Repurposing of a Thromboxane Receptor Inhibitor Based on a Novel Role in Metastasis Identified by Phenome Wide Association Study Authors: Thomas A

Home , Ifetroban, Terbogrel

Author Manuscript Published OnlineFirst on October 8, 2020; DOI: 10.1158/1535-7163.MCT-19-1106 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Repurposing of a thromboxane receptor inhibitor based on a novel role in metastasis identified by Phenome Wide Association Study Authors: Thomas A. Werfel1, 7, Donna J. Hicks1, Bushra Rahman1, Wendy E. Bendeman1, Matthew T. Duvernay2, Jae G. Maeng2, Heidi Hamm2, Robert R. Lavieri3, Meghan M. Joly3, Jill M. Pulley3, David L. Elion4, Dana M. Brantley-Sieders5,6, Rebecca S. Cook1, 4, 5, 8*

Affiliations: 1Department of Cell & Developmental Biology, Vanderbilt University School of Medicine, Nashville, TN 37232 USA 2Department of Pharmacology, Vanderbilt University Medical Center, Nashville, TN 37232 USA 3Vanderbilt Institute for Clinical and Translational Research, Vanderbilt University Medical Center, Nashville, TN 37232 USA 4Program in Cancer Biology, Vanderbilt University School of Medicine, Nashville, TN 37232 USA 5Breast Cancer Research Program, Vanderbilt Ingram Cancer Center, Vanderbilt University Medical Center, Nashville, TN 37232 USA 6Department of Medicine, Vanderbilt University Medical Center, Nashville, TN 37232 7Department of Chemical Engineering, University of Mississippi, University, MS 38677 USA 8Department of Biomedical Engineering, Vanderbilt University School of Engineering, Nashville, TN 37232 USA

*To whom correspondence should be addressed:

Rebecca S. Cook, Ph.D. 759 Preston Research Building Vanderbilt University 2220 Pierce Avenue Nashville, TN 37232 Email: [email protected] Office phone: 615-936-3813

Conflict of Interest: CPI211 used in this study was provided by Cumberland Pharmaceuticals, Inc. (CPI). CPI had no role in study design, experimental conduct, data collection and analysis, or preparation of the manuscript. Additionally, CPI provided no direct financial support for this work.

Compliance: BioVU data were utilized in accordance with all applicable institutional policies under IRB# 151121 Running title: Repurposing a TPr inhibitor to block cancer metastasis

Abstract:

While new drug discoveries are revolutionizing cancer treatments, repurposing existing drugs would accelerate the timeline and lower the cost for bringing treatments to cancer patients. Our goal was to repurpose CPI211, a potent and selective antagonist of the thromboxane A2- prostanoid receptor (TPr), a G-protein coupled receptor that regulates coagulation, blood pressure, and cardiovascular homeostasis. To identify potential new clinical indications for

CPI211, we performed a Phenome Wide Association Study (PheWAS) of the gene encoding

TPr, TBXA2R, using robust de-identified health records and matched genomic data from

>29,000 patients. Specifically, PheWAS was used to identify clinical manifestations correlating with a TBXA2R single nucleotide polymorphism (rs200445019), which generates a T399A substitution within TPr that enhances TPr signaling. Previous studies have correlated

200445019 with chronic venous hypertension, which was recapitulated by this PheWAS analysis. Unexpectedly, PheWAS uncovered a rs200445019 correlation with cancer metastasis across several cancer types. When tested in several mouse models of metastasis, TPr inhibition using CPI211 potently blocked spontaneous metastasis from primary tumors, without affecting tumor cell proliferation, motility, or tumor growth. Further, metastasis following intravenous tumor cell delivery was blocked in mice treated with CPI211. Interestingly, TPr signaling in vascular endothelial cells induced VE-cadherin internalization, diminished endothelial barrier function, and enhanced trans-endothelial migration by tumor cells, phenotypes that were decreased by CPI211. These studies provide evidence that TPr signaling promotes cancer metastasis, supporting the study of TPr inhibitors as anti-metastatic agents, and highlighting the use of PheWAS as an approach to accelerate drug repurposing.

Introduction

The use of de-identified human genetic data tied to robust electronic health records (EHRs) enables discovery of human genotypes correlating with specific clinical conditions. This approach, known as phenome-wide association study (PheWAS), (1,2), can discover clinical

‘phenotypic’ indications associating with a genotypic variation, enabling discoveries of novel pathophysiological gene functions. From a translational perspective, PheWAS could predict potential opportunities for repurposing an existing catalog of molecularly targeted therapies (3), diminishing the risk, cost, and time needed for bringing treatments to patients.

