clearly, there is an imposing need

to discover positive druggable targets in TNBC instead of accepting its triple negative non-

targetable status. Kinases play central roles in cancer cell signaling pathways and are druggable

targets for effective targeted therapies (7). In the past decade, numerous efforts have led to

successful development and FDA approval of inhibitors of various cancer-promoting kinases (8).

Therefore, we set out to identify activated and/or overexpressed kinases, as positive and druggable molecular targets, in TNBC with high potential for quick and efficient clinical

translation.

Our bioinformatics analysis of multiple patient-derived datasets identified that sphingosine kinase 1 (SPHK1), a lipid kinase, was expressed at significantly higher levels in

TNBC than in other breast cancer subtypes. SPHK1 catalyzes phosphorylation of sphingosine, an amino alcohol, to generate sphingosine-1-phosphate (S1P), a novel lipid signaling mediator with both intracellular (as a second messenger) and extracellular (as a ligand for G-protein-coupled receptors) functions (9). S1P regulates various cellular processes in mammalian cells, such as growth, survival, and migration. Exogenously overexpressing SPHK1 in 3T3 fibroblasts led to transformation in vitro and tumor formation in vivo, suggesting that SPHK1 acts as an oncogene

(10). SPHK1 is shown to be overexpressed in various cancers including breast cancer (11-14).

Importantly, a SPHK1 inhibitor, safingol, can effectively inhibit SPHK1 activities and is currently under multiple clinical trials (NCT00084812, NCT01553071).

In this study, we systematically tested the function of SPHK1 in TNBC progression and metastasis using multiple TNBC spontaneous metastasis models that recapitulate the entire cascade of biological steps of metastasis in patients and found that SPHK1 has a critical function in enhancing TNBC spontaneous metastasis. Mechanistically, SPHK1 upregulates FSCN1 (also known as fascin) transcription via activation of the NFκB transcriptional factor, and FSCN1 promotes metastasis. Clinically, SPHK1/NFκB/FSCN1 signaling pathway activation in patients’ TNBC tissues correlates with poor patient survival and increased metastases. To test the validity of SPHK1 pathway as druggable targets in TNBC, we therapeutically targeted

SPHK1 by safingol and/or NFκB with a clinically-applicable inhibitor bortezomib. Strikingly, combinatorial treatment with both SPHK1 and NFκB inhibitors significantly inhibited both

TNBC primary tumor growth and lung metastasis compared to either single agent treatment.

These data demonstrated that SPHK1/NFκB pathway can serve as a positive therapeutic targets for effective inhibition of TNBC and metastasis. These preclinical findings could be fast-track translated to the clinic for the treatment of TNBC and metastasis in patients.

Materials and methods

Cell culture. Human cancer cell lines (MCF-7, T47D, BT474, HCC1954, HCC70, Hs578T,

MDA-MB-231, MDA-MB-435, and MDA-MB-436) and a mouse breast cancer cell lines (4T1),

were obtained from the American Type Culture Collection. Mouse breast cancer cell line E0771 and Met-1fvb2 were purchased from CH3BioSystems and Lonza respectively. These cell lines were verified by the MD Anderson Cancer Center Cell Line Characterization Core Facility.

BC3-p53KD were provided by Dr. H. Piwnica-Worms (15). Cells were cultured in Dulbecco

modified Eagle medium (DMEM) with 10% fetal bovine serum (FBS) and 0.1% penicillin-

o streptomycin in 5% CO2 at 37 C. All cell lines were tested for mycoplasma contamination by

using the MycoAlert Mycoplasma Detection Kit (Lonza) and were negative.

Antibodies and reagents. Rabbit polyclonal antibody against Sphk1 (HPA022829) and FSCN1

(HPA005723) were purchased from Sigma-Aldrich. Mouse polyclonal antibody against FSCN1

1 (sc-46675) was bought from Santa Cruz Biotechnology, and rabbit monoclonal antibody

against SPHK1 (ab109522) was bought from Abcam. Mouse monoclonal anti-β-actin antibody

(A5441) was from Sigma-Aldrich, and anti-Ki67 antibody (M7240) was from Dako. Normal rabbit IgG (2729), NFκB p65 (8242), and H3K4me3 (9727) were all from Cell Signaling

Technology. p- NFκB p65 (S536) (ab86299) was purchased from Abcam. The in situ cell death

detection kit (TUNEL technology, 11684817910) was from Roche. The horseradish peroxidase–

linked secondary antibodies against mouse (NA931) and rabbit (NA934) were from GE

Healthcare. Actinomycin D (A9415) was from Sigma-Aldrich. A cell permeable peptide (CAS

213546-53-3) that inhibits translocation of the NFkB active complex into the nucleus and its

corresponding NFkB control peptide (sc-3060) were brought from Santa Cruz Biotechnology.

Safingol (CAS 15639-50-6) was purchased from Cayman Chemical. Bortezomib (CAS 179324-

69-7) was purchased from EMD Millipore. CAPTISOL® (20g) was kindly provided by Cydex

Pharmeceuticals.

Generation of stable cell-lines. To overexpress SPHK1, retroviral vector pWZL-Neo-Myr-Flag-

DEST containing the SPHK1 open reading frame (ORF) under the control of CMV promotor

with G418 (100 µg/ml) as selection marker was used (kindly provided by Dr. J. Zhao). Empty

vector was used as a control. To stably knock down SPHK1 in MDA-MB-435, Hs578T, and

BC3-p53 KD cells, we used two small hairpin RNA (shRNA) constructs, targeting the SPHK1 3’

untranslated region, cloned into the pGIPZ lentiviral vector (RefSeq NM_001142601, Open

Biosystems) with puromycin (2 µg/ml) as selection marker. To stably knock down FSCN1 in

MDA-MB-435 cells, we used three shRNA constructs, targeting the FSCN1 3’ untranslated

region, cloned into the pGIPZ lentiviral vector (RefSeq NM_003088.3, Open Biosystems) with

puromycin (2 µg/ml) as selection marker. Non-silencing shRNA was used as a control for both above mentioned shRNA knockdown experiments. To overexpress FSCN1, retroviral vector pLenti6/V5-DEST containing the FSCN1 ORF under the control of CMV promotor with

blasticidin (3 µg/ml) as selection marker was used (plasmid #31207, Addgene). Lentiviral vector

with mCherry sequence was used as a control. Lentiviral vectors (with ORFs or shRNA) were

transfected into the packaging cell line 293T, together with a packaging DNA plasmid (psPAX2)

and an envelope DNA plasmid (pMD2G), through Lipofectamine transfection. After 48 h,

viruses were collected, filtered, and incubated with target cells in the presence of 8-10 μg/mL

Polybrene for 24 h. The infected cells were selected with suitable selection markers, with

concentration mentioned above, to generate the stable clone.

siRNA knockdown. To knockdown FSCN1 in MDA-MB-231 cells, SMARTpool: ON-

TARGETplus human FSCN1 siRNA (DharmaconTM) was used to transfect the cells.

Lipofectamine® RNAiMAX transfection reagents (InvitogenTM) was used for siRNA transfection

and the protocol was followed per manufacturer’s instruction.

Site-directed mutagenesis. Nucleotides within the NFκB transcription factor binding sites of the

FSCN1 promoter were altered by site-directed mutagenesis by using Q5 site-directed

mutagenesis kit (NEB) according to the manufacturer’s instructions. Wild-type FSCN1 promoter

(-333/+147 bp) in the pGL3luc (basic) vector was used as a template to introduce mutation at

three nucleotides that affect both binding sites. The sense and anti-sense primers used were 5’-

GTCCGAGGTGATGGACATCAGGGG-3’ and 5’- ACCCCGACCCCAAGCCTC -3’,

respectively. Mutated promoter fragments were sequenced to verify the presence of mutations.

