Sphingosine Kinase 1 Signaling Promotes Metastasis of Triple-Negative Breast Cancer Sunil Acharya1,2, Jun Yao1, Ping Li1, Chenyu Zhang1, Frank J

Published OnlineFirst June 25, 2019; DOI: 10.1158/0008-5472.CAN-18-3803

Cancer Tumor Biology and Immunology Research

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, and Dihua Yu1,2

Abstract

Triple-negative breast cancer (TNBC) is the most aggres- with distance metastasis and poor clinical outcome in sive breast cancer subtype. To identify TNBC therapeutic patients with TNBC. Targeting SPHK1 and NFkBusing targets, we performed integrative bioinformatics analysis clinically applicable inhibitors (safingol and bortezomib, of multiple breast cancer patient-derived gene expression respectively) significantly inhibited aggressive mammary datasets and focused on kinases with FDA-approved or in- tumor growth and spontaneouslungmetastasisinortho- pipeline inhibitors. Sphingosine kinase 1 (SPHK1) was topic syngeneic TNBC mouse models. These findings high- identified as a top candidate. SPHK1 overexpression or light SPHK1 and its downstream target, NFkB, as promising downregulation in human TNBC cell lines increased or therapeutic targets in TNBC. decreased spontaneous metastasis to lungs in nude mice, respectively. SPHK1 promoted metastasis by transcription- Significance: SPHK1 is overexpressed in TNBC and pro- ally upregulating the expression of the metastasis- motes metastasis, targeting SPHK1 or its downstream target promoting gene FSCN1 via NFkB activation. Activation of NFkB with clinically available inhibitors could be effective the SPHK1/NFkB/FSCN1 signaling pathway was associated for inhibiting TNBC metastasis.

Introduction metastasis (4). It has been reported that TNBC tumors are about 2.5 times more likely to metastasize within 5 years than are Breast cancer, which arises mainly from mammary ducts or breast tumors of other subtypes (5). Because TNBC tumors lack lobules, is the leading cause of cancer-related death and most expression of hormone and HER2 receptors, that is, negative for commonly diagnosed cancer in women worldwide (1). therapeutic targets, TNBCs do not respond to, and patients Approximately 10%–20% of breast cancers are triple-negative, cannot benefit from, currently available hormonal and HER2- that is, they do not express estrogen receptor (ER), progesterone targeted therapies. receptor (PR), or HER2 (2, 3). Triple-negative breast cancer In contrast to the successful development of therapies for (TNBC) tends to occur at higher frequency in young women hormone receptors–positive, and/or HER2-positive breast can- and is particularly aggressive, with high recurrence and metas- cers, little progress has been made in identifying positively tasis rates (4). Compared with patients having other subtypes expressed molecular targets in TNBC that are druggable (6). of breast cancer, patients with TNBC have a poor overall Clearly, there is an imposing need to discover positive druggable prognosis, for example, the 5-year survival rate for patients targets in TNBC instead of accepting its triple-negative nontarge- with stage IV TNBC is about 22%, mainly due to early-onset of table status. Kinases play central roles in cancer cell signaling pathways and are druggable targets for effective targeted thera- 1Department of Molecular and Cellular Oncology, The University of Texas MD pies (7). In the past decade, numerous efforts have led to suc- Anderson Cancer Center, Houston, Texas. 2Cancer Biology Program, The Uni- cessful development and FDA approval of inhibitors of various versity of Texas MD Anderson Cancer Center UT Health Graduate School of cancer-promoting kinases (8). Therefore, we set out to identify Biomedical Sciences, Houston, Texas. 3Department of Pathology, The University activated and/or overexpressed kinases, as positive and druggable 4 of Texas MD Anderson Cancer Center, Houston, Texas. Department of Trans- molecular targets, in TNBC with high potential for quick and lational Molecular Pathology, The University of Texas MD Anderson Cancer efficient clinical translation. Center, Houston, Texas. 5Department of Experimental Radiation Oncology, The University of Texas MD Anderson Cancer Center, Houston, Texas. Our bioinformatics analysis of multiple patient-derived datasets identified that sphingosine kinase 1 (SPHK1), a lipid kinase, Note: Supplementary data for this article are available at Cancer Research was expressed at significantly higher levels in TNBC than in other Online (http://cancerres.aacrjournals.org/). breast cancer subtypes. SPHK1 catalyzes phosphorylation of Corresponding Author: Dihua Yu, PhD, Department of Molecular and Cellular sphingosine, an amino alcohol, to generate sphingosine-1-phos- Oncology, Unit 108, Rm Z11.5034, University of Texas MD Anderson Cancer phate (S1P), a novel lipid signaling mediator with both intracel- Center, 6565 MD Anderson Blvd., Houston, TX 77030. Phone: 713-792-3636; Fax: 713-792-4544; E-mail: [email protected] lular (as a second messenger) and extracellular (as a ligand for G-protein–coupled receptors) functions (9). S1P regulates vari- Cancer Res 2019;79:4211–26 ous cellular processes in mammalian cells, such as growth, sur- doi: 10.1158/0008-5472.CAN-18-3803 vival, and migration. Exogenously overexpressing SPHK1 in 3T3 2019 American Association for Cancer Research. fibroblasts led to transformation in vitro and tumor formation