To test this principle, we used PheWAS to investigate repurposing opportunities for the drug

CPI211 (4-6), a potent and highly selective small molecule inhibitor of thromboxane A2 and prostanoid receptor (TPr). CPI211 (PubChem ID 3037233) is safe and well tolerated, as shown in randomized, dose-escalating (≤1000 mg), placebo-controlled clinical trial (7). Decades of basic and translational science determined that TPr, expressed in platelets, endothelial cells

(ECs), and smooth muscle cells, regulates different aspects of cardiovascular homeostasis. TPr dysregulation contributes to venous hypertension, pulmonary hypertension, thrombosis and asthma, steering development of CPI211 and other TPr antagonists toward these clinical indications (8,9). However, aberrant activation of TPr may have yet undiscovered pathological consequences for which TPr inhibitors might be effective. Since pathologically increased TPr expression can be caused by naturally occurring single nucleotide polymorphisms (SNPs) within the TPr gene, TBXA2R, w propose that PheWAS analysis using these previously identified and characterized TBXA2R SNPs as a genomic proxy for increased TPr expression could identify clinical manifestations correlating with increased TPr expression (10).

Several previously described SNPs within TBXA2R are known to alter TPr expression, some that diminish TPr and thus correlate with decreased platelet function (11), and others that

Downloaded from mct.aacrjournals.org on September 26, 2021. © 2020 American Association for Cancer Research. Author Manuscript Published OnlineFirst on October 8, 2020; DOI: 10.1158/1535-7163.MCT-19-1106 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. increase TPr expression. As might be expected, the SNPs that increase TPr expression are known to correlate with advanced cardiovascular disease (12-14) or asthma (15). We investigated one TBXA2R SNP that increases TPr expression, rs200445019. This SNP was selected based on its occurrence within the TBXA2R coding region, generating a threonine-to- alanine alteration at residue 399 (T399A) within the C-terminus of the TPr splice variant, TPr-β

(https://www.ncbi.nlm.nih.gov/snp/rs200445019/), the domain of TPr-β driving TPr desensitization, internalization, trafficking, and proteosomal degradation (16-19). Following ligand activation, TPr signaling activates protein kinase C (PKC), a serine threonine kinase which then phosphorylates TPr-β T399, inducing TPr internalization and degradation. TPr T399 mutation causes loss of PKC-mediated TPr phosphorylation, and loss of ligand-induced receptor downregulation. Thus, the TPr-β T399A substitution generated by rs200445019 results in greater TPr expression and signaling, supporting use of rs200445019 as a genomic proxy for increased TPr in PheWAS analyses. Here, we report that PheWAS analysis of rs200445019 identified a novel and unexpected correlation between rs200445019 and metastatic cancer diagnoses, and that the TPr inhibitor CPI211 blocked metastasis in multiple cancer models.

Materials and Methods

The BioVU biorepository at Vanderbilt University Medical Center (VUMC) contains >250,000 de- identified DNA samples extracted from excess patient blood samples collected during routine clinical testing, linked to corresponding, longitudinal clinical and demographic data derived from

Synthetic Derivative, a de-identified electronic health record (EHR) built for research purposes

(20,21). BioVU data were utilized in accordance with VUMC IRB# 151121. The allelic variant of focus was the TBXA2R missense SNP rs200445019, selected based on the presence of meaningful validation signals in the PheWAS results to support inference of variant effects in vivo, including hypertension and cardiovascular indications, phenotypes expected to be observed if TPr function was enhanced.

PheWAS using previously reported methods (22) focused on 29,722 patients of European ancestry genotyped using the Illumina Infinium Exomechip (Illumina, Inc.), containing ∼250,000

SNPs across genomic protein coding regions discovered through exome and whole-genome sequencing in more than 12,000 individuals. All distinct phenotypic data (ICD9 billing codes representing approximately 1660 phenotypes) were captured from EHRs and translated into corresponding phenotype groupings. Cases for each phenotype (condition) were defined as patients having ≥ 2 ICD9 codes documented on separate days and mapping to a specific

PheCode. The PheCode ‘malignant secondary neoplasm’ (PheCode 198) included ICD9 codes for secondary malignant neoplasm of respiratory and digestive systems, secondary malignant neoplasms of other specified sites, malignant pleural effusion, and several others. Controls were defined as all patients who did not have any of the ICD9 codes defining a case. Only phenotypes occurring in 25 cases (0.42% of genotyped patients) were included. Physicians at

Vanderbilt University Medical Center with relevant qualifications and content-specific knowledge performed evidence reviews in cases of secondary malignancy. The PheWAS algorithm (22) was applied to calculate case and control genotype distribution, χ2 distribution, associated allelic P-value and allelic odds ratio (OR). For χ2 distribution cell counts below five, Fisher's exact test was used to calculate P-value using R software (http://www.r-project.org/).

CPI211. CPI211 (Supplemental Figure S1A, PubChem ID 3037233) was generously provided by Cumberland Pharmaceuticals, Inc. The chemical formula of CPI211 is C25H32N2O5, with a molecular weight of 440.5 g/mol. CPI211 is also known by the following aliases: Ifetroban,

143443-90-7, UNII-E833KT807K, BMS-180291.