Western blotting. Western blot analysis was performed as previously described (16).

Densitometry analyses were performed by evaluating band intensity mean gray value of

indicated protein and normalizing it with the mean gray value of corresponding lane’s loading control using ImageJ software.

RNA extraction, reverse transcriptase–polymerase chain reaction (RT-PCR), and

quantitative real-time PCR (qPCR). RNA extraction and RT-PCR were performed as

described previously (16). For the SYBR green-based qPCR assay, 1 µL of cDNA was used as a

template for quantitative real-time PCR with iQTM SYBR Green Supermix (Biorad) and the

StepOnePlusTM (Applied Biosystem) instrument according to the manufacturer’s instruction. The

RNA expression rate was quantified by the relative quantification (2-Δ ΔCt) method, and 18S

expression was used as the internal control. The primers that were used are listed below:

Genes Primers

SPHK1 F: 5’-AACTACTTCTGGATGGTCAG -3’

R: 5’-TCCTGCAAGTAGACACTAAG -3’

FSCN1 F: 5’-CCAGGGTATGGACCTGTCTG-3’

R: 5’-CGCCACTCGATGTCAAAGTA-3’

18S F: 5’-AACCCGTTGAACCCCATT-3’

R: 5’-CCATCCAATCGGTAGTAGCG-3’

ChIP assay. Procedures for chromatin isolation and immunoprecipitation were performed as

previously described (16). Normal IgG, NFκB p65, and H3K4me3 antibodies were used at 2 µg

per reaction in immunoprecipitation. Co-precipitated DNA (2 µl) was analyzed by quantitative

PCR. The forward and reverse primers used for amplification of the NFκB binding region in the

FSCN1 promoter (-333/+147 bp) were as follows: forward: 5’-

CTCAAACCTCGCTCGTCCTT-3’ and reverse: 5’- CATCACCCCTCACAACCCC -3’.

Three-dimensional (3D) cell culture. 3D culture was performed in either an 8-well chamber slide (BD Falcon) or in Costar 6-well plate with ultra-low attachment surface (Corning). For the

8-well chamber, 100 µl of Matrigel was added to the bottom of each chamber and incubated at

37oC for 20 min. Cells of interest were mixed in culture medium with 5% Matrigel and added to

each well to a final concentration of 1500 cells/well. For 6-well plates with low attachment, 2 ×

105 cells of interest that were mixed into the culture medium with 5% Matrigel were added to

each well.

Quantification of 3D invasiveness. Indicated cells were grown in 3D culture in 8-well chamber

culture slides. Images of the spheroid structures at multiple fields were obtained with use of a

microscope at the indicated time, and invasive structures per field were counted. Structures that

had projections coming out from the main spheroid body were counted as invasive structures.

mRNA stability assay. Equal numbers of 435 shSCR and 435 shSPHK1.1 cells were plated in a

6-well low-attachment plate with 5% Matrigel and incubated for 3 d at 37oC. Cells were treated

with 5 µg/mL actinomycin D for 1, 2, 4, 8, 12, and 16 h. Total RNA was extracted after each

time-point by using TRIzol reagent (Invitrogen), and quantitative PCR was performed to

determine the relative mRNA level of FSCN1.

Immunohistochemical analysis. The excised MFP tumors were fixed in 10% neutral buffered

formalin and embedded in paraffin for immunohistochemical (IHC) staining. IHC staining was

performed similarly as previously described (16). TUNEL staining was done in paraffin sections

with use of an in situ cell death detection kit, POD (11684817910, Roche), according to the

manufacturer’s instructions.

SPHK1 kinase activity. SPHK1 activity in cytosol was determined as described previously (17).

The intracellular level of S1P in SPHK1 modulated cells were quantified using S1P ELISA kit

(Echelon Biosciences, K-1900) and protocol was followed as per manufacturer’s instructions.

Animal experiments. All procedures and experimental protocols involving mice were approved by the Institutional Animal Care and Use Committee at The University of Texas MD Anderson

Cancer Center.

Female nude mice (6 weeks old, 4-7 mice per group as indicated in figures and/or figure legends) were orthotopically injected with human cancer cells (2 × 105 cells for MDA-MB-435

and MDA-MB-231 cells, 1 × 106 cells for BC3-p53KD cells; cells were re-suspended in 50:50 mixture of Matrigel in PBS) into mammary fat pads (mfps), and tumors were allowed to develop for an indicated number of days. Tumor sizes were measured with digital calipers twice a week,

and tumor volumes were calculated with use of a modified ellipsoidal formula: 1/2 × (length ×

width2). MFP tumors were surgically excised with survival surgery, and the mice were further monitored for an indicated number of weeks for spontaneous metastasis. All mice were

euthanized at indicated times, and lungs were harvested, fixed and paraffin embedded. After

lungs were H&E stained, the number of metastatic lesions were enumerated by pathologist using

brightfield microscopy. Detailed description of in vivo treatment experiments in mice is provided

in Supplementary Methods.

cDNA microarray and analysis. Unbiased platform, HumanHT-12_v4 (Illumina), was applied

for gene profiling of MFP tumors and matched spontaneous lung metastasis formed by control

and Sphk1 knockdown MDA-MB-435 cells in collaboration with the cDNA microarray core

facility at MD Anderson Cancer Center. The raw and normalized microarray data have been

deposited in the GEO database under accession number GSE128624. Gene cluster maps for MFP

and lung metastasis samples were generated by using sequence analysis of microarray (SAM)

analysis. To identify SPHK1-regulated genes in 435 cells, R software and limma software

packages were used to identify differentially expressed genes using a 1.5-fold change threshold

and an adjusted p value cutoff at 0.01. Ingenuity Pathway Analysis (IPA) software

(http://www.ingenuity.com) was used to perform the functional annotation and pathway analysis

of the differentially expressed genes. Gene set enrichment analysis was performed on MFP

microarray data with use of an online tool (http://software.broadinstitute.org/gsea/index.jsp), as

described previously (18).

Case Selection, Tissue Microarray (TMA) Construction and analysis. We obtained archival, formalin-fixed and paraffin-embedded (FFPE) material from surgically resected breast cancer specimens from the Breast Tumor Bank at M. D. Anderson Cancer Center from 2001 to 2013

(Houston, TX). Tumor tissue specimens obtained from 117 triple negative breast cancers were histologically examined, classified using the World Health Organization (WHO) classification of

Breast Tumors and selected for TMA construction. After histologic examination, tumor TMAs

were prepared using triplicate 1-mm-diameter cores per tumor. All the archival paraffin-

embedded tumor samples were coded with no patient identifiers. Detailed clinical and pathologic

information, including demographic, pathologic TNM staging, overall survival, and time of

recurrence were collected. Detailed descriptions of IHC staining/quantification and survival

analysis are provided in Supplementary Methods.

Bioinformatics, statistics, and survival analysis. GEO2R analysis, a Web-based application for

analyzing gene expression in Gene Expression Omnibus (GEO) data sets, was performed as

described elsewhere (19). The Kaplan-Meier plotter (20), a Web-based tool, was used to assess

the effect of the SPHK1 and the FSCN1 genes on survival of TNBC patients. To select the

TNBC patients, following selection criteria were used: ER negative, PR negative, HER2

negative, grade 3 and intrinsic subtype basal. Survival rates were compared by using the log-rank

test, and hazard ratios were calculated by using a multivariable Cox proportional hazards model.