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in vivo, suggesting that SPHK1 acts as an oncogene (10). SPHK1 is control peptide (sc-3060) were brought from Santa Cruz Biotech- shown to be overexpressed in various cancers including breast nology. Safingol (CAS 15639-50-6) was purchased from Cayman cancer (11–14). Importantly, a SPHK1 inhibitor, safingol, can Chemical. Bortezomib (CAS 179324-69-7) was purchased from effectively inhibit SPHK1 activities and is currently under multiple EMD Millipore. CAPTISOL (20 g) was kindly provided by CyDex clinical trials (NCT00084812 and NCT01553071). Pharmaceuticals. In this study, we systematically tested the function of SPHK1 in TNBC progression and metastasis using multiple TNBC sponta- Generation of stable cell lines neous metastasis models that recapitulate the entire cascade of To overexpress SPHK1, retroviral vector pWZL-Neo-Myr-Flag- biological steps of metastasis in patients and found that SPHK1 DEST containing the SPHK1 open reading frame (ORF) under the has a critical function in enhancing TNBC spontaneous metasta- control of CMV promotor with G418 (100 mg/mL) as selection sis. Mechanistically, SPHK1 upregulates FSCN1 (also known as marker was used (kindly provided by Dr. J. Zhao, Department fascin) transcription via activation of the NFkB transcriptional of Biological Chemistry and Molecular Pharmacology, Harvard factor and FSCN1 promotes metastasis. Clinically, SPHK1/NFkB/ Medical School, Boston, MA). Empty vector was used as a control. FSCN1 signaling pathway activation in patients' TNBC tissues To stably knockdown SPHK1 in MDA-MB-435, Hs578T, and BC3- correlates with poor patient survival and increased metastases. To p53KD cells, we used two short hairpin RNA (shRNA) constructs, test the validity of SPHK1 pathway as druggable targets in TNBC, targeting the SPHK1 30 untranslated region, cloned into the pGIPZ we therapeutically targeted SPHK1 by safingol and/or NFkB with lentiviral vector (RefSeq NM_001142601, Open Biosystems) with a clinically applicable inhibitor bortezomib. Strikingly, combi- puromycin (2 mg/mL) as selection marker. To stably knockdown natorial treatment with both SPHK1 and NFkB inhibitors signif- FSCN1 in MDA-MB-435 cells, we used three shRNA constructs, icantly inhibited both TNBC primary tumor growth and lung targeting the FSCN1 30 untranslated region, cloned into the pGIPZ metastasis compared with either single-agent treatment. These lentiviral vector (RefSeq NM_003088.3, Open Biosystems) with data demonstrated that SPHK1/NFkB pathway can serve as pos- puromycin (2 mg/mL) as selection marker. Nonsilencing shRNA itive therapeutic targets for effective inhibition of TNBC and was used as a control for both above mentionedshRNA knockdown metastasis. These preclinical findings could be fast-track translat- experiments. To overexpress FSCN1, retroviral vector pLenti6/ ed to the clinic for the treatment of TNBC and metastasis in V5-DEST containing the FSCN1 ORF under the control of CMV patients. promotor with blasticidin (3 mg/mL) as selection marker was used (plasmid #31207, Addgene). Lentiviral vector with mCherry sequence was used as a control. Lentiviral vectors (with ORFs or Materials and Methods shRNA) were transfected into the packaging cell line 293T, together Cell culture with a packaging DNA plasmid (psPAX2) and an envelope DNA Human cancer cell lines (MCF-7, T47D, BT474, HCC1954, plasmid (pMD2G), through Lipofectamine transfection. After HCC70, Hs578T, MDA-MB-231, MDA-MB-435, and MDA-MB- 48 hours, viruses were collected, filtered, and incubated with target 436) and a mouse breast cancer cell line (4T1) were obtained from cells in the presence of 8–10 mg/mL polybrene for 24 hours. The the ATCC. Mouse breast cancer cell lines E0771 and Met-1fvb2 infected cells were selected with suitable selection markers, with were purchased from CH3BioSystems and Lonza, respectively. concentration mentioned above, to generate the stable clone. These cell lines were verified by the MD Anderson Cancer Center Cell Line Characterization Core Facility. BC3-p53KD was provid- siRNA knockdown ed by H. Piwnica-Worms (15). Cells were cultured in DMEM with To knockdown FSCN1 in MDA-MB-231 cells, SMARTpool: 10% FBS and 0.1% penicillin–streptomycin in 5% CO2 at 37 C. ON-TARGETplus human FSCN1 siRNA (Dharmacon) was used All cell lines were tested for Mycoplasma contamination by using to transfect the cells. Lipofectamine RNAiMAX Transfection the MycoAlert Mycoplasma Detection Kit (Lonza) and were Reagents (Invitrogen) was used for siRNA transfection and the negative. protocol was followed per the manufacturer's instruction.

Antibodies and reagents Site-directed mutagenesis Rabbit polyclonal antibody against Sphk1 (HPA022829) and Nucleotides within the NFkB transcription factor binding sites FSCN1 (HPA005723) were purchased from Sigma-Aldrich. of the FSCN1 promoter were altered by site-directed mutagenesis Mouse polyclonal antibody against FSCN1 1 (sc-46675) was by using Q5 Site-Directed Mutagenesis Kit (NEB) according to bought from Santa Cruz Biotechnology, and rabbit mAb against the manufacturer's instructions. Wild-type FSCN1 promoter SPHK1 (ab109522) was bought from Abcam. Mouse monoclonal (333/þ147 bp) in the pGL3luc (basic) vector was used as a anti-b-actin antibody (A5441) was from Sigma-Aldrich and anti- template to introduce mutation at three nucleotides that affect Ki67 antibody (M7240) was from Dako. Normal rabbit IgG both binding sites. The sense and antisense primers used were 50- (2729), NFkB p65 (8242), and H3K4me3 (9727) were all from GTCCGAGGTGATGGACATCAGGGG-30 and 50-ACCCCGACCC- Cell Signaling Technology. p-NFkB p65 (S536) (ab86299) was CAAGCCTC-30, respectively. Mutated promoter fragments were purchased from Abcam. The In Situ Cell Death Detection Kit sequenced to verify the presence of mutations. [terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling (TUNEL) technology, 11684817910] was from Roche. Western blotting The horseradish peroxidase–linked secondary antibodies against Western blot analysis was performed as described previous- mouse (NA931) and rabbit (NA934) were from GE Healthcare. ly (16). Densitometry analyses were performed by evaluating Actinomycin D (A9415) was from Sigma-Aldrich. A cell perme- band intensity mean gray value of indicated protein and normal- able peptide (CAS 213546-53-3) that inhibits translocation of the izing it with the mean gray value of corresponding lane's loading NFkB active complex into the nucleus and its corresponding NFkB control using ImageJ software.

