Asporin Restricts Mesenchymal Stromal Cell Differentiation, … · Asporin Restricts Mesenchymal Stromal Cell Differentiation, Alters the Tumor Microenvironment, and Drives Metastatic

Tumor Biology and Immunology

Asporin Restricts Mesenchymal Stromal CellDifferentiation, Alters the TumorMicroenvironment, and Drives MetastaticProgressionRobert M. Hughes1,2,3, Brian W. Simons1, Hamda Khan1,2,3, Rebecca Miller1,2,3,Valentina Kugler1,2,3, Samantha Torquato1,2,3, Debebe Theodros2,3, Michael C. Haffner2,3,4,Tamara Lotan2,3,4, Jessie Huang5, Elai Davicioni6, Steven S. An2,3,5,7, Ryan C. Riddle8,Daniel L.J. Thorek9, Isla P. Garraway10, Elana J. Fertig2,3, John T. Isaacs1,2,3,W. Nathaniel Brennen2,3, Ben H. Park2,3,7, and Paula J. Hurley1,2,3

Abstract

Tumor progression to metastasis is not cancer cell autono-mous, but rather involves the interplay of multiple cell typeswithin the tumormicroenvironment. Herewe identify asporin(ASPN) as a novel, secreted mesenchymal stromal cell (MSC)factor in the tumor microenvironment that regulates meta-static development. MSCs expressed high levels of ASPN,which decreased following lineage differentiation. ASPN lossimpaired MSC self-renewal and promoted terminal cell dif-ferentiation. Mechanistically, secreted ASPN bound to BMP-4and restricted BMP-4–induced MSC differentiation prior tolineage commitment. ASPN expression was distinctly con-served between MSC and cancer-associated fibroblasts (CAF).ASPN expression in the tumor microenvironment broadlyimpacted multiple cell types. Prostate tumor allografts in

ASPN-null mice had a reduced number of tumor-associatedMSCs, fewer cancer stem cells, decreased tumor vasculature,and an increased percentage of infiltratingCD8þT cells. ASPN-null mice also demonstrated a significant reduction in lungmetastases compared with wild-type mice. These data estab-lish a role for ASPN as a critical MSC factor that extensivelyaffects the tumor microenvironment and induces metastaticprogression.

Significance: Thesefindings show that asporin regulates keyproperties of mesenchymal stromal cells, including self-renewal andmultipotency, and asporin expression by reactivestromal cells alters the tumor microenvironment and pro-motes metastatic progression.

IntroductionTumors do not develop in isolation and components of the

tumor microenvironment demonstrably influence many cancerhallmarks including metastasis (1, 2). A growing body of evidencenow indicates that cells of mesenchymal origin including mesen-chymal stem cells, mesenchymal stromal cells, and fibroblasts,collectively referred to herein as MSCs, reside in the tumor micro-environment and contribute to cancer progression (2–5).MSCs area heterogeneous population of multipotent stromal cells that giverise to physiologic cell lineages including osteoblasts, adipocytes,and chondrocytes (6). MSCs also give rise in part to pathologic celllineages including cancer-associated fibroblasts (CAF) and fibro-blasts associatedwith injury or inflammation (2, 3, 7, 8). Similar toMSCs, CAFs are highly heterogeneous and dynamic, which mayaccount for the reported pleiotropic roles of MSCs and CAFs incancer progression as either potentiators of primary andmetastaticcancer development (4, 5, 9–14) or restrainers of cancergrowth (15–17). The seminal properties of MSCs in the tumormicroenvironment have yet to be fully established. Because of theconservation between gene expression programs in developmentand cancer (18, 19), key factors may be similar between MSCs indevelopment and MSCs in the tumor microenvironment.