For correlation analysis, expression values of SPHK1 and FSCN1 from patient samples were

downloaded from The Cancer Genome Atlas (TCGA) and Curtis breast dataset (21). GraphPad

Prism (Prism 6; GraphPad Software Inc.) was used to generate a correlation graph and calculate

the Pearson coefficient (r) from the downloaded data. All statistical analyses were performed by

using GraphPad Prism. The data were analyzed by either one-way analysis of variance (multiple

groups) or a t test (two groups). Differences with p < 0.05 (two-sided) were considered

statistically significant. *p<0.05, **p<0.01, ***p<0.001 by two-tailed t-test.

More methods (Plasmids construction; Cell proliferation assay; Migration assay and invasion

assay; Transient transfection and luciferase reporter assay; and Flow cytometry) with detailed

descriptions are provided in Supplementary Methods.

Results SPHK1 is highly expressed in TNBCs and promotes spontaneous lung metastasis. To

identify kinase gene(s) that are particularly overexpressed in TNBC to serve as potential

therapeutic targets, we performed GEO2R analysis (GSE27447) between TNBC and non-TNBC

tumor samples from patients (22). Only four kinase genes were among the top 100 differentially

expressed genes, of which SPHK1 was the only kinase that was overexpressed in TNBC tumors

compared with non-TNBC tumors (Supplementary Fig.S1A-S1B). Our further analysis of breast

cancer microarray data of TCGA validated that expression of SPHK1 is significantly higher in

basal subtype when compared with normal breast tissues and other subtypes of breast cancers

(Fig.1A).

Survival analysis by Kaplan-Meier (KM)-plotter indicated that expression of SPHK1 is

significantly associated with poorer relapse-free survival in TNBC patients (Fig.1B). SPHK1

expression is also higher in TNBC-derived cell-lines than in cell-lines of other subtypes at both

mRNA (Fig.1C and Supplementary Fig.S1C-S1D) and protein (Fig.1D and Supplementary

Fig.S1E) levels. Thus, SPHK1 is mostly overexpressed in TNBC tumors and cell-lines.

To determine the function of SPHK1 in TNBC progression and metastasis, we first

performed gain-of-function studies using MDA-MB-231 human TNBC cell-line that express an

intermediate level of endogenous SPHK1 compared with other breast cancer cell-lines, and

generated control (231vec) and SPHK1-overexpressing (231SPHK1) stable sublines (Fig.1E-1F).

231SPHK1 cells showed increased levels of intracellular S1P compared to 231vec cells (Fig.

1G). 231SPHK1 cells showed increased phosphorylation of sphingosine leading to increased

S1P as detected by a semi in vitro kinase assay compared to 231vec cells (Fig.1H). 231SPHK1

cells also showed increased migration (Supplementary Fig.S2A) and invasion (Supplementary

Fig.S2B) potential in vitro, but no significant differences in cell proliferation in vitro

(Supplementary Fig.S2C) compared to 231vec cells.

To examine whether SPHK1 promotes TNBC tumorigenesis and spontaneous metastasis,

231vec control and 231SPHK1 cells were orthotopically injected into mammary fat pads (mfp)

of nude mice. The mfp tumors were surgically excised 28 days post-injection, and the mice were

monitored for about 10 more weeks for development of spontaneous metastasis (Supplementary

Fig.S2D). There was no significant difference in mfp tumor size between 231vec and 231SPHK1

groups by day 28 (Fig.1I). Remarkably, the number of metastatic lesions in the lungs was

significantly higher in mice bearing 231SPHK1 mfp tumors than in control mice bearing 231vec

mfp tumors (Fig.1J). These data showed that despite having no significant effect on mfp tumor

growth, SPHK1 overexpression enhanced spontaneous lung metastasis of MDA-MB-231 human

TNBC cells.

SPHK1 knockdown decreases spontaneous lung metastasis. To determine whether SPHK1 is

required for TNBC progression and metastasis, we stably knocked down SPHK1 in TNBC

patient-derived xenograft (PDX) cells (BC3-p53KD) (15) using lentiviral vector expressing two

distinct SPHK1-targeting shRNAs (shSPHK1.1 and shSPHK1.2). Lentiviral vector expressing

non-targeting scrambled shRNA (shScr) was used to generate the control cell line. Additionally,

since PDX cells are not amendable for some in vitro assays, we also knocked down SPHK1 in

two other TNBC cell-lines [MDA-MB-435 (23) and Hs578T] expressing relatively high levels of

endogenous SPHK1. SPHK1 knockdown by shSPHK1.1 was highly effective at the mRNA level

detected by qRT-PCR (Supplementary Fig.S3A) and at the protein level determined by western

blotting (Fig.2A and Supplementary Fig.S3B). Knocking down SPHK1 had no significant effect

on cell proliferation in vitro in any of the three cell-lines (Supplementary Fig.S3C). SPHK1

knockdown in MDA-MB-435 cells showed decreased levels of intracellular S1P compared to

control (Supplementary Fig.3D). SPHK1 knockdown in MDA-MB-435 and Hs578T cells led to

decreased phosphorylation of sphingosine leading to decreased S1P as detected by a semi in vitro

kinase assay (Supplementary Fig.S3E), and inhibited the migration and invasion potential in vitro (Supplementary Fig.3SF-SH).

To determine whether SPHK1 is required for TNBC tumorigenesis and spontaneous

metastasis, SPHK1 knockdown BC3-p53KD.shSPHK1.1 and MDA-MB-435 (shSPHK1.1 and

shSPHK1.2) cells along with respective control cells were orthotopically injected into mfp of

nude mice. The mfp tumors were resected by survival surgery on day 54 for mice injected with

BC3-p53KD sublines, and on day 28 for mice injected with MDA-MB-435 sublines

(Supplementary Fig.S4A-S4B). There was no significant difference in primary tumor size

(Fig.2B-2C) or in tumor cell proliferation (Supplementary Fig.S4C-S4D) between control and

SPHK1 knockdown mfp tumors in either models, although the mfp tumors of SPHK1

knockdown cells were more apoptotic as detected by TUNEL staining than did tumors formed by control cells (Supplementary Fig.S4E-S4F).

To evaluate the impact of SPHK1 knockdown on metastasis, lungs were harvested from

mice bearing BC3-p53KD mfp tumors at 187 days after mfp tumor resection, and from mice

bearing highly aggressive MDA-MB-435 mfp tumors at 47 days after primary tumor resection

(Supplementary Fig.S4A-S4B). A significant decrease in the number of spontaneous lung

metastases were observed in mice injected with SPHK1 knockdown cells compared with mice

injected with control cells in both models (Fig.2D-2E). Together, these data indicate that SPHK1

is essential for TNBC’s development of aggressive spontaneous lung metastasis and the SPHK1

overexpression may serve as a positive therapeutic target for inhibition of TNBC lung metastasis.