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Table 1. Primers used viously (16). TUNEL staining was done in paraffin sections with Genes Primers use of an In Situ Cell Death Detection Kit, POD (11684817910, 0 0 SPHK1 F: 5 -AACTACTTCTGGATGGTCAG -3 Roche), according to the manufacturer's instructions. R: 50-TCCTGCAAGTAGACACTAAG -30 FSCN1 F: 50-CCAGGGTATGGACCTGTCTG-30 R: 50-CGCCACTCGATGTCAAAGTA-30 SPHK1 kinase activity 18S F: 50-AACCCGTTGAACCCCATT-30 SPHK1 activity in cytosol was determined as described previ- R: 50-CCATCCAATCGGTAGTAGCG-30 ously (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 the manufacturer's RNA extraction, RT-PCR, and qPCR instructions. RNA extraction and RT-PCR were performed as described previously (16). For the SYBR Green–based qPCR assay, 1 mLof Animal experiments cDNA was used as a template for qRT-PCR with iQ SYBR Green All procedures and experimental protocols involving mice were Supermix (Bio-Rad) and the StepOnePlus (Applied Biosystems) approved by the Institutional Animal Care and Use Committee at instrument according to the manufacturer's instruction. The RNA The University of Texas MD Anderson Cancer Center (Houston, expression rate was quantified by the relative quantification TX). DDC (2 t) method, and 18S expression was used as the internal Female nude mice (6 weeks old, 4–7 mice per group as control. The primers that were used are listed Table 1. indicated in figures and/or figure legends) were orthotopically injected with human cancer cells (2 105 cells for MDA-MB-435 Chromatin immunoprecipitation assay and MDA-MB-231 cells, and 1 106 cells for BC3-p53KD cells; Procedures for chromatin isolation and immunoprecipitation cells were resuspended in 50:50 mixture of Matrigel in PBS) into were performed as described previously (16). Normal IgG, NFkB MFPs, and tumors were allowed to develop for an indicated p65, and H3K4me3 antibodies were used at 2 mg per reaction in number of days. Tumor sizes were measured with digital calipers immunoprecipitation. Coprecipitated DNA (2 mL) was analyzed twice a week and tumor volumes were calculated with use of a by qPCR. The forward and reverse primers used for amplification modified ellipsoidal formula: 1/2 (length width2). MFP of the NFkB binding region in the FSCN1 promoter (333/þ147 tumors were surgically excised with survival surgery, and the mice bp) were as follows: forward: 50-CTCAAACCTCGCTCGTCCTT-30 were further monitored for an indicated number of weeks for and reverse: 50-CATCACCCCTCACAACCCC-30. spontaneous metastasis. All mice were euthanized at indicated times, and lungs were harvested, fixed, and paraffin embedded. Three-dimensional cell culture After lungs were hematoxylin and eosin stained, the number Three-dimensional (3D) culture was performed in either an of metastatic lesions were enumerated by pathologist using 8-well chamber slide (BD Falcon) or in Costar 6-well plate with brightfield microscopy. Detailed description of in vivo treatment ultra-low attachment surface (Corning). For the 8-well chamber, experiments in mice is provided in Supplementary Materials and 100 mL of Matrigel was added to the bottom of each chamber and Methods. incubated at 37C for 20 minutes. Cells of interest were mixed in culture medium with 5% Matrigel and added to each well to a cDNA microarray and analysis final concentration of 1,500 cells/well. For 6-well plates with low Unbiased platform, HumanHT-12_v4 (Illumina), was applied attachment, 2 105 cells of interest that were mixed into the for gene profiling of MFP tumors and matched spontaneous lung culture medium with 5% Matrigel were added to each well. metastasis formed by control and Sphk1 knockdown MDA-MB- 435 cells in collaboration with the cDNA microarray core facility Quantification of 3D invasiveness at MD Anderson Cancer Center. The raw and normalized micro- Indicated cells were grown in 3D culture in 8-well chamber array data have been deposited in the Gene Expression Omnibus culture slides. Images of the spheroid structures at multiple fields (GEO) database under accession number GSE128624. Gene were obtained with use of a microscope at the indicated time, and cluster maps for MFP and lung metastasis samples were generated invasive structures per field were counted. Structures that had by using sequence analysis of microarray analysis. To identify projections coming out from the main spheroid body were SPHK1-regulated genes in 435 cells, R software and limma soft- counted as invasive structures. ware packages were used to identify differentially expressed genes using a 1.5-fold change threshold and an adjusted P value cutoff at mRNA stability assay 0.01. Ingenuity Pathway Analysis (IPA) software (http://www. Equal numbers of 435 scrambled shRNA (shSCR) and 435 ingenuity.com) was used to perform the functional annotation shSPHK1.1 cells were plated in a 6-well low-attachment plate with and pathway analysis of the differentially expressed genes. Gene 5% Matrigel and incubated for 3 days at 37C. Cells were treated set enrichment analysis (GSEA) was performed on MFP micro- with 5 mg/mL actinomycin D for 1, 2, 4, 8, 12, and 16 hours. Total array data with use of an online tool (http://software.broadinsti RNA was extracted after each timepoint by using TRIzol Reagent tute.org/gsea/index.jsp), as described previously (18). (Invitrogen), and qPCR was performed to determine the relative mRNA level of FSCN1. Case selection, tissue microarray construction, and analysis We obtained archival, formalin-fixed, and paraffin-embedded IHC analysis material from surgically resected breast cancer specimens from the The excised mammary fat pad (MFP) tumors were fixed in 10% Breast Tumor Bank at M.D. Anderson Cancer Center from 2001 neutral-buffered formalin and embedded in paraffin for IHC to 2013. Tumor tissue specimens obtained from 117 TNBC staining. IHC staining was performed similarly as described pre- were histologically examined, classified using the World Health

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Organization classification of breast tumors, and selected for MDA-MB-231 human TNBC cell line that express an intermediate tissue microarray (TMA) construction. After histologic examina- level of endogenous SPHK1 compared with other breast cancer tion, tumor TMAs were prepared using triplicate 1-mm diameter cell lines, and generated control (231vec) and SPHK1-overexpres- cores per tumor. All the archival paraffin-embedded tumor sam- sing (231SPHK1) stable sublines (Fig. 1E and F). The 231SPHK1 ples were coded with no patient identifiers. Detailed clinical and cells showed increased levels of intracellular S1P compared with pathologic information, including demographic, pathologic 231vec cells (Fig. 1G). The 231SPHK1 cells showed increased tumor–node–metastasis staging, overall survival (OS), and time phosphorylation of sphingosine leading to increased S1P as of recurrence were collected. Detailed descriptions of IHC stain- detected by a semi in vitro kinase assay compared with 231vec ing/quantification and survival analysis are provided in Supple- cells (Fig. 1H). The 231SPHK1 cells also showed increased migra- mentary Materials and Methods. tion (Supplementary Fig. S2A) and invasion (Supplementary Fig. S2B) potential in vitro, but no significant differences in cell Bioinformatics, statistics, and survival analysis proliferation in vitro (Supplementary Fig. S2C) compared with GEO2R analysis, a web-based application for analyzing gene 231vec cells. expression in GEO datasets, was performed as described else- To examine whether SPHK1 promotes TNBC tumorigenesis where (19). The Kaplan–Meier plotter (20), a web-based tool, was and spontaneous metastasis, 231vec control and 231SPHK1 cells used to assess the effect of SPHK1 and FSCN1 genes on survival of were orthotopically injected into MFPs of nude mice. The MFP patients with TNBC. To select the patients with TNBC, following tumors were surgically excised 28 days postinjection, and the mice selection criteria were used: ER negative, PR negative, HER2 were monitored for about 10 more weeks for development of negative, grade 3, and intrinsic subtype basal. Survival rates were spontaneous metastasis (Supplementary Fig. S2D). There was no compared by using the log-rank test and HRs were calculated by significant difference in MFP tumor size between 231vec and using a multivariable Cox proportional hazards model. For cor- 231SPHK1 groups by day 28 (Fig. 1I). Remarkably, the number of relation analysis, expression values of SPHK1 and FSCN1 from metastatic lesions in the lungs was significantly higher in mice patient samples were downloaded from The Cancer Genome Atlas bearing 231SPHK1 MFP tumors than in control mice bearing (TCGA) and Curtis breast dataset (21). GraphPad Prism (Prism 6; 231vec MFP tumors (Fig. 1J). These data showed that despite GraphPad Software Inc.) was used to generate a correlation graph having no significant effect on MFP tumor growth, SPHK1 over- and calculate the Pearson coefficient (r) from the downloaded expression enhanced spontaneous lung metastasis of MDA-MB- data. All statistical analyses were performed by using GraphPad 231 human TNBC cells. Prism. The data were analyzed by either one-way ANOVA (multiple groups) or a t test (two groups). Differences with P < 0.05 SPHK1 knockdown decreases spontaneous lung metastasis (two-sided) were considered statistically significant (, P < 0.05; To determine whether SPHK1 is required for TNBC progression , P < 0.01; , P < 0.001 by two-tailed t test). and metastasis, we stably knocked down SPHK1 in TNBC patient- More methods (plasmids construction, cell proliferation derived xenograft (PDX) cells (BC3-p53KD; ref. 15) using lenti- assay, migration assay and invasion assay, transient transfec- viral vector expressing two distinct SPHK1-targeting shRNAs tion and luciferase reporter assay, and flow cytometry) with (shSPHK1.1 and shSPHK1.2). Lentiviral vector expressing non- detailed descriptions are provided in Supplementary Materials targeting shScr was used to generate the control cell line. In and Methods. addition, because 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 Results of endogenous SPHK1. SPHK1 knockdown by shSPHK1.1 was SPHK1 is highly expressed in TNBCs and promotes highly effective at the mRNA level detected by qRT-PCR (Supple- spontaneous lung metastasis mentary Fig. S3A) and at the protein level determined by Western To identify kinase gene(s) that are particularly overexpressed in blotting (Fig. 2A; Supplementary Fig. S3B). Knocking down TNBC to serve as potential therapeutic targets, we performed SPHK1 had no significant effect on cell proliferation in vitro in GEO2R analysis (GSE27447) between TNBC and non-TNBC any of the three cell lines (Supplementary Fig. S3C). SPHK1 tumor samples from patients (22). Only four kinase genes were knockdown in MDA-MB-435 cells showed decreased levels of among the top 100 differentially expressed genes, of which, intracellular S1P compared with control (Supplementary Fig. SPHK1 was the only kinase that was overexpressed in TNBC S3D). SPHK1 knockdown in MDA-MB-435 and Hs578T cells tumors compared with non-TNBC tumors (Supplementary Fig. led to decreased phosphorylation of sphingosine, leading to S1A and S1B). Our further analysis of breast cancer microarray decreased S1P as detected by a semi in vitro kinase assay (Sup- data of TCGA validated that expression of SPHK1 is significantly plementary Fig. S3E), and inhibited the migration and invasion higher in basal subtype when compared with normal breast potential in vitro (Supplementary Fig. S3F–S3H). tissues and other subtypes of breast cancers (Fig. 1A). To determine whether SPHK1 is required for TNBC tumor- Survival analysis by Kaplan–Meier plotter indicated that expres- igenesis and spontaneous metastasis, SPHK1-knockdown sion of SPHK1 is significantly associated with poorer relapse-free BC3-p53KD.shSPHK1.1 and MDA-MB-435 (shSPHK1.1 and survival in patients with TNBC (Fig. 1B). SPHK1 expression is also shSPHK1.2) cells along with respective control cells were higher in TNBC-derived cell lines than in cell lines of other orthotopically injected into MFP of nude mice. The MFP subtypes at both mRNA (Fig. 1C; Supplementary Fig. S1C and tumors were resected by survival surgery on day 54 for mice S1D) and protein (Fig. 1D; Supplementary Fig. S1E) levels. Thus, injected with BC3-p53KD sublines, and on day 28 for mice SPHK1 is mostly overexpressed in TNBC tumors and cell lines. injected with MDA-MB-435 sublines (Supplementary Fig. S4A To determine the function of SPHK1 in TNBC progression and and S4B). There was no significant difference in primary metastasis, we first performed gain-of-function studies using tumorsize(Fig.2BandC)orintumorcellproliferation