Cell culture. Cells purchased from American Tissue Type Collection [4T1: (CRL-2539) HEK-

293T/17 (CRL-11268), MDA-MB-231 (HTB-26), MiaPaCa-2 (CRL-1420), A549 (CRM-CCL-185)]

(GenTarget Inc.) followed by puromycin selection. LVP encoding human TPr-β and TPr-

β(T399A) were custom synthesized in Lentifect (GeneCopoeia). 293T cells were transduced with empty LVP or those expressing TPrβ(WT) and TPr-β(T399A), followed by blasticidin selection. Mouse pulmonary microvascular ECs (MPMECs) were harvested and cultured as described (23). For MPMEC growth assays, cells (0.5 X 104) were seeded on gelatin-coated 35- mm dishes, and cultured 7d in growth media, U46619 (0.5 µM), CPI211 (0.1 µM), or 4T1 conditioned media (serum-free media conditioned for 48 h culture of 4T1 cells, passed through a 0.2 µm syringe filter). Cells were stained with 0.01% crystal violet, rinsed with water and dried, then imaged on a flatbed scanner. Human umbilical vein ECs (HUVEC) were purchased from

Lonza and maintained in EGM-2 medium (Lonza). All cells used were cultured for <15 passages.

Platelet preparation. Human platelets were collected from blood donated by healthy anonymous volunteers, and prepared as previously described (24). The platelet pellet was suspended in Tyrode’s buffer containing 0.1% bovine serum albumin (BSA) and counted on a

Beckman Z1 Coulter counter.

Western blotting. Cells were homogenized in lysis buffer [50 mM Tris pH 7.4, 100 mM NaF,

400 mM NaCl, 0.5% NP-40, 100 µM Na3VO4, 1X protease inhibitor cocktail (Roche)] and cleared by centrifugation. Platelets were homogenized with 0.6 N perchloric acid and cleared by centrifugation. For immunoprecipitation, cell lysates (1000 μg) cleared with protein A/G+ agarose were incubated with anti-VE-cadherin antibody (5 μg) and protein A/G+ agarose.

Samples were resolved on 4-12% polyacrylamide gels (Novex), transferred to nitrocellulose membranes (iBlot), and western analysis was performed as described (25) using the following

Endothelial-Cadherin, phospho-tyrosine horseradish peroxidase (PTyr-100)].

Animals. All animals were housed under pathogen-free conditions. Experiments were performed in accordance with AAALAC guidelines and Vanderbilt University Institutional Animal

Care and Use Committee approval. Where indicated, CPI211 was delivered daily at 50 mg/kg by oral gavage in 25 µl vehicle (4% sucrose in sterile water). 1 x 106 tumor cells (4T1, MDA-

MB-231, A549, and MiaPaCa2) were injected via tail vein of athymic (nu/nu) Balb/C female mice. 4T1 tumor cells (1 x 106) were injected into the left inguinal mammary fatpad of athymic

(nu/nu) Balb/C or WT Balb/C female mice. Tumor dimensions were measured using calipers.

Tumor volume was calculated as: volume = length x width2 x 0.52). For bioluminescence imaging, mice were injected intraperitoneally with D-luciferin monosodium substrate

(ThermoFisher, 150 mg/kg) and imaged on IVIS Lumina III (Xenogen Corporation) 30 minutes post-injection.

Histological analysis. Tissue processing, staining, and immunohistochemistry (IHC) was performed by the Vanderbilt Translational Pathology Shared Resource. IHC on 5-m paraffin- embedded sections was performed as described previously (26) using anti-CD31 (Abcam), and anti-Ki67 (Santa Cruz Biotechnologies). Photomicrographs were acquired on an Olympus CK40 inverted microscope through an Optronics DEI-750C camera using CellSens software for capture and morphometric analyses (27).

Trans-endothelial cell migration assay.

MPMECs or HUVECs (1 X 105) were seeded on Matrigel-coated transwell lower surfaces and cultured 1 week before labeling with CellTracker Red (Molecular Probes; 0.5 mg/ml),

Downloaded from mct.aacrjournals.org on September 26, 2021. © 2020 American Association for Cancer Research. Author Manuscript Published OnlineFirst on October 8, 2020; DOI: 10.1158/1535-7163.MCT-19-1106 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. transferred to 24-wells dishes in 2% serum ± U46619 (0.5 μM) and/or CPI211 (0.1 μM) for 6 hours. 4T1-GFP cells (2 X 105) were seeded into the upper chamber for 16 hours, swabbed from upper transwell surfaces then visualized on the lower filter surface using fluorescence microscopy.

Statistics. Treatment groups were compared using either two-tailed student’s t-test or one-way

ANOVA test coupled with Tukey means comparison test, where a p-value < 0.05 was deemed representative of a significant difference between treatment groups.

Results

Unbiased PheWAS correlates a naturally occurring TBXA2R SNP with metastasis.

The TPr-T399A alteration generated by rs200445019 reportedly impairs TPr desensitization and degradation following ligand-induced stimulation. To confirm this, we expressed wild-type TPr-β

(TPr-WT) and TPr-β T399A (TPr-T399A) in 293T cells, which express low endogenous TPr levels (Supplemental Figure S1B). Under serum-starved conditions, similar protein expression of TPr was seen in cells expressing TPr-WT and TPr-T399A. As expected, addition of the TPr agonist U46619 (0.5 µM) increased PKC activity and MLC phosphorylation (Figure 1A), which occurred at similar levels in cells expressing TPr-WT and TPr-T399A. However, TPr-WT protein levels diminished within 5 minutes of U46619 treatment, while TPr-T399A levels remained unchanged, consistent with previous reports that TPr T399 drives ligand-induced TPr down regulation. This was confirmed by treating cells with U46619 for 5 minutes, then chasing with U46619-free media for up to 60 minutes, revealing a sustained down-regulation of TPr-

WT, but persistently elevated TPr-T399A (Figure 1B). These findings confirm use rs200445019 as a proxy for increased TPr expression.