FSCN1 upregulation contributes to SPHK1-driven metastasis. SPHK1 is a critical lipid

kinase with pleiotropic effects on various cellular functions (9), however, little is known about

how SPHK1 promotes metastasis. To attain insights on the molecular mechanism underlying

SPHK1-driven spontaneous metastasis, the primary mammary tumor tissues and matched spontaneous lung metastasis tissues from mice injected with 435.shScr versus 435.shSPHK1.1

cells (see Fig.2C and 2E) were profiled for SPHK1-modulated genes (Supplementary Fig.S5A-

S5B). The list of SPHK1-regulated differentially expressed genes (435.shSPHK1.1 vs

435.shSCR) was generated for mfp tumor samples and was subjected to Ingenuity Pathway

Analysis (IPA). IPA identified FSCN1 as a top SPHK1-regulated gene involved in cancer cell

migration, invasion, and metastasis (Fig.3A and Supplementary Fig.S5C). Indeed, the FSCN1 gene expression was high in SPHK1 high-expressing tissue sample (i.e. 435.shScr) and low in

SPHK1 low-expressing tissue samples (i.e. 435.shSPHK1.1) of both primary tumors and

spontaneous lung metastases as validated by qRT-PCR (Fig.3B and Supplementary Fig.S5D) and

by western blotting (Fig.3C and Supplementary Fig.S5E). Similarly, other SPHK1 high-

expressing mfp tumor samples (i.e. 231SPHK1 and BC3-p53KD.shScr) also had higher FSCN1

expression than SPHK1 low-expressing mfp tumors (i.e. 231vec and BC3-p53KD.shSPHK1.1,

respectively) as shown by western blotting (Fig.3D-3E). Furthermore, in two different breast

cancer patients’ datasets [TCGA and Curtis breast datasets (21)], SPHK1 gene expression

correlated with FSCN1 gene expression, and high expressions of both SPHK1 and FSCN1 genes

was observed in TNBC patients’ compared to other subtypes (Fig.3F and Supplementary

Fig.S5F). FSCN1 expression, like SPHK1 expression, was also upregulated in basal subtype

compared with other subtypes of breast cancer in TCGA dataset (Fig.3G); moreover, FSCN1

expression correlated with a poor survival rate in TNBC patients (Fig.3H).

The FSCN1 proteins organize F-actin into parallel bundles and are required for the

formation of actin-based cellular protrusions (24). It plays a critical role in cell migration, motility, and adhesion and in cellular interactions (25, 26). To test whether FSCN1 can recover

metastasis from SPHK1 knockdown cells, FSCN1 was ectopically expressed in 435.shSPHK1.1

and Hs578T.shSPHK1.1 cells (Supplementary Fig.S6A-S6D). Clearly, ectopic expression of

FSCN1 in SPHK1 knockdown cells increased their migration (Fig.4A and Supplementary

Fig.S6E-S6F) and invasion (Fig. 4B and Supplementary Fig.S6G-S6H) potentials in vitro, but it

had no significant effect on cell proliferation in vitro (Supplementary Fig.S6I-S6J). Importantly,

ectopic expression of FSCN1 in 435shSPHK1.1 cells rescued the spontaneous metastatic

potential in vivo (Fig.4C). On the other hand, knocking down FSCN1 gene in SPHK1

overexpressing 231SPHK1 cells (Supplementary Fig.S7A) and in MDA-MB-435 cells having

high endogenous SPHK1 expression (Supplementary Fig.S7B-S7C) significantly reduced their

migration (Fig.4D and Supplementary Fig.S7D) and invasion (Fig.4E and Supplementary

Fig.S7E), indicating that the SPHK1-induced metastasis-related functions are mediated, at least

partially, by FSCN1.

Regarding FSCN1-mediated metastasis-related functions, various studies have shown that FSCN1 is important for invadopodia assembly to promote protrusive invasion of cancer cells

(27, 28). Thus, we tested whether SPHK1 high-expressing cells with high FSCN1 expression

may induce more protrusive invasion in three dimensional (3D) culture. Indeed, SPHK1 high-

expressing BC3-p53KD.shScr and 435.shScr cells with high FSCN1 expression formed many protrusive structures projecting into the surrounding matrix after 10 days in standard 3D culture; whereas BC3-p53KD.shSPHK1.1 and 435.shSPHK1.1 cells with low FSCN1 expression mostly formed round shaped structures with very few protrusions (Fig.4F and Supplementary Fig.S7F).

Importantly, knocking down FSCN1 gene in SPHK1-overexpressing 231SPKH1 cells (Fig.4G)

and MDA-MB-435 cells (Supplementary Fig.S7G) reduced their protrusive structures projecting

into the surrounding matrix in standard 3D culture. These data indicate that FSCN1 upregulation

could contribute to SPHK1-driven invasion and metastasis by inducing protrusive invasion.

SPHK1 upregulates FSCN1 transcription via activation of NFκB. The critical function of

FSCN1 in SPHK1-driven invasion and metastasis impelled us to dissect how FSCN1 is

upregulated by SPHK1. Interestingly, we found that knockdown SPHK1 down-regulated FSCN1

expression in standard 3D culture but not in two dimensional (2D) culture at both mRNA

(Fig.5A and Supplementary Fig.S8A) and protein (Fig.5B and Supplementary Fig.S8B) levels

(29). Next, we tested whether SPHK1 regulates FSCN1 mRNA via modulating mRNA stability or transcription. The control and SPHK1 knockdown MDA-MB-435 cells in 3D culture were

treated with actinomycin D to block transcription and were detected for FSCN1 mRNA stability,

which showed no significant difference (Supplementary Fig.S8C), indicating that SPHK1 does

not modulate FSCN1 mRNA stability.

To investigate whether SPHK1 regulates FSCN1 mRNA transcription, we cloned the -

1376 to +147 base pair (bp) FSCN1 promoter region into the pGL3 basic vector expressing a

luciferase reporter gene, and transfected into either 435.shScr or 435.shSPHK1.1 cells. FSCN1

promoter-driven luciferase activities were reduced by SPHK1 knockdown in 3D cultured, but not

in 2D cultured 435.shSPHK1.1 cells (Fig.5C), indicating SPHK1 is essential for FSCN1 mRNA

transcription. Subsequently, all studies on SPHK1-induced FSCN1 transcription were performed

in the 3D culture system.

To determine the FSCN1 promoter region responsible for SPHK1-mediated transcriptional

upregulation, we made a series of 5’ deletions in the -1376/+147 bp FSCN1 promoter construct

in the pGL3 basic vector and generated -333/+147 bp and -93/+147 bp constructs

(Supplementary Fig.S8D). We transfected these deletion constructs of FSCN1 promoter into

either 435.shScr or 435.shSPHK1.1 cells and compared their activities in driving luciferase

reporter gene expression. The -333/+147 bp FSCN1 promoter region was sufficient to induce

transcription in SPHK1 high expressing cells, whereas both basal transcription and SPHK1-

induced transcription were significantly inhibited when FSCN1 promoter was deleted to -93

/+147 bp region (Fig.5D). The data indicate that the -333/-93 bp region in FSCN1 promoter is

important for basal and SPHK1-mediated FSCN1 transcription. Using online software (PROMO), we identified that the -333bp to -93 bp region of FSCN1 promoter harbors binding sites of

various transcription factors, one of which was NFκB (Fig.5E), that can be activated by SPHK1

(30). Indeed, NFκB pathway was activated more in SPHK1-overexpressing 231SPHK1 tumor

lysates and 435.shScr cells growing in 3D culture compared to respective controls

(Supplementary Fig.S8E-S8F). Intracellular S1P, downstream of SPHK1, was known to act as a cofactor of TRAF2 leading to IKKα/β activation resulting in IĸBα phosphorylation and

degradation, consequently, NF-kB activation upon TNFα stimulation (31). Therefore, we examined the phosphorylation status of IKKα/β and IĸBα in our SPHK1-modulated MDA-MB-

435 cells in both 2D and 3D cultures. Compared to SPHK1-overexpressing 435.shScr control cells, phosphorylation of IKKα/β and IĸBα are decreased in SPHK1 knocked down

435.shSPHK1 cells in 3D culture, not in 2D culture, confirming that SPHK1/S1P regulates NF- kB activation via IKKα/β/IĸBα pathway (Supplementary Fig.S8G). Additionally, gene set

enrichment analysis (GSEA) of our cDNA microarray data revealed that tumor necrosis factor

(TNF) signaling was upregulated in mfp tumors of SPHK1-high expressing 435.shScr cells

relative to the SPHK1-low expressing 435.shSPHK1.1 mfp tumors (Supplementary Fig.S8H)

(32). Thus, SPHK1 may induce FSCN1 upregulation via activation of the NFκB transcription

factor.