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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 patients with TNBC (total n ¼ 112), which were stratified on the basis of the SPHK1 expression. C, 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 S1P in indicated cells. I, In vivo MFP tumor growth in nude mice with orthotopic injection of 231vec and 231SPHK1 cells. J, Hematoxylin and eosin–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 imagesof hematoxylin and eosin–stained lung sections are shown (right). Scale bar, 200 mm. Lum, luminal; NC, negative control; PC, positive control. Data are represented as mean SD. , P < 0.01; , P < 0.001; n.s., nonsignificant.

(Supplementary Fig. S4C and S4D) between control and at day 187 after MFP tumor resection and from mice bearing SPHK1-knockdown MFP tumors in either models, although highly aggressive MDA-MB-435 MFP tumors at day 47 after the MFP tumors of SPHK1-knockdown cells were more apo- primary tumor resection (Supplementary Fig. S4A and S4B). A ptotic as detected by TUNEL staining than did tumors formed signiﬁcant decrease in the number of spontaneous lung metas- by control cells (Supplementary Fig. S4E and S4F). tases was observed in mice injected with SPHK1-knockdown cells To evaluate the impact of SPHK1 knockdown on metastasis, compared with mice injected with control cells in both models lungs were harvested from mice bearing BC3-p53KD MFP tumors (Fig. 2D and E). Together, these data indicate that SPHK1 is

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Figure 2. SPHK1 knockdown decreases 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.shSPHK1.1 cells. C, MFP tumor growth in nude mice with orthotopic injection of 435.shcr, 435. shSPHK1.1, and 435.shSPHK1.2 cells. D, Hematoxylin and eosin–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 hematoxylin and eosin–stained lung sections are shown (right). E, Hematoxylin and eosin–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 hematoxylin and eosin–stained lung sections are shown (right). Data are represented as mean SD. , P < 0.05; , P < 0.001; n.s., nonsignificant.