To detect phenotypes associated with rs200445019, we leveraged the BioVU database (21) to query 29,722 de-identified patient EHRs and their associated genotyping, scanning for novel associations between PheWAS codes and rs200445019. As expected, our unbiased PheWAS approach identified associations of rs200445019 with chronic venous hypertension, pulmonary heart disease, and primary pulmonary hypertension (Table 1) (14). Unexpectedly, PheWAS also identified a novel association between rs200445019 and metastatic disease (coded as

Secondary Malignancy) at multiple tissue sites, including lymph nodes, respiratory organs, digestive systems, brain/spine, and others. Chart review by medical experts confirmed the diagnoses of secondary malignancies, and revealed that these metastatic lesions were derived from multiple solid tumor types, including breast, colon, lung, head and neck, renal, gastric, ovarian cancers, melanomas, and others (Figure 1C). However, PheWAS did not detect a specific correlation of rs200445019 with any primary cancer diagnosis, per se, suggesting that increased TPr expression and/or activity might have a selective role in tumor metastasis.

Next, we assessed the relationship between TBXA2R expression and cancer patient outcome.

Analysis of METABRIC-curated invasive breast cancers assessed by gene expression array

(N=2051)(28) revealed decreased overall survival (OS) of patients whose tumors harbored high

TBXA2R (Figure 1D). High TBXA2R expression also correlated with decreased OS in lung adenocarcinomas and gastric cancers (29). We extended this analysis across 14 additional cancer types, using publicly available tumor RNA-Seq data tied to disease free progression

(DFP) outcomes [(29)Supplemental Table S1], finding that 12 of 15 solid cancer types displayed an inverse correlation between TBXA2R expression and DFP, with strong statistical significance in 11 of these, including breast cancer, ovarian cancer, renal papillary cell carcinoma, sarcoma, and others. Interestingly, 2 /14 cancer types assessed showed increased

DFP in patients expressing increased TBXA2R [bladder cancer and hepatocellular carcinoma

(HCC)]. The reasons underlying why bladder cancer and HCC might show improved survival upon increased TBXA2R expression are unclear, but may relate to unique pathophysiologies of

Downloaded from mct.aacrjournals.org on September 26, 2021. © 2020 American Association for Cancer Research. Author Manuscript Published OnlineFirst on October 8, 2020; DOI: 10.1158/1535-7163.MCT-19-1106 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. these cancers, routes of metastatic spread, or tissue-selective roles of TPr. For the 12 cancers demonstrating a correlation between TBXA2R expression and worse outcome, these findings support the notion that TBXA2R / TPr may have an underappreciated role in tumor metastasis, and that targeted TPr inhibition might suppress metastasis.

To confirm TPr inhibition by the TPr antagonist CPI211, we assessed 293T cells expressing exogenous TPr-β. Western analysis confirmed that CPI211 blocked MLC phosphorylation in

U46619-treated cells expressing TPr-WT and TPr-T399A (Figure 1E). We also confirmed

TPr-α inhibition by CPI211 using washed human platelets, which almost exclusively express

TPr-α. U46619-induced P-MLC was blocked robustly by CPI211 (Figure 1F). Dilutions of

CPI211 down to 5 nM blocked U46619-induced platelet aggregation (Figure 1G) confirming potent TPr inhibition by CPI211.

TPr inhibition reduces cancer metastasis in several pre-clinical cancer models

We assessed the impact of TPr inhibition on hematogenous metastasis using tumor cells [4T1

(mouse mammary cancer), MDA-MB-231 (human breast cancer), MiaPaCa2 (human pancreatic cancer) and A549 (human lung cancer)] delivered directly into circulation by intravenous (i.v.) injection. Western analysis revealed only very low TPr expression in each tumor cell line, and modest or no P-MLC in U46619-treated cells (Figure 2A). Athymic (nu/nu) mice were pre- treated with CPI211 (50 mg/kg) before tumor cell delivery, continuing treatment for the following

28 days (Figure 2B). 4T1 lung metastases were seen in 100% of vehicle-treated mice, but only

70% of CPI211-treated mice (N = 10, Figure 2C). Similarly, CPI211 treatment decreased the percentage of mice harboring MDA-MB-231 lung metastases from 90% to 20% (N = 10), and mice with A549 lung metastases from 60% to 10% (N = 10). Although 90% of mice inoculated with MiaPaca2 cells developed lung lesions regardless of vehicle or CPI211 treatment, the average number of MiaPaca2 metastases per mouse was diminished in CPI211-treated mice by