To investigate whether the NFκB binding site in the FSCN1 promoter is critical for

SPHK1-induced FSCN1 upregulation, we generated NFκB binding site mutant (NFκB BSmut)

from the -333/+147 bp wild type FSCN1 promoter-driven luciferase reporter construct (NFκB

BSwt) (Fig.5E and Supplementary Fig.S8I). The NFκB BSwt and the NFκB BSmut constructs

were transfected into SPHK1 high-expressing (MDA-MB-435 and Hs578T) cells and compared

for promoter activities. Compared to NFκB BSwt construct, NFκB BSmut construct with

mutation of the NFκB binding site had significantly reduced FSCN1 promoter activity in both

cell models (Fig.5F). Additionally, a cell permeable peptide that inhibits translocation of the

NFkB active complex into the nucleus significantly inhibited NFκB BSwt FSCN1 promoter

activities compared to the control peptide in MDA-MB-435 cells (Fig.5G). These data indicate

that nuclear NFκB binding to the NFκB binding site of FSCN1 promoter is critical for FSCN1

transcriptional upregulation in SPHK1 high-expressing TNBC cells. Next, we examined whether

NFκB transcription factor binding to the NFκB binding region (-263/-253 bp) of FSCN1

promoter are higher in SPHK1 high-expressing TNBC cells than that in SPHK1 low-expressing

cells. We performed a ChIP assay using NFκB antibody to bring down NFκB by

immunoprecipitation (IP) and followed by qPCR with primers flanking NFκB binding region (-

263/-253 bp) of FSCN1 promoter. Clearly, the binding of NFκB is more enriched in the -263/-

253 bp region of FSCN1 promoter in SPHK1 high-expressing (435.shScr and Hs578T.shScr)

cells than in their corresponding SPHK1 knockdown cells, whereas the binding of tri-methylated

H3K4 showed no significant difference (Fig.5H). Together, these data indicate that SPHK1

upregulates FSCN1 gene expression at the transcriptional level via activation of NFκB

transcription factor.

SPHK1/pNFκB/FSCN1 expressions in patients’ TNBC tissues correlate with poor survival.

To determine the clinically relevance of our above findings, we examined whether

SPHK1/NFκB/FSCN1 pathway activation in patients’ TNBC tissues is associated with increased

metastasis and poor clinical outcome. We performed immunohistochemistry (IHC) analyses of

SPHK1, p-NFκB and FSCN1 expressions in tissue microarrays (TMAs) of patients’ TNBC

tissue samples. Patients (N=117) whose TNBC tissues are included in the TMAs have various

clinical and pathological features (Supplementary Table S1). IHC analyses revealed high

expression of SPHK1, pNFκB and FSCN1 in 59.2% (68/115), 67% (71/106) and 62.7 %

(69/110) of TNBC tissue samples, respectively (Figs.6A-6B). To examine whether

SPHK1/pNFκB/FSCN1 signaling pathway activation is critical for TNBC progression and

metastasis in patients, we examined the relationships of high expressions of SPHK1, pNFκB and

FSCN1 with TNBC progression and metastasis. Since IHC staining of phospho-proteins in

archived tissues of patients may be unreliable, we analyzed whether co-expressions of SPHK1

and FSCN1 may correlates with increased metastasis and poor clinical outcome. Patients were

divided into two groups: 1) low expression of one or both markers (i.e. SPHK1 and/or FSCN1

low), and 2) high expression of both markers (i.e. SPHK1 and FSCN1 high) in their TNBCs.

Compared to group-1 patients, high expressions of SPHK1 and FSCN1 in group-2 patients are

significantly associated with poorer distance metastasis-free survival (DMFS) (p=0.045) as well

as worse overall survival (OS) (p=0.003) (Fig.6C-6D). Thus, clinically, high expression of

SPHK1 and FCSN1 in TNBC tissues correlate with increased distant metastasis and poor

survival in TNBC patients.

Targeting SPHK1 and the NFκB axis impedes tumor progression and spontaneous lung

metastasis. The above data from TNBC animal models and TNBC patients’ tissues prompted us

to test whether therapeutically targeting SPHK1/pNFκB/FSCN1 axis could inhibit TNBC

progression and metastasis in an aggressive TNBC animal model. Since SPHK1 regulates

FSCN1 via the NFκB transcription factor, and clinically applicable inhibitors of both SPHK1 and

NFκB are available, we tested the efficacy of SPHK1 and NFκB inhibitors, either as a single

agent or in combination, for deterring TNBC progression and metastasis. SPHK1 were targeted

by safingol, a SPHK1 inhibitor tested in multiple clinical trials (NCT00084812, NCT01553071),

and NFκB were targeted with bortezomib, an FDA-approved proteasome inhibitor drug for

multiple myeloma, but shown to inhibit NFκB activity (33). Safingol treatment decreased

SPHK1 protein expression in both human MDA-MB-435 cells and 4T1 mouse TNBC cells in

vitro (Supplementary Fig.S9A-S9B). Similarly, bortezomib treatment in 4T1 cells decreased

phospho-NFκB expression in 2D and, more prominently, in 3D culture (Supplementary

Fig.S9C).

To test whether safingol alone, bortezomib alone, or their combination could deter TNBC

progression and metastasis, we orthotopically injected highly aggressive 4T1 mouse TNBC cells

(50,000 cells/mice) into BALB/c mice to induce mfp tumors. On day 7, when the primary tumors

were palpable, we randomized the mice into four groups and treated them by intraperitoneal

(i.p.) injection of one of the following for 3 weeks: (a) vehicle (n=10), (b) safingol (5 mg/kg,

every 3 days, n=11), (c) bortezomib (0.5 mg/kg, every 3 days, n=11), or (d) safingol plus

bortezomib (n=15). Tumor growth and body weight of mice were monitored every three days.

The single treatment of either safingol or bortezomib had no significant effects on primary tumor

growth, whereas the combination treatment significantly reduced primary tumor growth (Fig.7A

and Supplementary Fig.S9D). Interestingly, safingol only, or bortezomib only significantly

prolonged overall survival of mice compared with vehicle treatment (median survival of 37 days,

or 40 days, versus 31 days, respectively) (Fig.7B). Strikingly, safingol plus bortezomib

combination treatment dramatically increased the median survival of mice from 31 days to 47

days (Fig.7B). There was no significant difference in mouse body weight, and no dramatic difference in their blood counts of lymphocytes and myeloid cells among four treatment groups,

suggesting that drug treatment, either as a single agent or in combination, did not induce any

acute toxicity (Supplementary Fig.S9E-S9F).