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essential for TNBC's development of aggressive spontaneous lung Thus, we tested whether SPHK1-high–expressing cells with high metastasis and the SPHK1 overexpression may serve as a positive FSCN1 expression may induce more protrusive invasion in 3D therapeutic target for inhibition of TNBC lung metastasis. culture. Indeed, SPHK1-high–expressing BC3-p53KD.shScr and 435.shScr cells with high FSCN1 expression formed many pro- FSCN1 upregulation contributes to SPHK1-driven metastasis trusive structures projecting into the surrounding matrix after SPHK1 is a critical lipid kinase with pleiotropic effects on 10 days in standard 3D culture; whereas BC3-p53KD.shSPHK1.1 various cellular functions (9), however, little is known about and 435.shSPHK1.1 cells with low FSCN1 expression mostly how SPHK1 promotes metastasis. To attain insights on the formed round shaped structures with very few protrusions molecular mechanism underlying SPHK1-driven spontaneous (Fig. 4F; Supplementary Fig. S7F). Importantly, knocking down metastasis, the primary mammary tumor tissues and matched FSCN1 gene in SPHK1-overexpressing 231SPKH1 cells (Fig. 4G) spontaneous lung metastasis tissues from mice injected with 435. and MDA-MB-435 cells (Supplementary Fig. S7G) reduced their shScr versus 435.shSPHK1.1 cells (see Fig. 2C and E) were profiled protrusive structures projecting into the surrounding matrix in for SPHK1-modulated genes (Supplementary Fig. S5A and S5B). standard 3D culture. These data indicate that FSCN1 upregulation The list of SPHK1-regulated differentially expressed genes (435. could contribute to SPHK1-driven invasion and metastasis by shSPHK1.1 vs. 435.shSCR) was generated for MFP tumor samples inducing protrusive invasion. and was subjected to IPA. IPA identified FSCN1 as a top SPHK1- regulated gene involved in cancer cell migration, invasion, and SPHK1 upregulates FSCN1 transcription via activation of NFkB metastasis (Fig. 3A; Supplementary Fig. S5C). Indeed, the FSCN1 The critical function of FSCN1 in SPHK1-driven invasion and gene expression was high in SPHK1-high–expressing tissue sam- metastasis impelled us to dissect how FSCN1 is upregulated by ple (i.e., 435.shScr) and low in SPHK1-low–expressing tissue SPHK1. Interestingly, we found that knockdown of SPHK1 down- samples (i.e., 435.shSPHK1.1) of both primary tumors and spon- regulated FSCN1 expression in standard 3D culture but not in taneous lung metastases as validated by qRT-PCR (Fig. 3B; Sup- two-dimensional (2D) culture at both mRNA (Fig. 5A; Supple- plementary Fig. S5D) and by Western blotting (Fig. 3C; Supple- mentary Fig. S8A) and protein (Fig. 5B; Supplementary Fig. S8B) mentary Fig. S5E). Similarly, other SPHK1-high–expressing MFP levels (29). Next, we tested whether SPHK1 regulates FSCN1 tumor samples (i.e., 231SPHK1 and BC3-p53KD.shScr) also had mRNA via modulating mRNA stability or transcription. The higher FSCN1 expression than SPHK1-low–expressing MFP control and SPHK1-knockdown MDA-MB-435 cells in 3D culture tumors (i.e., 231vec and BC3-p53KD.shSPHK1.1, respectively) were treated with actinomycin D to block transcription and were as shown by Western blotting (Fig. 3D and E). Furthermore, in two detected for FSCN1 mRNA stability, which showed no significant different breast cancer patients' datasets (TCGA and Curtis breast difference (Supplementary Fig. S8C), indicating that SPHK1 does datasets; ref. 21), SPHK1 gene expression correlated with FSCN1 not modulate FSCN1 mRNA stability. gene expression, and high expressions of both SPHK1 and FSCN1 To investigate whether SPHK1 regulates FSCN1 mRNA tran- genes was observed in patients with TNBC compared with other scription, we cloned the 1,376 to þ147 bp FSCN1 promoter subtypes (Fig. 3F; Supplementary Fig. S5F). FSCN1 expression, region into the pGL3 basic vector expressing a luciferase reporter like SPHK1 expression, was also upregulated in basal subtype gene, and transfected into either 435.shScr or 435.shSPHK1.1 compared with other subtypes of breast cancer in TCGA dataset cells. FSCN1 promoter–driven luciferase activities were reduced (Fig. 3G); moreover, FSCN1 expression correlated with a poor by SPHK1 knockdown in 3D cultured, but not in 2D cultured 435. survival rate in patients with TNBC (Fig. 3H). shSPHK1.1 cells (Fig. 5C), indicating SPHK1 is essential for The FSCN1 proteins organize F-actin into parallel bundles and FSCN1 mRNA transcription. Subsequently, all studies on are required for the formation of actin-based cellular protru- SPHK1-induced FSCN1 transcription were performed in the 3D sions (24). It plays a critical role in cell migration, motility, and culture system. adhesion and in cellular interactions (25, 26). To test whether To determine the FSCN1 promoter region responsible for FSCN1 can recover metastasis from SPHK1-knockdown cells, SPHK1-mediated transcriptional upregulation, we made a series FSCN1 was ectopically expressed in 435.shSPHK1.1 and of 50 deletions in the 1,376/þ147 bp FSCN1 promoter construct Hs578T.shSPHK1.1 cells (Supplementary Fig. S6A–S6D). Clearly, in the pGL3 basic vector and generated 333/þ147 bp and 93/ ectopic expression of FSCN1 in SPHK1-knockdown cells þ147 bp constructs (Supplementary Fig. S8D). We transfected increased their migration (Fig. 4A; Supplementary Fig. S6E and these deletion constructs of FSCN1 promoter into either 435. S6F) and invasion (Fig. 4B; Supplementary Fig. S6Gand S6H) shScr or 435.shSPHK1.1 cells and compared their activities in potentials in vitro, but it had no significant effect on cell prolif- driving luciferase reporter gene expression. The 333/þ147 bp eration in vitro (Supplementary Fig. S6I and S6J). Importantly, FSCN1 promoter region was sufficient to induce transcription in ectopic expression of FSCN1 in 435shSPHK1.1 cells rescued the SPHK1-high–expressing cells, whereas both basal transcription spontaneous metastatic potential in vivo (Fig. 4C). On the other and SPHK1-induced transcription were significantly inhibited hand, knocking down FSCN1 gene in SPHK1-overexpressing when FSCN1 promoter was deleted to 93 /þ147 bp region 231SPHK1 cells (Supplementary Fig. S7A) and in MDA-MB- (Fig. 5D). The data indicate that the 333/93 bp region in 435 cells having high endogenous SPHK1 expression (Supple- FSCN1 promoter is important for basal and SPHK1-mediated mentary Fig. S7B and S7C) significantly reduced their migration FSCN1 transcription. Using online software (PROMO), we iden- (Fig. 4D; Supplementary Fig. S7D) and invasion (Fig. 4E; Sup- tified that the 333bp to 93 bp region of FSCN1 promoter plementary Fig. S7E), indicating that the SPHK1-induced metas- harbors binding sites of various transcription factors, one of tasis-related functions are mediated, at least partially, by FSCN1. which was NFkB (Fig. 5E), that can be activated by SPHK1 (30). Regarding FSCN1-mediated metastasis-related functions, var- Indeed, NFkB pathway was activated more in SPHK1-overexpres- ious studies have shown that FSCN1 is important for invadopodia sing 231SPHK1 tumor lysates and 435.shScr cells growing in 3D assembly to promote protrusive invasion of cancer cells (27, 28). culture compared with respective controls (Supplementary Fig.

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Figure 3. Positive correlation between expression of SPHK1 and expression of FSCN1 in orthotopic MFP tumors and patients with TNBC. 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. 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 versus 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 patients with breast cancer from TCGA dataset. Gray circle, patients with TNBC; black circle, 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 patients with TNBC (total n ¼ 112), which were stratiﬁed on the basis of the SPHK1 expression. Data are represented as mean SD. , P < 0.001.

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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, Hematoxylin and eosin–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 hematoxylin and eosin–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 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 < 0.01; , P < 0.001; n.s., nonsignificant. Scale bar, 200 mm.

S8E and S8F). Intracellular S1P, downstream of SPHK1, was we examined the phosphorylation status of IKKa/b and IkBa in known to act as a cofactor of TRAF2, leading to IKKa/b activation, our SPHK1-modulated MDA-MB-435 cells in both 2D and 3D resulting in IkBa phosphorylation and degradation, and conse- cultures. Compared with SPHK1-overexpressing 435.shScr con- quently, NFkB activation upon TNFa stimulation (31). Therefore, trol cells, phosphorylation of IKKa/b and IkBa are decreased in

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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 2D and 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 (1,376/þ147 bp) cultured in 2D and 3D culture system. Firefly luciferase activity was reported after normalizing to Renilla activity, which was 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 50 deletion constructs were transduced into these cell lines as indicated. NT, nontransfecting. E, Schematic diagram showing various transcription factor binding sites in FSCN1 promoter region (333/93 bp; top). Nucleotide sequences of wild-type NFkB binding site (NFkB BSwt) and NFkB binding site with mutation (NFkB 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/þ147 bp FSCN1 promoter with NFkB BSwt or NFkB BSmut sequence. G, Relative promoter activity assay in MDA-MB-435 cells in 3D culture, transduced with luciferase reporter plasmid containing 333/þ147 bp FSCN1 promoter with NFkB BSwt. Cells were treated with either NFkB control (C) peptide or NFkB inhibitor peptide (I). H, MDA-MB-435 (left) and Hs578T (right) cells transduced with either shScr or shSPHK1.1 in 3D culture system. ChIP was performed with antibodies against IgG (negative control), NFkB, 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; , P < 0.001; n.s., nonsignificant.