Asporin (ASPN), a member of the small leucine-rich proteo-glycan (SLRP) family of extracellular proteins, is markedly

1The James Buchanan Brady Urological Institute, Department of Urology,Johns Hopkins School of Medicine, Baltimore, Maryland. 2The Department ofOncology, Johns Hopkins School of Medicine, Baltimore, Maryland. 3TheSidney Kimmel Comprehensive Cancer Center, Johns Hopkins School ofMedicine, Baltimore, Maryland. 4The Department of Pathology, JohnsHopkins School of Medicine, Baltimore, Maryland. 5The Department ofEnvironmental Health and Engineering, Johns Hopkins Bloomberg Schoolof Public Health, Baltimore, Maryland. 6Genome Dx Biosciences, Inc.,Vancouver, British Columbia, Canada. 7The Whiting School of Engineering,Department of Chemical and Biomolecular Engineering, Johns HopkinsUniversity, Baltimore, Maryland. 8The Department of Orthopedic Surgery,Johns Hopkins School of Medicine, Baltimore, Maryland. 9The Department ofRadiology, Johns Hopkins School of Medicine, Baltimore, Maryland. 10TheDepartment of Urology, David Geffen School of Medicine at UCLA, LosAngeles, California.

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

Corresponding Author: Paula J. Hurley, Johns Hopkins University, CRBII 154,1550 Orleans Street, Baltimore, MD 21231. Phone: 410-614-9453; Fax: 410-502-5742; E-mail: [email protected]

Cancer Res 2019;79:3636–50

doi: 10.1158/0008-5472.CAN-18-2931

�2019 American Association for Cancer Research.

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enriched in fetal prostate mesenchyme during develop-ment (18, 20). ASPN is similarly expressed in reactive stromalcells, including CAFs, in the tumor microenvironment ofprostate (20–22), breast (23), scirrhous gastric (24), and pancre-atic (25) cancers. The role of ASPN in prostate MSCs and in thetumor microenvironment is poorly understood. Intriguingly,ASPN has been reported to regulate mediators of MSC differen-tiation including BMP-2 (26) and TGFb1 (27). Yet, the roles ofASPN in MSC self-renewal and multipotency have not beendefinitively determined.

ASPN has been shown to induce migration of scirrhous gas-tric (24, 28) and pancreatic (25) cancer cells; nevertheless, ASPNexpression in the tumor microenvironment may have a dual rolein regulating cancer progression (21–25, 29), possibly due topolymorphisms in the ASPN aspartic acid (D)-repeat domainlength (21). ASPN expression in prostate reactive stromal cells isassociated with adjacent local cancer aggressiveness and withworse oncologic outcomes (21, 22). However, two, of the over10 reported, germline polymorphisms in the ASPN D-repeatdomain length have been shown to be differentially associatedwith the development of metastatic prostate cancer followingsurgery (21).ASPNwith 14D-repeats (ASPND14)was associatedwith an increased risk while ASPN D13 was associated with areduced risk of metastatic progression (21).

Herein, we report that ASPN functions as a novel, secretedMSCfactor and a key driver of metastatic development. We establish arole for ASPN in regulating fundamental properties of MSCsincluding self-renewal, differentiation, and migration. We dem-onstrate that ASPN expression is highly enriched in MSCs, and itsexpression decreases during differentiation to connective tissuelineages. Our data show that ASPN regulates MSC self-renewaland restricts MSC differentiation through regulation of BMP-4signaling. ASPN-null mice have fewer MSCs in the bone marrowand an enriched population of intermediate (or more differen-tiated) MSCs in the prostate. While most MSC-derived progenyhave decreased ASPN expression, high ASPN expression is con-served between MSCs and CAFs in both primary and metastatictumors. Prostate allograft tumors in ASPN null-mice have analtered tumor microenvironment with fewer tumor-associatedMSCs, decreased vasculature, and an increased percentage ofinfiltrating CD8þ T cells. Tumors in ASPN-null mice also havea reduced number of cancer stem cells and a marked decrease inmetastatic potential. These findings suggest that ASPN is animportant regulator of MSC multipotency and metastaticdevelopment.

