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

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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 data- sets 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 Ó2019 American Association for Cancer Research. fibroblasts led to transformation in vitro and tumor formation www.aacrjournals.org 4211 Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 2019 American Association for Cancer Research. Published OnlineFirst June 25, 2019; DOI: 10.1158/0008-5472.CAN-18-3803 Acharya et al. 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.
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