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SPHK1 knocked down 435.shSPHK1 cells in 3D culture, not in 2D and FSCN1 may correlate with increased metastasis and poor culture, confirming that SPHK1/S1P regulates NFkB activation via clinical outcome. Patients were divided into two groups: IKKa/b/IkBa pathway (Supplementary Fig. S8G). In addition, (i) low expression of one or both markers (i.e., SPHK1 and/ GSEA of our cDNA microarray data revealed that TNF signaling or FSCN1 low), and (ii) high expression of both markers (i.e., was upregulated in MFP tumors of SPHK1-high–expressing 435. SPHK1 and FSCN1 high) in their TNBCs. Compared with group shScr cells relative to the SPHK1-low–expressing 435.shSPHK1.1 1 patients, high expressions of SPHK1 and FSCN1 in group MFP tumors (Supplementary Fig. S8H; ref. 32). Thus, SPHK1 may 2 patients are significantly associated with poorer distance induce FSCN1 upregulation via activation of the NFkB transcrip- metastasis-free survival (P ¼ 0.045), as well as worse OS tion factor. (P ¼ 0.003; Fig. 6C and D). Thus, clinically, high expression To investigate whether the NFkB binding site in the FSCN1 of SPHK1 and FCSN1 in TNBC tissues correlated with increased promoter is critical for SPHK1-induced FSCN1 upregulation, we distant metastasis and poor survival in patients with TNBC. generated NFkB binding site mutant (NFkB BSmut) from the 333/þ147 bp wild-type FSCN1 promoter–driven luciferase Targeting SPHK1 and the NFkB axis impedes tumor reporter construct (NFkB BSwt; Fig. 5E; Supplementary Fig. progression and spontaneous lung metastasis S8I). The NFkB BSwt and the NFkB BSmut constructs were The above data from TNBC animal models and TNBC transfected into SPHK1-high–expressing (MDA-MB-435 and patients' tissues prompted us to test whether therapeutically Hs578T) cells and compared for promoter activities. Compared targeting SPHK1/pNFkB/FSCN1 axis could inhibit TNBC pro- with NFkB BSwt construct, NFkB BSmut construct with mutation gression and metastasis in an aggressive TNBC animal model. of the NFkB binding site had significantly reduced FSCN1 pro- Because SPHK1 regulates FSCN1 via the NFkBtranscription moter activity in both cell models (Fig. 5F). In addition, a cell factor, and clinically applicable inhibitors of both SPHK1 and permeable peptide that inhibits translocation of the NFkB active NFkB are available, we tested the efficacy of SPHK1 and NFkB complex into the nucleus significantly inhibited NFkB BSwt inhibitors, either as a single agent or in combination, for FSCN1 promoter activities compared with the control peptide in deterring TNBC progression and metastasis. SPHK1 were tar- MDA-MB-435 cells (Fig. 5G). These data indicate that nuclear geted by safingol, a SPHK1 inhibitor tested in multiple clinical NFkB binding to the NFkB binding site of FSCN1 promoter is trials (NCT00084812 and NCT01553071), and NFkBwere critical for FSCN1 transcriptional upregulation in SPHK1-high– targeted with bortezomib, an FDA-approved proteasome inhib- expressing TNBC cells. Next, we examined whether NFkB tran- itor drug for multiple myeloma, but shown to inhibit NFkB scription factor binding to the NFkB binding region (263/253 activity (33). Safingol treatment decreased SPHK1 protein bp) of FSCN1 promoter are higher in SPHK1-high–expressing expression in both human MDA-MB-435 cells and 4T1 mouse TNBC cells than that in SPHK1-low–expressing cells. We per- TNBC cells in vitro (Supplementary Fig. S9A and S9B). Simi- formed a chromatin immunoprecipitation (ChIP) assay using larly, bortezomib treatment in 4T1 cells decreased phospho- NFkB antibody to bring down NFkB by immunoprecipitation NFkBexpressionin2Dand,moreprominently,in3Dculture and followed by qPCR with primers flanking NFkB binding (Supplementary Fig. S9C). region (263/253 bp) of FSCN1 promoter. Clearly, the binding To test whether safingol alone, bortezomib alone, or their of NFkB is more enriched in the 263/253 bp region of FSCN1 combination could deter TNBC progression and metastasis, we promoter in SPHK1-high–expressing (435.shScr and Hs578T. orthotopically injected highly aggressive 4T1 mouse TNBC cells shScr) cells than in their corresponding SPHK1-knockdown cells, (50,000 cells/mice) into BALB/c mice to induce MFP tumors. On whereas the binding of trimethylated H3K4 showed no significant day 7, when the primary tumors were palpable, we randomized difference (Fig. 5H). Together, these data indicate that SPHK1 the mice into four groups and treated them by intraperitoneal upregulates FSCN1 gene expression at the transcriptional level via injection of one of the following for 3 weeks: (i) vehicle (n ¼ 10), activation of NFkB transcription factor. (ii) safingol (5 mg/kg, every 3 days, n ¼ 11), (iii) bortezomib (0.5 mg/kg, every 3 days, n ¼ 11), or (iv) safingol plus bortezomib SPHK1/pNFkB/FSCN1 expressions in patients' TNBC tissues (n ¼ 15). Tumor growth and body weight of mice were monitored correlate with poor survival every 3 days. The single treatment of either safingol or bortezomib To determine the clinical relevance of our above findings, we had no significant effects on primary tumor growth, whereas the examined whether SPHK1/NFkB/FSCN1 pathway activation in combination treatment significantly reduced primary tumor patients' TNBC tissues is associated with increased metastasis growth (Fig. 7A; Supplementary Fig. S9D). Interestingly, safingol and poor clinical outcome. We performed IHC analyses of only, or bortezomib only significantly prolonged OS of mice SPHK1, p-NFkB,andFSCN1expressionsinTMAsofpatients' compared with vehicle treatment (median survival of 37 days, or TNBC tissue samples. Patients (N ¼ 117) whose TNBC tissues 40 days, vs. 31 days, respectively; Fig. 7B). Strikingly, safingol plus are included in the TMAs have various clinical and pathologic bortezomib combination treatment dramatically increased the features (Supplementary Table S1). IHC analyses revealed high median survival of mice from 31 days to 47 days (Fig. 7B). There expression of SPHK1, pNFkB, and FSCN1 in 59.2% (68/115), was no significant difference in mouse body weight, and no 67% (71/106), and 62.7 % (69/110) of TNBC tissue samples, dramatic difference in their blood counts of lymphocytes and respectively (Fig. 6A and B). To examine whether SPHK1/ myeloid cells among four treatment groups, suggesting that drug pNFkB/FSCN1 signaling pathway activation is critical for TNBC treatment, either as a single agent or in combination, did not progression and metastasis in patients, we examined the rela- induce any acute toxicity (Supplementary Fig. S9E and S9F). tionships of high expressions of SPHK1, pNFkB, and FSCN1 To evaluate the inhibitory effects of safingol, bortezomib, or with TNBC progression and metastasis. Because IHC staining their combination on tumor growth and metastasis, 5 mice under of phospho-proteins in archived tissues of patients may be each of the four treatments were euthanized at day 21 post unreliable, we analyzed whether coexpressions of SPHK1 treatment and their MFP tumors, as well as lungs were harvested