Materials and MethodsIHC (30), immunofluorescence (30), immunoblotting (21),

RNA isolation and quantitative real-time PCR (30), colony-forming unit assay (31), cell proliferation (32), migration (32),cytoskeletal remodeling (32), MSC isolation and differentia-tion (5, 30, 32–36), the PELICAN study (37), and the CP1 E. colimodel of prostate inflammation (38) have been previouslydescribed and are detailed in the Supplementary Materials andMethods.

Cell lines and cell culturePC-3, DU-145, WPMY-1, TRAMP-C2, and HEK293T cell lines

were obtained from the ATCC. The B6MycCaP cancer cell line wasa kind gift fromDr. LeighEllis (Roswell ParkCancer Institute,New

York, NY). All cell lines were maintained in either DMEM (DU-145, WPMY-1, TRAMP-C2, B6MycCaP) or RPMI1640 (PC-3)supplemented with 10% FBS (Corning), and penicillin/strepto-mycin (Life Technologies). After thawing from frozen stock, celllines were used prior to passage 7. The WPMY-1-ASPN variant–expressing cell lines were generated and cultured as describedpreviously (21). Human MSCs were isolated from tissue andcultured in RoosterNourish-MSC (RoosterBio) as described pre-viously (5, 39). Mouse MSCs were cultured in DMEM supple-mented with 10% FBS (Corning), Glutamax (Life Technologies)and penicillin/streptomycin (Life Technologies). B6CaP orga-noids were generated from C57BL/6J Hi-Myc allografts and cul-tured using an adapted protocol from prior reports (40, 41).Briefly, B6CaP allograft tumors were finely minced with a scalpel,digested in DMEM/F12 þ 10% FBS þ 1:10 dilution of collage-nase/hyaluronidase for 1 hour at 37�C, triturated in prewarmed1� PBS þ DNAse I, and filtered through a 40-mm cell strainer.Cells were embedded in growth factor–reduced (GFR) Matrigel,plated on ultra-low attachment plates (Corning), and cultured inadvanced DMEM/F12 supplemented with 10% charcoal-strippedFBS, B-27, GlutaMAX, HEPES, and penicillin/streptomycin,recombinant mouse EGF (10 ng/mL), TGFb inhibitor A83-01(200 nmol/L), ROCK inhibitor Y-27632 (10 mmol/L), and DHT(100 nmol/L). For harvest and passage, Matrigel-embedded orga-noids were incubated in prewarmed Dispase (5 U/mL) andsubsequently trypsinized for single-cell isolation. Cell lines wereauthenticated by short tandem repeat analysis and confirmedMycoplasma free by PCR testing [Johns Hopkins University (JHU)Genetic Resources Core Facility].

Prostate cancer and inflammation studyTissue from radical prostatectomies performed at Johns Hop-

kins School ofMedicine (Baltimore,MD) from2009 to 2011wereexamined for ASPN expression in cancer-adjacent stroma and ininflammation-adjacent stroma. Four-micron–thick radical pros-tatectomy sections were stained for ASPN (Sigma) by IHC. Caseswere scored by a urologic pathologist for ASPN expression instroma adjacent to cancer and in distinct areas of stroma adjacentto chronic inflammation. Chronic inflammation was defined byclusters of 20 or more lymphocytes. Of the 15 cases selected, 13cases contained both cancer and distinct areas of chronic inflam-mation. Using established scoring schemes (21), ASPN intensitywas evaluated and assigned an incremental score of 0 (negative), 1(weak), 2 (moderate), or 3 (strong). The extent of staining wasassigned a percentage from 0% to 100%. An ASPN score wascalculated by multiplying the intensity score and the extent score(H-score).

Aspn�/� miceAll animal procedures were performed under a JHU-approved

Institutional Animal Care and Use Committee protocol. Micewere purchased from The Jackson Laboratory and maintainedunder standard pathogen-free conditions. B6;129S5-Aspntm1Lex/Mmucd were generated through the Mutant Mouse RegionalResource Center (MMRRC) at University of California Davis(Davis, CA), a NIH-funded strain repository. B6;129S5-Aspntm1Lex

were donated to MMRRC by Genentech, Inc. Exon 2 was targeted(NCBI accession AF316825.1) by homologous recombination.The embryonic stem cell (ESC) line was derived from 129S5/SvEvBrd from Lexicon Genetics. B6;129S5-Aspntm1Lex mice werethen fully backcrossed at least nine generations to the C57BL/6J

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background. Mice were genotyped by PCR of genomic DNA withwild-type and mutation-specific primers.