To evaluate the inhibitory effects of safingol, bortezomib, or their combination on tumor

growth and metastasis, five mice under each of the four treatments were euthanized at day 21

post treatment and their mfp tumors as well as lungs were harvested for examination. Consistent

with tumor volume detected above in mice, primary tumor weights were significantly reduced by

combination treatment, but not the single-treatments (Fig.7C). IHC analyses of SPHK1 and

pNFκB in these primary tumor samples showed that safingol treatment decreased the SPHK1 expression and bortezomib treatment decreased the nuclear pNFκB compared to vehicle

treatment, suggesting that the drugs were effectively inhibiting their molecular targets (Fig.7D-

7E and Supplementary Fig.S9G). Safingol treatment also decreased its downstream nuclear

pNFκB compared to vehicle treatment (Fig.7E). Either safingol or bortezomib treatment reduced

FSCN1 expression (Fig.7F and Supplementary Fig.S9G). Remarkably, combination treatment

significantly decreased SPHK1 expression, nuclear pNFκB level, and FSCN1 expression

compared to vehicle or single treatments (Fig.7D-7F and Supplementary Fig.S9G). Furthermore,

combination treatment decreased tumor cell proliferation detected by Ki67 staining and

increased tumor cell apoptosis as shown by TUNEL staining, respectively (Fig.7G-7H and

Supplementary Fig.S9G). More importantly, safingol alone and bortezomib alone significantly

reduced spontaneous metastasis to the lungs compared to vehicle treatment (Fig.7I and

Supplementary Fig.S9H). Combination treatment further reduced lung metastasis significantly

compared to each single treatment, inducing ~80% inhibition of spontaneous lung metastasis in

mice compared to vehicle treatment (Fig.7I and Supplementary Fig.S9H).

To evaluate whether or not the inhibition of metastasis by combination treatment resulted from reduced primary tumor growth, we examine the effect on metastasis after primary tumors

were removed at similar sizes in vehicle and combination treatment groups. (Supplementary

Fig.S10A-S10B). . The lungs of these mice were harvested for examination of metastatic lesions

11 days post primary tumor resection in both vehicle and combination treatment groups.

Combination treatment significantly reduced spontaneous metastasis to the lungs compared to

vehicle treatment when the primary mfp tumors were removed at similar sizes, suggesting that

inhibition of metastasis by combination treatment is not only the consequences of reduced

primary tumor growth (Supplementary Fig.S10C).

Discussion

TNBCs are negative of therapeutic targets and do not respond to current hormonal and

HER2-targeted therapies (4). In this study, we embark on identifying positive therapeutic

target(s) in TNBCs to develop effective targeted therapies for TNBC, especially TNBC

metastasis. Our integrative bioinformatics analysis of breast cancer patient-derived gene

expression datasets revealed that expression of SPHK1 is significantly higher in TNBCs and is associated with poor clinical outcome of TNBC patients. Further, we found that SPHK1 plays a prominent role in promoting TNBC spontaneous metastasis in multiple mouse models.

Mechanistically, SPHK1 upregulates the expression of FSCN1 at the transcriptional level via

activation of NFκB. Clinically, high expression of SPHK1 and FCSN1 in patients’ TNBC tissues correlates with increased distant metastasis and poor survival. Furthermore, we demonstrated

that targeting SPHK1 and NFκB by clinically-applicable inhibitors effectively inhibited tumor

progression and spontaneous metastasis to the lungs in a highly aggressive TNBC mouse model.

Together, our data indicate that SPHK1 and NFκB could serve as positive therapeutic targets for

inhibiting TNBC metastasis.

We demonstrated that SPHK1 expression was higher in TNBCs compared to other

subtypes of breast cancer. Abnormal expression of SPHK1 has been strongly associated with the

development and progression of various cancers (9, 14). Several studies of breast cancer and

other cancer types have also shown that SPHK1 plays a role in cancer cell proliferation (34-36).

In contrast, our data showed that SPHK1 cannot drive TNBC cell proliferation in vitro nor tumor

growth in vivo in multiple TNBC models and in both gain- and loss-of-function studies.

Consistent with our findings, recent studies of SPHK1-specific inhibitor, PF-543, showed that

inhibiting SPHK1 had no effect on the proliferation of various cancer cells, including a TNBC

cell line MDA-MB-231 (37, 38). Notably, although SPHK1 knockdown had no significant

impact on cell proliferation, MFP tumors of SPHK1 knockdown cells had increased apoptosis

compared to MFP tumors of control cells, similar to a previous report (39). Most importantly,

our data from both gain- and loss-of-function studies showed that SPHK1 functions to promote

metastasis-related properties of TNBC cells in vitro and TNBC spontaneous metastasis in vivo.

Furthermore, we dissected the FSCN1 upregulation as a critical molecular mechanism of

SPHK1-induced TNBC cell invasion and metastasis.

In our study, we used spontaneous metastasis models to determine whether SPHK1

functions in metastasis, since the spontaneous metastasis model recapitulates all of the steps

involved in the multistep process of the metastatic cascade (40). Exogenous expression of

SPHK1 significantly increased, whereas knocking down SPHK1 significantly decreased spontaneous metastasis to the lungs in nude mice, suggesting SPHK1 expression is vital for the

metastatic potential of TNBC cells. Despite some reports suggesting that the SPHK1/S1P axis

modulates the MAPK/ERK pathway and EGFR pathways to promote invasion and metastasis in some cancer types, the mechanism by which SPHK1 promotes TNBC metastasis was unclear

(41, 42). Understating molecular pathways driving TNBC metastasis process, with fast-track

clinical translation potentials, is needed for developing effective therapeutic intervention of

TNBC metastasis. In this study, using unbiased gene expression profiling of in vivo tumor

samples, we identified FSCN1 as a top candidate gene modulated by SPHK1 and involved in

TNBC cell migration, invasion, and metastasis. FSCN1 is a cytoskeletal actin bundling protein

that binds and packages actin filaments into tertiary structures to enhance cell motility,

migration, and adhesion (24, 25, 43). FSCN1 is overexpressed in various cancers (44-46),

whereas its expression is either absent or very low in normal epithelial cells (43). Tumor cells

with high expression of FSCN1 have increased cell membrane protrusions such as filopodia and

invadopodia, which help tumor cells’ migration and extracellular matrix invasion, critical steps

for metastasis development (25, 27, 43, 46). Although it is known that FSCN1 high-expression in

cancer cells facilitates metastasis, the upstream regulators of FSCN1 were not well known. Here,

we identified that SPHK1 high expression in TNBC cells increases FSCN1 mRNA transcription

by activating NFκB. Additionally, we demonstrated that FSCN1 is a novel downstream effector

of SPHK1 in promoting TNBC cell migration, invasion, and metastasis.

Previous studies have shown that the SPHK1/S1P signaling can induce activation or inhibition of various transcription factors, including NFκB, E2F, c-Myc, and Sp1, and

consequently impact on cell proliferation, apoptosis, and/or inflammation (36, 47, 48). SPHK1

was reported to induce NFkB activation via intracellular S1P that serves as a cofactor of TRAF2

to activate IKKα/β, then IKKα/β phosphorylates IĸBα resulting in its degradation to allow NFkB

activation upon TNFα stimulation (31). Consistently, in 3D-cultured 435.shSPHK1 cells, both

IKKα/β and IĸBα phosphorylations were decreased compared to SPHK1-highexpressing

435.shScr cells. Thus, SPHK1-induced NFkB activation is mostly mediated by intracellular S1P

function, although it may also involve S1P’s extracellular function.

Here, we made the unique finding that SPHK1 activates NFκB to upregulate FSCN1

expression. Our data showed that FSCN1 promoter activity was significantly decreased in

SPHK1 knockdown cells, indicating SPHK1 upregulates FSCN1 at the transcriptional level. We

identified a 240-bp region within FSCN1 promoter and a NFκB binding site herein that is

responsible for this SPHK1-induced FSCN1 upregulation. We also showed that binding of NFκB

to the FSCN1 promoter region was inhibited in SPHK1 knockdown cells compared with control

cells (Fig. 5H). Interestingly, SPHK1 activated NFκB more effectively in 3D than 2D cultures

(Supplementary Fig. S8F and S9C) and we readily detected SPHK1 upregulation of FSCN1

expression in 3D culture. Together, SPHK1 transcriptionally upregulates FSCN1 expression via

activation of NFκB.