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Figure 6. Combined expression of SPHK1, pNFkB, and FSCN1 in patients with TNBC correlates with poor survival. A, Representative images of IHC staining showing the high and low expression of SPHK1, pNFkB, and FSCN1. Figure objective magnification, 10 and inset objective magnification, 20. B, Graph showing the percentage of patients with TNBC with high and low expression of SPHK1, pNFkB, and FSCN1. Kaplan–Meier survival plot showing distance metastasis-free survival (C) and OS (D) among patients with TNBC 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 ¼ 66); group 2: patients with high expression of both markers (SPHK1 and FSCN1 high, n ¼ 42).

for examination. Consistent with tumor volume detected above in tasis to the lungs compared with vehicle treatment (Fig. 7I; mice, primary tumor weights were significantly reduced by com- Supplementary Fig. S9H). Combination treatment further bination treatment, but not the single treatments (Fig. 7C). IHC reduced lung metastasis significantly compared with each single analyses of SPHK1 and pNFkB in these primary tumor samples treatment, inducing approximately 80% inhibition of spontane- showed that safingol treatment decreased the SPHK1 expression ous lung metastasis in mice compared with vehicle treatment and bortezomib treatment decreased the nuclear pNFkB com- (Fig. 7I; Supplementary Fig. S9H). pared with vehicle treatment, suggesting that the drugs were To evaluate whether or not the inhibition of metastasis by effectively inhibiting their molecular targets (Fig. 7D and E; combination treatment resulted from reduced primary tumor Supplementary Fig. S9G). Safingol treatment also decreased its growth, we examined the effect on metastasis after primary downstream nuclear pNFkB compared with vehicle treatment tumors were removed at similar sizes in vehicle and combina- (Fig. 7E). Either safingol or bortezomib treatment reduced FSCN1 tion treatment groups (Supplementary Fig. S10A and S10B). expression (Fig. 7F; Supplementary Fig. S9G). Remarkably, com- The lungs of these mice were harvested for examination of bination treatment significantly decreased SPHK1 expression, metastatic lesions 11 days post primary tumor resection in both nuclear pNFkB level, and FSCN1 expression compared with vehicle and combination treatment groups. Combination treat- vehicle or single treatments (Fig. 7D–F; Supplementary Fig. ment significantly reduced spontaneous metastasis to the lungs S9G). Furthermore, combination treatment decreased tumor cell compared with vehicle treatment when the primary MFP proliferation detected by Ki67 staining and increased tumor cell tumors were removed at similar sizes, suggesting that inhibi- apoptosis as shown by TUNEL staining, respectively (Fig. 7G and tion of metastasis by combination treatment is not only the H; Supplementary Fig. S9G). More importantly, safingol alone consequences of reduced primary tumor growth (Supplemen- and bortezomib alone significantly reduced spontaneous metas- tary Fig. S10C).

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Figure 7. Targeting SPHK1 and NFkB 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 four groups for treatment: vehicle, safingol only, bortezomib only, and combination of both drugs (saf þ bor). B, Survival curve of the mice in each treatment group as mentioned in A. C, MFP tumor weight at day 28 from mice in each treatment group as mentioned in A. At day 28, 5 mice were euthanized from each group as mentioned in A for time-matched experiment and tumor weight was taken. Quantification of IHC staining of SPHK1 (D), nuclear pNFkB(E), FSCN1 (F), Ki67 (G), and TUNEL (H) in MFP tumor samples of mice from time-matched experiment as mentioned in C. I, Hematoxylin and eosin–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; , P < 0.001; n.s., nonsignificant.