Because venous tumor cell delivery allows tumor cells to bypass steps of intravasation, we used a rigorous model of spontaneous metastasis from primary orthotopic 4T1 mouse mammary tumors. Mice harboring luciferase-expressing 4T1 tumors were randomized into treatment groups receiving vehicle or CPI211 when tumors reached 50 mm3. Mice were treated daily for

28 days, although tumors were surgically resected on treatment day 12. By treatment day 21, luminescence was detected in the thoracic region of vehicle-treated mice, but not CPI211- treated mice (Figure 2E). By day 28, average luminescence in vehicle-treated mice was approximately 18-fold higher than in CPI211-treated mice. Notably, 3 of 10 mice treated with

CPI211 had no measurable luminescence above baseline (Figure 2E, right panel). Histological examination of lungs confirmed reduced lung metastases in CPI211-treated mice versus vehicle-treated mice, including an absence of metastases in 3 of 10 CPI211-treated mice.

TPr inhibition did not affect tumor cell proliferation.

We measured volume of tumors grown in athymic mice and treated for 12 days with CPI211 beginning when tumors were 50 mm3, revealing no difference in tumor size between vehicle and

CPI211-treated groups (Figure 3A). Immunohistochemistry (IHC) for the cellular proliferation marker Ki67 revealed similar Ki67 staining in tumors harvested from vehicle-treated and

CPI211-treated mice (Figure3B-3C). These findings were supported by measurements of tumor cell growth in culture, showing that neither TPr activation nor TPr inhibition affected growth of

4T1 or MDA-MB-231 cells (Figure 3D).

CPI211 decreased tumor vasculature.

An abundance of large vascular structures seen in vehicle-treated tumors were substantially diminished in CPI211-treated tumors, as shown by staining with the vessel endothelial marker

CD31 (Figure 3E). Software-based morphometric analyses of CD31-stained tumor sections revealed a decreased total area of CD31+ vessels in CPI211-treated tumors (Figure 3F) and fewer CD31+ structures exceeding 50 μm2 (Figure 3G). Thus, it is possible that TPr signaling affects the tumor vasculature in a manner that promotes metastasis.

TPr signaling supports increased trans-endothelial migration of tumor cells.

We investigated the impact of TPr signaling on endothelial cells (ECs) using primary cultures of mouse pulmonary microvascular ECs (MPMECs), which abundantly expressed TPr (Figure

4A). U46619 induced PKC activity in MPMECs. As expected, TPr expression was not diminished in U46619-treated MPMECs, since mouse cells express only TPr-α, which, unlike

TPr-β, is not internalized in response to ligand activation (19). Importantly, PKC substrate phosphorylation was potently inhibited by CPI211. Similar PKC substrate phosphorylation was seen in western analyses of primary human umbilical vein ECs (HUVECs) treated with U46619, which was blocked upon treatment with CPI211. Notably, TPr expression diminished in U46619- treated HUVECs, which abundantly express TPr-β(30). Agonist-induced TPr diminution was blocked by CPI211 in HUVECs.

Growth of MPMECs seeded at low density over 7 days was unaffected by U46619 and/or

CPI211 (Figure 4B). Interestingly, serum-free 4T1 tumor cell-conditioned media increased

MPMEC growth, although this, too, was unaffected by CPI211.

Accumulating evidence shows that EC barrier function becomes compromised in cancers (31-

33). We measured tumor cell migration across an endothelial barrier using a modified transwell approach (Figure 4C). CellTracker Red-labeled MPMECs seeded on Matrigel on the lower side of a transwell filter were grown into a barrier monolayer, then treated with U46619 prior to adding 4T1-GFP cells into the upper chamber, assessing trans-endothelial migrations by

Similarly, an increased number of MDA-MB-231 cells migrated across U46619-treated HUVECs as compared to vehicle-treated HUVECs. In both cases, CPI211 blocked tumor cell migration across the EC barrier. However, in the absence of ECs, 4T1 cell migration across Matrigel- coated transwells was unaffected by U46619 or by CPI211 (Supplemental Figure S2A), as was MDA-MB-231 migration (Supplemental Figure S2B). These findings suggest that TPr signaling may not affect tumor cell migration, per se, but may enhance trans-endothelial migration of tumor cells.

Next we measured the barrier function of MPMEC monolayers cultured on transwells and treated with U46619 and/or CPI211. Transwell filters were transferred to fresh wells containing only PBS after 6 hours, at which point albumin (50 µg/ml) was added to the upper transwell chamber. After 2 minutes, the lower chamber for was sampled for albumin distribution (Figure

4F). U46619-treated MPMECs showed increased albumin passage to the lower chamber, which was blocked by CPI211. These results suggest that TPr signaling may increase permeability of an endothelial barrier, and are consistent with previous reports suggesting that thromboxane signaling may decrease vascular barrier function (34,35).

Vascular endothelial (VE)-Cadherin localization at EC junctions is critical for endothelial barrier function. Notably, VE-cadherin tyrosine phosphorylation induces VE-cadherin internalization, compromising endothelial barrier function (36), enabling metastatic dissemination of tumor cells

(37,38). Using immunofluorescence (IF) to assess VE-cadherin localization in MPMECs cultured on transwell filters, we identified punctate patterns of VE-cadherin staining along cell membranes at or near cell-cell contacts in vehicle-treated MPMECs (Figure 4G), whereas

U46619-treated MPMECs displayed diffuse intracellular VE-Cadherin staining. While U46619 did not affect total VE-cadherin expression (Figure 4H), U46619-induced VE-Cadherin tyrosine

Neoadjuvant and adjuvant models of CPI211 treatment diminish metastatic spread from primary tumors in immune-competent mice.