The SPHK1/S1P axis is known to regulate immune responses by regulating lymphocytes

trafficking, innate immune response, and inflammation (49). For these reasons, we used a syngeneic mouse model for testing therapeutic efficacy of SPHK1 targeting in vivo. Also we

decided to use drugs that are already in clinical trials or have been FDA-approved for fast track

clinical translation. Specifically, we targeted SPHK1 by safingol, which is currently used in

clinical trials and target NFκB by bortezomib, which is a FDA-approved drug against multiple

myeloma. Although a few drugs have been developed against FSCN1, none have been tested so

far in a clinical setting (50). Even tough, single agent treatment of safingol or bortezomib had no

significant effect on primary tumor growth, combination treatment significantly delayed tumor

progression (Fig. 7A), suggesting a complex interaction between these two drugs that warrants

further investigation. Excitingly, treatment with single agent of either safingol or bortezomib

significantly decreased the spontaneous metastasis of aggressive TNBC to the lungs, which was

further decreased with combination treatment and the safingol plus bortezomib also significantly

increased mice survival (Fig. 7B). Our data indicate that combining safingol and bortezomib may

be effective in treating TNBC tumors and metastasis, which warrants further clinical testing.

Additionally, understanding of the impact of these drugs on the tumor microenvironment in vivo

will enable us to further improve these drugs’ efficacy in the clinic. It would also be interesting

to test the benefits of using these drug combination as an adjuvant treatment regimen after

surgery to prevent or intervene with metastasis and/or recurrences, especially for those patients whose primary tumor express high levels of SPHK1 and/or FSCN1. Our study also suggests that expression of SPHK1/pNFκB/FSCN1 axis in TNBC primary tumors could be a predictive biomarker for development of metastasis, which needs to be further validated. Taken together, we have shown that the SPHK1/pNFκB/FSCN1 axis is activated in TNBCs and can serve as positive therapeutic targets that can be inhibited by clinically applicable kinases inhibitors, and our preclinical testing demonstrated that targeting SPHK1/pNFκB effectively inhibited metastasis from highly aggressive TNBC tumors. We foresee that these findings may be speedily translated into the clinic to benefit TNBC patients in great need of effective therapies.

Acknowledgements

This work is supported by Susan G. Komen Breast Cancer Foundation promise grant

KG091020 (D.Y.), NIH grants P30-CA 16672 (MDACC), RO1-CA112567-06 (D.Y.), RO1-

CA184836 (D.Y.), Breast Cancer Moon Shot funding from The University of Texas MD

Anderson Cancer Center and China Medical University Research Fund (D.Y.). Dr. D. Yu is the

Hubert L. & Olive Stringer Distinguished Chair in Basic Science at MD Anderson Cancer

Center.

We would like to thank MD Anderson Cancer Center (MDACC) Breast Tumor Bank, for

providing us with TMA slides; Dr. Jean J. Zhao for providing us with SPHK1 overexpression

plasmid; and Dr. Emily Powell for her assistance in obtaining PDX cell line. We would also like

to thank MDACC Functional Genomics Core, Research Histology Core, and Small Animal Core

Facility for technical support; Department of Scientific Publications of MDACC for manuscript

revision; and members from Dr. Dihua Yu’s laboratory for insightful discussions.

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Figure Legends

Figure 1: Sphk1 is overexpressed in TNBC tumors and cell-lines and SPHK1

overexpression promotes spontaneous metastatic spread to lungs. (A) Box-and-whisker plot

showing the expression of SPHK1 in normal breast tissue and various subtypes of breast cancers.

(B) Kaplan-Meier plotter was used to generate a survival curve of TNBC patients (total n = 112),

which were stratified based on the SPHK1 expression. (C) Quantitative reverse transcriptase

PCR (qRT–PCR) showing the relative expression of SPHK1 in various human breast cancer cell-

lines. (D) Western blotting analysis showing the SPHK1 expression in various human breast

cancer cell-lines as indicated. (E) qRT-PCR showing the relative expression of SPHK1 in

mRNA level in MDA-MB-231 cells transduced with empty vector or SPHK1 overexpressing

vector. (F) A western blot showing the expression of SPHK1 in protein level in MDA-MB-231

cells transduced with empty vector or SPHK1 overexpressing vector. (G) ELISA of intracellular

S1P Levels in MDA-MB-231 cells transduced with empty vector or SPHK1 overexpressing

vector. (H) In vitro kinase assay for the detection of sphingosine-1-phosphate (S1P) in indicated

cells. (I) In vivo mammary fat pad (MFP) tumor growth in nude mice with orthotopic injection of

231vec and 231SPHK1 cells. (J) H&E stained lung sections were quantified for the number of

spontaneous metastatic lesions from nude mice with orthotopic injection of 231vec and

231SPHK1 cells (left). Representative images of H&E stained lung sections are shown (right).

Scale bar: 200 µm. Lum, Luminal; HR, Hazard ratio; NC, negative control; PC, Positive control.

Data are represented as mean ± SD. *, p < 0.05, **, p < 0.01 and ***, p < 0.001, n.s., non-

significant.

Figure 2: SPHK1 knockdown decrease spontaneous metastatic spread to lungs. (A) A

western blot showing the expression of SPHK1 in BC3-p53KD and MDA-MB-435 cells

transduced with lentiviral vectors expressing control shRNA (shScr) or SPHK1 targeting

shRNA (shSPHK1.1 or shSPHK1.2). (B) MFP tumor growth curve in nude mice with orthotopic

injection of BC3-p53KD.shScr and BC3-p53KD.shSPHK1.1cells. (C) MFP tumor growth in

nude mice with orthotopic injection of 435.shcr, 435.shSPHK1.1 and 435.shSPHK1.2 cells. (D)

H&E stained lung sections were quantified for the number of spontaneous metastatic lesions from nude mice with orthotopic injection of BC3-p53KD.shScr and BC3-

p53KD.shSPHK1.1cells (left). Representative images of H&E stained lung sections are shown

(right). (E) H&E stained lung sections were quantified for the number of spontaneous metastatic

lesions from nude mice with orthotopic injection of 435.shScr, 435.shSPHK1.1 and

435.shSPHK1.2 cells (left). Representative images of H&E stained lung sections are shown

(right). Data are represented as mean ± SD. *, p < 0.05, **, p < 0.01 and ***, p < 0.001, n.s.,

non-significant.