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Discussion cell migration, invasion, and metastasis. FSCN1 is a cytoskeletal TNBCs are negative of ER, PR, and HER2 expression, thus, do actin bundling protein that binds and packages actin filaments not respond to current hormonal- and HER2-targeted thera- into tertiary structures to enhance cell motility, migration, and pies (4). In this study, we embark on identifying positive thera- adhesion (24, 25, 43). FSCN1 is overexpressed in various peutic target(s) in TNBCs to develop effective targeted therapies cancers (44–46), whereas its expression is either absent or very for TNBC, especially TNBC metastasis. Our integrative bioinfor- low in normal epithelial cells (43). Tumor cells with high matics analysis of breast cancer patient–derived gene expression expression of FSCN1 have increased cell membrane protrusions datasets revealed that expression of SPHK1 is significantly higher such as filopodia and invadopodia, which help tumor cells' in TNBCs and is associated with poor clinical outcome of patients migration and extracellular matrix invasion, critical steps for with TNBC. Furthermore, we found that SPHK1 plays a promi- metastasis development (25, 27, 43, 46). Although it is known nent role in promoting TNBC spontaneous metastasis in multiple that FSCN1 high-expression in cancer cells facilitates metasta- mouse models. Mechanistically, SPHK1 upregulates the expres- sis, the upstream regulators of FSCN1 were not well known. sion of FSCN1 at the transcriptional level via activation of NFkB. Here, we identifiedthatSPHK1highexpressioninTNBCcells Clinically, high expression of SPHK1 and FCSN1 in patients' increases FSCN1 mRNA transcription by activating NFkB. In TNBC tissues correlates with increased distant metastasis and addition, we demonstrated that FSCN1 is a novel downstream poor survival. Furthermore, we demonstrated that targeting effector of SPHK1 in promoting TNBC cell migration, invasion, SPHK1 and NFkB by clinically applicable inhibitors effectively and metastasis. inhibited tumor progression and spontaneous metastasis to the Previous studies have shown that the SPHK1/S1P signaling can lungs in a highly aggressive TNBC mouse model. Together, our induce activation or inhibition of various transcription factors, data indicate that SPHK1 and NFkB could serve as positive including NFkB, E2F, c-Myc, and Sp1, and consequently impact therapeutic targets for inhibiting TNBC metastasis. on cell proliferation, apoptosis, and/or inflammation (36, 47, 48). We demonstrated that SPHK1 expression was higher in TNBCs SPHK1 was reported to induce NFkB activation via intracellular compared with other subtypes of breast cancer. Abnormal expres- S1P that serves as a cofactor of TRAF2 to activate IKKa/b, then sion of SPHK1 has been strongly associated with the development IKKa/b phosphorylates IkBa, resulting in its degradation to allow and progression of various cancers (9, 14). Several studies of NFkB activation upon TNFa stimulation (31). Consistently, in breast cancer and other cancer types have also shown that SPHK1 3D-cultured 435.shSPHK1 cells, both IKKa/b and IkBa phos- plays a role in cancer cell proliferation (34–36). In contrast, our phorylations were decreased compared with SPHK1-high– data showed that SPHK1 cannot drive TNBC cell proliferation in expressing 435.shScr cells. Thus, SPHK1-induced NFkB activation vitro nor tumor growth in vivo in multiple TNBC models and in is mostly mediated by intracellular S1P function, although it may both gain- and loss-of-function studies. Consistent with our also involve S1P's extracellular function. findings, recent studies of SPHK1-specific inhibitor, PF-543, Here, we made the unique finding that SPHK1 activates NFkB showed that inhibiting SPHK1 had no effect on the proliferation to upregulate FSCN1 expression. Our data showed that FSCN1 of various cancer cells, including a TNBC cell line MDA-MB- promoter activity was significantly decreased in SPHK1-knock- 231 (37, 38). Notably, although SPHK1 knockdown had no down cells, indicating SPHK1 upregulates FSCN1 at the transcrip- significant impact on cell proliferation, MFP tumors of SPHK1- tional level. We identified a 240-bp region within FSCN1 pro- knockdown cells had increased apoptosis compared with MFP moter and a NFkB binding site herein that is responsible for this tumors of control cells, similar to a previous report (39). Most SPHK1-induced FSCN1 upregulation. We also showed that bind- importantly, our data from both gain- and loss-of-function stud- ing of NFkB to the FSCN1 promoter region was inhibited in ies showed that SPHK1 functions to promote metastasis-related SPHK1-knockdown cells compared with control cells (Fig. 5H). properties of TNBC cells in vitro and TNBC spontaneous metas- Interestingly, SPHK1 activated NFkB more effectively in 3D than tasis in vivo. Furthermore, we dissected the FSCN1 upregulation as 2D cultures (Supplementary Fig. S8F and S9C) and we readily a critical molecular mechanism of SPHK1-induced TNBC cell detected SPHK1 upregulation of FSCN1 expression in 3D culture. invasion and metastasis. Together, SPHK1 transcriptionally upregulates FSCN1 expression In our study, we used spontaneous metastasis models to via activation of NFkB. determine whether SPHK1 functions in metastasis, because the The SPHK1/S1P axis is known to regulate immune responses by spontaneous metastasis model recapitulates all of the steps regulating lymphocytes trafficking, innate immune response, and involved in the multistep process of the metastatic cascade (40). inflammation (49). For these reasons, we used a syngeneic mouse Exogenous expression of SPHK1 significantly increased, where- model for testing therapeutic efficacy of SPHK1 targeting in vivo. as knocking down SPHK1 significantly decreased spontaneous Also we decided to use drugs that are already in clinical trials or metastasis to the lungs in nude mice, suggesting SPHK1 expres- have been FDA-approved for fast-track clinical translation. Spe- sion is vital for the metastatic potential of TNBC cells. Despite cifically, we targeted SPHK1 by safingol, which is currently used in some reports suggesting that the SPHK1/S1P axis modulates the clinical trials and target NFkB by bortezomib, which is a FDA- MAPK/ERK pathway and EGFR pathways to promote invasion approved drug against multiple myeloma. Although a few drugs and metastasis in some cancer types, the mechanism by which have been developed against FSCN1, none have been tested so far SPHK1 promotes TNBC metastasis was unclear (41, 42). in a clinical setting (50). Even though single-agent treatment of Understating molecular pathways driving TNBC metastasis safingol or bortezomib had no significant effect on primary tumor process, with fast-track clinical translation potentials, is needed growth, combination treatment significantly delayed tumor pro- for developing effective therapeutic intervention of TNBC gression (Fig. 7A), suggesting a complex interaction between these metastasis. In this study, using unbiased gene expression pro- two drugs that warrants further investigation. Excitingly, treat- filing of in vivo tumor samples, we identified FSCN1 as a top ment with single agent of either safingol or bortezomib signifi- candidategenemodulatedbySPHK1andinvolvedinTNBC cantly decreased the spontaneous metastasis of aggressive TNBC

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SPHK1 Enhances TNBC Metastasis

to the lungs, which was further decreased with combination Acquisition of data (provided animals, acquired and managed patients, treatment and the safingol plus bortezomib also significantly provided facilities, etc.): S. Acharya, P. Li, C. Zhang, F.J. Lowery, Q. Zhang, increased mice survival (Fig. 7B). Our data indicate that combin- F. Yang, I.I. Wistuba, H. Piwnica-Worms, A.A. Sahin fi Analysis and interpretation of data (e.g., statistical analysis, biostatistics, ing sa ngol and bortezomib may be effective in treating TNBC computational analysis): S. Acharya, J. Yao, C. Zhang, H. Guo, D. Yu tumors and metastasis, which warrants further clinical testing. In Writing, review, and/or revision of the manuscript: S. Acharya, J. Yao, addition, understanding of the impact of these drugs on the tumor C. Zhang, J. Qu, H. Piwnica-Worms, D. Yu microenvironment in vivo will enable us to further improve these Administrative, technical, or material support (i.e., reporting or organizing drugs' efficacy in the clinic. It would also be interesting to test the data, constructing databases): J. Qu benefits of using these drug combination as an adjuvant treatment Study supervision: D. Yu regimen after surgery to prevent or intervene with metastasis and/ or recurrences, especially for those patients whose primary tumor Acknowledgments express high levels of SPHK1 and/or FSCN1. Our study also This work is supported by Susan G. Komen Breast Cancer Foundation suggests that expression of SPHK1/pNFkB/FSCN1 axis in TNBC promise grant KG091020 (to D. Yu), NIH grants P30-CA 16672 (MD Anderson primary tumors could be a predictive biomarker for development Cancer Center, MDACC), RO1-CA112567-06 (to D. Yu), and RO1-CA184836 of metastasis, which needs to be further validated. Taken together, (to D. Yu), Breast Cancer Moon Shot funding from The University of Texas MD we have shown that the SPHK1/pNFkB/FSCN1 axis is activated in Anderson Cancer Center, and China Medical University Research Fund (to D. Yu). D. Yu is the Hubert L. & Olive Stringer Distinguished Chair in Basic TNBCs and can serve as positive therapeutic targets that can be Science at MD Anderson Cancer Center. We would like to thank MDACC Breast inhibited by clinically applicable kinases inhibitors, and our Tumor Bank for providing us with TMA slides, Dr. Jean J. Zhao for providing us preclinical testing demonstrated that targeting SPHK1/pNFkB with SPHK1 overexpression plasmid, and Dr. Emily Powell for her assistance in effectively inhibited metastasis from highly aggressive TNBC obtaining PDX cell line. We would also like to thank MDACC Functional tumors. We foresee that these findings may be speedily translated Genomics Core, Research Histology Core, and Small Animal Core Facility for fi into the clinic to benefit patients with TNBC in great need of technical support, Department of Scienti c Publications of MDACC for article revision, and members from D. Yu's laboratory for insightful discussions. effective therapies.

fl The costs of publication of this article were defrayed in part by the Disclosure of Potential Con icts of Interest payment of page charges. This article must therefore be hereby marked No potential conflicts of interest were disclosed. advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Authors' Contributions Conception and design: S. Acharya, F.J. Lowery, A.A. Sahin, D. Yu Received December 3, 2018; revised April 16, 2019; accepted June 19, 2019; Development of methodology: S. Acharya, F.J. Lowery, I.I. Wistuba, D. Yu published first June 25, 2019.

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4226 Cancer Res; 79(16) August 15, 2019 Cancer Research

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

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

Cancer Res 2019;79:4211-4226. Published OnlineFirst June 25, 2019.

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