Next, we established models of spontaneous metastasis from orthotopic 4T1 tumors grown in immune-competent isogenic Balb/C mice, with two separate CPI211 treatment schemes assessed. The first approach modeled neoadjuvant CPI211 treatment (i.e., without tumor resection), in which tumor-bearing mice were randomized into treatment groups when tumors reached 50 mm3. Mice were treated daily for 28 days with vehicle or CPI211 (Figure 5A). While lung metastases were seen in all vehicle-treated mice (N = 10), only 60% of CPI211-treated mice harbored lung metastases (Figure 5B). Of the mice that developed lung metastases, fewer lung metastases were seen CPI211-treated samples versus vehicle, demonstrating that

TPr inhibition diminishes spontaneous metastases in the neoadjuvant setting.

We next tested a model of adjuvant TPr inhibition following surgical resection of 300 mm3 tumors. Mice were randomized for treatment 24 hours after tumor resection (Figure 5C), receiving daily treatment with vehicle or CPI211 for 21 days. While 100% of vehicle treated mice and 90% of CPI211-treated mice developed lung metastases (Figure 5D), the number of lung metastases per mouse was reduced in CPI211-treated mice to half of what was seen in controls. These studies reveal a potential benefit for reducing metastatic burden through TPr inhibition, even after tumor cells may have entered circulation.

Discussion

We identified a novel and unexpected correlation between TPr and tumor metastasis using a combination of PheWAS analyses in large population datasets, gene expression studies in

(Supplemental Figure S3). Importantly, the TPr inhibitor CPI211 decreased both experimental

(hematogenous) and spontaneous tumor metastasis.

The metastatic process requires tumor cells to overcome a series of physiological barriers (2).

Tumor cells must invade through primary tumor extracellular matrix to reach the vasculature, migrate across the vascular endothelium into circulation, survive in circulation, evade immune recognition, and adhere to and migrate across the endothelium into distant tissue, where tumor cells must survive and grow (39). Findings shown herein highlight an underappreciated role for

TPr signaling in disrupting the endothelial barrier in the metastatic process, facilitating tumor metastasis. These studies build upon published studies demonstrating that platelets, a rich source of the TPr ligand TxA2, are potent mediators of metastasis through a range of pathways involving interactions between tumor cells, endothelial cells, and platelets (40-42). Platelet-CTC aggregation may enhance metastasis by providing important survival signals to the tumor cell, by shielding CTCs from immune surveillance, and by promoting adhesion to the vascular endothelium. In fact, previous studies report that TBXA2R-null mice display reduced metastases in experimental (tail vein) models of metastasis(42). Thus, CPI211 may reduce platelet-assisted metastatic processes. Although we have not tested this hypothesis specifically, we confirmed that CPI211 inhibits platelet aggregation (Figure 1).

While platelet TPr likely has a prominent role in facilitating tumor metastasis, we show that endothelial TPr signaling increases trans-endothelial tumor cell migration, even in the absence of platelets (Figure 4). Endothelial TPr signaling diminished VE-cadherin at endothelial cell-cell junctions. Interestingly, published studies show that hypoxia contributes to VE-cadherin endocytosis in endothelial cells, resulting in endothelial barrier disruption and vascular dysfunction, while increasing TPr expression and sensitivity (38,43), together suggesting that

Recent studies highlighted a role for COX-1 in generation of a pre-metastatic niche within the vasculature of colorectal cancers, in large part through its ability to increase TxA2 production, thus enhancing platelet-tumor cell aggregation upon vessel walls. Inhibition of COX-1 using aspirin decreased metastasis through down-regulation of platelet TxA2 (41), implicating TPr signaling in metastasis. These studies hold great clinical significance, as the Add-Aspirin study

(NCT02804815) is currently testing if daily aspirin use (100mg, 300 mg, or placebo, for 5 years) after SOC treatment prevents recurrence and improves survival in patients lacking clinically evident metastases at the conclusion of SOC treatment.

It is important to note that COX-1 produces both TxA2 and gut-protecting prostaglandin E2

(PGE2). As such, aspirin may have dangerous consequences for some patients. It is exciting to speculate TPr inhibition could provide the anti-metastatic benefits of COX1 pathway inhibition, but without the potentially harmful side effects in aspirin-sensitive patients. However, given the potent antagonism of platelet coagulation by CPI211, studies going forward should proceed with awareness of bleeding or other potential adverse events that may arise in CPI211-treated patients.