Figure 3: Positive correlation between expression of SPHK1 and expression of FSCN1 in

orthotopic MFP tumors and TNBC patients. (A) Venn diagram representing the number of

differentially regulated genes in MFP tumor samples from 435.shScr that are involved in

migration, invasion and metastasis pathways as identified by IPA (Ingenuity pathway analysis)

analysis. Fifteen differentially regulated and common genes that are involved in metastasis

related properties are listed according to the fold change difference between MFP tumor samples

from 435.shSPHK1.1 vs. 435.shScr cells. (B) qRT-PCR showing the relative expression of

FSCN1 in mRNA level in MFP tumor samples from 435.shScr and 435.shSPHK1.1 cells. (C) A

western blot showing the expression of FSCN1 in protein level in MFP tumor samples from

435.shScr and 435.shSPHK1.1 cells. (D) A western blot showing the expression of FSCN1 and

SPHK1 in protein level in MFP tumor samples from 231vec and 231SPHK1 cells. (E) A western

blot showing the expression of FSCN1 and SPHK1 in protein level in MFP tumor samples from

BC3-p53KD.shScr and BC3-p53KD.shSPHK1.1 cells. (F) Correlation between SPHK1

expression and FSCN1 expression in primary tumor samples of breast cancer patients from

TCGA dataset. Gray circle represents TNBC patients and black circle represents patients with

other subtypes. (G) Box-and-whisker plot showing the expression of FSCN1 in normal breast

tissue and various subtypes of breast cancers. (H) Kaplan-Meier plotter was used to generate a

survival curve of TNBC patients (total n = 112), which were stratified based on the SPHK1

expression. Data are represented as mean ± SD. *, p < 0.05, **, p < 0.01 and ***, p < 0.001, n.s.,

non-significant.

Figure 4: Fascin might mediate SPHK1 metastasis function. (A) Quantification of transwell

migration assay for indicated cells. (B) Quantification of transwell invasion assay using matrigel

for indicated cells. (C) H&E stained lung sections were quantified for the number of spontaneous

metastatic lesions from nude mice with orthotopic injection of 435 shSPHK1.1 cells transduced with either mCherry or FSCN1 (left). Representative images of H&E stained lung sections are

shown (right). (D) 231SPHK1 cells were transfected with either siCtrl or siFSCN1 (pooled) and

were subjected to transwell migration assay. Cells migrating through transwell membrane were

imaged and representative images are shown (right) along with quantification (left). (E)

231SPHK1 cells were transfected with either siCtrl or siFSCN1 (pooled) and were subjected to transwell invasion assay. Cells invaded through transwell membrane were imaged and

representative images are shown (right) along with quantification (left). (F) BC3-p53KD cells, transduced with either control shRNA (shScr) or SPHK1 targeting shRNA (shSPHK1.1) were grown in three dimensional (3D) culture system and cell colonies with invasive structures and

projections were quantified (left) on both cell-lines and representative brightfield images are

shown (right). (G) 231SPHK1 cells, transduced with either control siRNA (shCtrl) or FSCN1

targeting pooled siRNA (siFSCN1) were grown in 3D culture system and cell colonies with

invasive structures and projections were quantified (left) on both cell-lines and representative

brightfield images are shown (right). Data are represented as mean ± SD. *, p < 0.05, **, p <

0.01 and ***, p < 0.001, n.s., non-significant. Scale bar: 200 µm.

Figure 5: SPHK1 upregulates FSCN1 gene expression at transcriptional level through

NFkB transcription factor. (A) qRT-PCR showing the relative mRNA expression of FSCN1 in

BC3-p53KD (left) and MDA-MB-435 (right) cells transduced with either shScr or shSPHK1.1 in two dimensional (2D) and three dimensional (3D) culture system. (B) A western blot showing

the expression of FSCN1 in BC3-p53KD (top) and MDA-MB-435 (bottom) cells transduced with either shScr or shSPHK1.1 in 2D and 3D culture system. (C) Relative promoter activity

assay in 435.shScr and 435.shSPHK1.1 cells transduced with luciferase reporter plasmid

containing FSCN1 promoter (-1376/+147 bp) cultured in 2D and 3D culture system. Firefly

luciferase activity is reported after normalizing to renilla activity, which is used as internal

control for transfection variability. (D) Promoter activity assay in MDA-MB-435 (left) and

Hs578T (right) cells transduced with either control shRNA (shScr) or shRNA targeting SPHK1

(shSPHK1.1) in 3D culture system. Various 5’ deletion constructs were transduced into these

cell-lines as indicated. NT, Non-transfecting. (E) Top: Schematic diagram showing various

transcription factor binding sites in FSCN1 promoter region (-333/-93 bp). Bottom: nucleotide

sequences of wild-type NFκB binding site (NFκB BSwt) and NFκB binding site with mutation

(NFκB BSmut). Mutated nucleotides are shown in gray. (F) Relative promoter activity assay in

MDA-MB-435 and Hs578T cells in 3D culture, transduced with luciferase reporter plasmid

containing -333/+147 bp FSCN1 promoter with NFκB BSwt or NFκB BSmut sequence. (G)

Relative promoter activity assay in MDA-MB-435 cells in 3D culture, transduced with luciferase

reporter plasmid containing -333/+147 bp FSCN1 promoter with NFκB BSwt. Cells were treated

with either NFκB control (C) peptide or NFκB inhibitor peptide (I). (H) MDA-MB-435 (left) and

Hs578T (right) cells transduced with either shScr or shSPHK1.1 in 3D culture system.

Chromatin immunoprecipitation (ChIP) was performed with antibodies against IgG (negative-

control), NFκB and H3K4me3 (positive-control). Binding to the FSCN1 promoter region was

quantified by qPCR from immunoprecipitated DNA, and fold-enrichment was calculated relative

to IgG. Data are representative of at least three independent experiments and are represented as

mean ± SD. *, p < 0.05, **, p < 0.01 and ***, p < 0.001, n.s., non-significant.

Figure 6: Combined Expression of SPHK1, pNFκB and FSCN1 in TNBC patients

correlates with poor survival. (A) Representative images of immunohistochemical staining

showing the high and low expression of SPHK1, pNFκB and FSCN1. Figure objective

magnification: 10X and inset objective magnification: 20X (B) Graph showing the percentage of

TNBC patients with high and low expression of SPHK1, pNFκB and FSCN1. (C-D) Kaplan–

Meier survival plot showing distance metastasis free survival (C) and overall survival (D) among

TNBC patients classified into two different groups based on the combined expression of SPHK1

and FSCN1. Group 1: patients with low expression of one or both markers (SPHK1 and/or

FSCN1 low, n=66); and Group 2: patients with high expression of both markers (SPHK1 and

FSCN1 high, n=42).

Figure 7: Targeting SPHK1 and NFκB signaling pathway delayed tumor progression and

reduced spontaneous metastasis to lungs. (A) MFP tumor growth curve in BALB/c mice with orthotopic injection of 4T1 cells. At day 7, when tumor reached the palpable size, mice were randomized into 4 groups for treatment: vehicle, safingol only, bortezomib only and combination of both drugs (saf + bor). (B) Survival curve of the mice on each treatment groups as mentioned

in (A). (C) MFP tumor weight at day 28, from mice on each treatment groups as mentioned in

(A). At day 28, five mice were euthanized from each group as mentioned in (A) for time

matched experiment and tumor weight were taken. (D - H) Quantification of

immunohistochemical staining of SPHK1 (D), nuclear pNFκB (E), FSCN1 (F), Ki67 (G) and

TUNEL (H) on mfp tumor samples of mice from time matched experiment as mentioned in (C).

(I) H&E stained lung sections were quantified for the number of spontaneous metastatic lesions

from of BALB/c mice euthanized for time matched experiment as mentioned in (c). Data are

representative as mean ± SD. *, p < 0.05, **, p < 0.01 and ***, p < 0.001, n.s., non-significant.

Sphingosine-kinase-1 signaling promotes metastasis of triple-negative breast cancer

Sunil Acharya, Jun Yao, Ping Li, et al.

Cancer Res Published OnlineFirst June 25, 2019.

Updated version Access the most recent version of this article at: doi:10.1158/0008-5472.CAN-18-3803

Supplementary Access the most recent supplemental material at: Material http://cancerres.aacrjournals.org/content/suppl/2019/06/25/0008-5472.CAN-18-3803.DC1

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