Since TPr inhibition diminished metastasis without impacting tumor growth, it is anticipated that

TPr inhibition could be effective in combination with SOC cytotoxic therapies in the adjuvant or neoadjuvant setting. Data presented herein suggest that TPr inhibitors could provide greatest benefit as a preventive treatment, a clinical course of action not frequently used. However, this approach is being tested in at least one clinical trial (NCT03694249), in which patients diagnosed with aggressive cancers with a high risk of recurrence (e.g., gastric cancer; triple

Downloaded from mct.aacrjournals.org on September 26, 2021. © 2020 American Association for Cancer Research. Author Manuscript Published OnlineFirst on October 8, 2020; DOI: 10.1158/1535-7163.MCT-19-1106 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. negative breast cancer and others) but lacking clinically identifiable metastases will be randomized post-surgically into treatment groups receiving CPI211 or placebo for 12 months, and followed for an additional 12 months. This landmark clinical trial will test the feasibility of a pharmaco-preventive strategy that may diminish metastatic burden in certain cancer patients.

The notion that TPr inhibitors will not eliminate existing metastases must be considered.

However, TPr inhibition may reduce spread of tumor cells from secondary to tertiary tumor sites.

Timing TPr inhibitor administration requires additional study, as does testing of other TPr inhibitors in clinical development (terutroban, ridogrel, terbogrel, and others) (44). Further, it will be essential to identify which cancer types are responsive to TPr inhibition, necessitating a greater understanding of how different cancers use different metastatic routes.

In summary, we introduce TPr as an anti-metastatic target across many types of cancers.

Findings herein support the continued investigation of agents interfering with steps of the metastatic cascade, and support the use of PheWAS analysis to accelerate the pace of drug repurposing.

Acknowledgments. We kindly thank Cumberland Pharmaceuticals, Inc and Dr. Ines Macias-

Perez for providing CPI211. We acknowledge Drs. Ingrid Meier and Justin Balko for thoughtful discussion. We acknowledge Vanderbilt Shared Resources: the VICC Breast SPORE Pathology

(Dr. Melinda Sanders), Translational Pathology, Digital Histology, and VANTAGE shared resources. This work was supported by grants NIH P50 CA098131, NIH P30 CA68485, National

Center for Advancing Translational Sciences CTSA UL1TR000445, and Congressionally

Directed Medical Research Program W81XWH-161-0063.

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PheWAS Condition Code P value Odds Ratio Case Carriers Total Cases Controls Chronic venous hypertension (validation phenotype) 453 9.1E-06 31.89 2 15 22,276 Pulmonary heart disease 415 0.003 2.11 18 1512 24,713 Primary pulmonary hypertension 415.21 0.01 3.28 5 261 24,713 Secondary malignant neoplasm 198 3.8E-03 1.95 32 3568 20,663 Secondary malignancy of respiratory organs 198.2 6.5E-03 2.42 13 1190 20,663 Secondary malignancy of lymph nodes 198.1 7.0E-03 2.20 17 1698 20,663 Secondary malignant neoplasm of digestive system 198.3 1.2E-02 2.85 7 520 20,663 Secondary malignancy of brain/spine 198.5 2.1E-02 2.68 7 587 20,663 Table 1. PheWAS of 29,722 patients of European ancestry in BioVU genotyped using the Illumina Infinium Exomechip and phenotyped for approximately 1660 ICD9 codes show expected associations of rs200445019 with hypertension, pulmonary hypertension, and pulmonary heart disease. Novel associates of rs200445019 with secondary malignancy (i.e., metastasis) was identified. A case was defined as an EHR with two or more ICD9 codes mapping to one of the phenotypes used for this analysis. A case carrier is a defined as a case testing positive for rs200445019. Patients with records lacking the identified ICD9 codes were categorized as controls. The PheWAS algorithm was applied to calculate case and control genotype distribution, the χ2 distribution, associated allelic P-value and allelic odds ratio (OR). For those χ2 distributions in which observed cell counts fell below five, Fisher's exact test was used to calculate the P-value using the R statistical package (http://www.r-project.org/). Only phenotypes occurring in a minimum of 25 cases (0.42% of genotyped patients) were included in the analysis.

Figure Legends.

Figure 1. PheWAS and expression analyses correlate TBXA2R with metastasis and decreased progression-free survival.

A-B. Western analysis of serum starved cells treated 5 minutes with U46619 (0.5 µM) using the antibodies shown to the left of each panel. U46619-treated cells were washed after 5 minutes, then cultured up to 60 minutes with serum-free media (B).

C. Distribution of cancer types producing secondary malignancies in PheWAS of >29,000 patients.

D. Kaplan-Meier analysis of METABRIC-curated breast cancers assessing the relationship between TBXA2R and patient OS using cBio software (left panel). Cutoff between low/high: 2

S.D. above the average TBXA2R. Kaplan-Meier meta-analysis of NSCLC (center panel) and gastric cancer (right panel) gene expression array data assessing the relationship between PFS and TBXA2R using Kmplot software. Cutoff between low/high: median TBXA2R expression.

E. Western analysis of cells treated with CPI211 (100 nM) and U46619 (0.5 µM).

F. Western analysis of washed human platelets treated ex vivo with CPI211 (0.1 nM) and

U46619 (0.5 µM).

G. Aggregometry of washed human platelets treated ex vivo with U46619 (0.5 µM) ± CPI211 (5 nM to 500 nM).

Figure 2. The TPr inhibitor CPI211 decreases metastasis in mouse metastasis models.

A. Western analysis of lysates collected from cells treated with U46619 (0.5 µM).

B. Schematic timeline used for treating mice with CPI211 (50 mg/kg) for 2 days prior to, through

28 days after, tail vein delivery of tumor cells.

C. Lungs were assessed histologically for metastases. Values shown are the number of lung lesions per mouse. Midlines are average, and error bars are S.D. P values, Student’s T-test.

D. Treatment schematic for 4T1-Luc tumor-bearing mice with CPI211 for 28 days. Primary tumors were resected on treatment day 12.

E. Left panel: Average lung bioluminescence was determined weekly by IVIS. Each bar shows average bioluminescence per group. Error bars = S.D. Center panel: Raw bioluminescence

(photons/second) for each mouse on treatment day 14 and day 28. Right panel: Values shown are number of lung metastases in each mouse on day 28. Midline represents the average, error bars represent S.D.

Figure 3. The TPr inhibitor CPI211 does not affect primary tumor growth but decreases tumor vessels. 4T1 mouse mammary tumors were grown in athymic mice until reaching 50 mm3. Mice were treated daily with CPI211 (50 mg/kg) for 12 days.

A. Tumor volume was measured on treatment day 12. Each data point is the tumor volume in a single mouse. Midlines = average tumor volume, error bars = S.D.

B. Tumor sections were stained with hematoxylin and eosin (H&E) or by IHC to detect Ki67.

C. The percentage of Ki67+ tumor cells was quantitated in 5 random fields per sample. Midline

= average of biological replicates.

D. 4T1 and MDA-MB-231 cells were treated with CPI211 and/or U46619 in serum-free media for 3 days. 10% serum was included as a positive control for growth. Value points represent the average of three technical replicates ± S.D, N = 3,

E. IHC to detect CD31+ vessels in 4T1 tumors. Inset represents the area shown in higher power.

F-G. Quantitation of total CD31+ vessel area (F) and number of CD31+ vessels with a lumen area > 50 µm2 (G). Data points = average value measured in three random fields per section, N

= 4. Midlines = average ± S.D.

Figure 4. The TPr inhibitor CPI211 increases barrier function of vascular endothelial cells.

A. Western analysis of MPMECs and HUVECs treated with CPI211 (100 nM) ± U46619 (500 nM).

B. Crystal violet staining and quantitation measuring growth of MPMECs treated with U46619

(500 nM) and CPI211 (100 nM).

C. Trans-endothelial tumor cell migration assay. ECs (CellTracker Red+) monolayers on the underside of Matrigel-coated transwells were treated 6 h with U46619 (500 nM) and CPI211

(100 nM) before adding GFP+ tumor cells to upper chambers. GFP+ cell migration to the lower transwell surface was visualized by fluorescence microscopy.

D. Representative images of trans-endothelial migration of GFP+ 4T1 and MDA-MB-231 across mouse MPMECs and human HUVECs, respectively.

E. Quantitation of GFP+ 4T1 (N = 8) and MDA-MB-231 (N = 4) cells migrating across an endothelial barrier treated with U46619 ± CPI211. Data points are average ± S.D.

F. Albumin leakage across an MPMEC monolayer grown on Matrigel-coated transwell, N = 3, each assessed in triplicate. Average ± S.D. is shown. Student’s T-test.

G. IF staining for VE-Cadherin in MPMECs grown on Matrigel-coated transwell filter and treated

6 hours with U46619.

H. Western analysis of whole cell lysates (left panel) or VE-Cadherin IP (right panel) from

MPMECs treated 6h with U46619 ± CPI211.

Figure 5. CPI211 diminishes spontaneous metastases.

A-B. 4T1 tumor-bearing wild-type (Balb/C) mice were treated 28 days with neoadjuvant CPI211

(50 mg/kg daily) or vehicle when tumors reached 50 mm3 (A). Representative low power images

(20X) of H&E-stained sections of lungs collected at day 28 (B, left panel). Lung metastases were enumerated (B, right panel). Datapoints are the number of metastases in each mouse.

Midlines are average ± S.D, N = 10.

C-D. 4T1 tumor bearing wild-type (Balb/C) mice were treated with adjuvant CPI211 (50 mg/kg daily) or vehicle beginning after surgical resection of 300 mm3 tumors (C). Representative low

Downloaded from mct.aacrjournals.org on September 26, 2021. © 2020 American Association for Cancer Research. Author Manuscript Published OnlineFirst on October 8, 2020; DOI: 10.1158/1535-7163.MCT-19-1106 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. power images (20X) of H&E-stained sections of lungs collected at day 28 are shown (D, left panel). The inset shows the area of increased magnification. Lung metastases were enumerated (D, right panel). Midlines are the average ± S.D, N = 8.

Repurposing of a thromboxane receptor inhibitor based on a novel role in metastasis identified by Phenome Wide Association Study

Thomas A Werfel, Donna J Hicks, Bushra Rahman, et al.

Mol Cancer Ther Published OnlineFirst October 8, 2020.

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