B6CaP model of metastasisThe B6CaP allograft model was derived from a C57BL/6J Hi-

Mycmousemetastatic lesion.Cells from themetastatic tissuewereisolated and serially passaged in C57BL/6J mice. When injectedsubcutaneously with B6CaP allograft cells, 60%–70% of C57BL/6J mice developed lungmetastases. For this study, we injected 5.5� 106 B6CaP cells in 100 mL PBS subcutaneously into Aspnþ/þ,Aspnþ/�, and Aspn�/� mice (n ¼ 6 mice per group), resectedtumors once they reached 1 cm3, waited 11 weeks postresection,euthanized the mice, and inspected the lungs for metastases.Lungs were formalin-fixed, paraffin-embedded, sectioned, andstained as described previously (42).

Flow cytometryTo assess the in vivoMSC population in adult mouse prostates

or B6CaP tumors by flow cytometry, the tissue of interestwas isolated, mechanically dissociated, digested in RPMI þ10% FBSþ 1:10 dilution of collagenase/hyaluronidase for 1 hourat 37�C, triturated in prewarmed PBS þ DNAse I, and filteredthrough a 40-mm cell strainer. If necessary, red blood cells werelysed by briefly incubating the cell pellet in ice-cold ACK lysisbuffer (Quality Biological). Cells were Fc-blocked (Miltenyi Bio-tec) for 10minutes in4�C.Dead cellswere discriminatedusing theLIVE/DEAD Fixable Aqua Dead Cell Stain Kit (Thermo FisherScientific) and samples were stained with the following antibo-dies from BioLegend: CD45, CD29, CD105, Sca-1, Ly6G, Ly6C,CD11b, CD11c, CCR2, F4/80, MHC II, CD3, CD4, CD8a, FoxP3,CD44, CD62L, as well as CD3 from BD Pharmingen. Stainedsamples were analyzed on an LSR II flow cytometer (BD Bios-ciences) and quantified using FlowJo.

Coimmunoprecipitation assaysHEK293T cells were transfected with a murine ASPN-Myc-

FLAG tagged expression vector (Origene) or vehicle control usingLipofectamine 3000 (Thermo Fisher Scientific) according to themanufacturer's protocol. Two days posttransfection, cells werewashed with PBS and incubated in serum-free DMEM overnight.HEK293T cells were then treated with 0.3 mg/mL recombinantmurineBMP-4protein (R&DSystems)or vehicle and incubated in5% CO2 at 37�C for 30 minutes. After BMP-4 incubation, con-ditioned media was filtered through a 0.45-mm filtration unit(Millipore) andone thirdwas set aside for the "input" sample. Theremaining two thirds was treated with a protease inhibitor cock-tail (Sigma), precleared with mouse IgG agarose (Sigma) for1 hour rotating at 4�C, and then incubated with anti-FLAG M2magnetic beads (Sigma) overnight rotating at 4�C. Beads werewashed three times with ice-cold TBS and proteins were elutedfrom the beads with 3XFLAG peptide (Sigma). Input conditionedmedia was concentrated using an AmiconUltra-4 centrifugal filterunit with a 10 kDa NMWL (Millipore) according to the manu-facturer's protocol.

Gene expression analysisGene expression profiling was performed for Aspnþ/þ and

Aspn�/� fetal MSCs expanded to passage 3 and cultured undernormal growth conditions to 70%–80% confluency prior to RNAisolation. Three biological replicates were analyzed per experi-mental group and Affymetrix mouse Clariom D arrays were run.Bioinformatics analyses were performed in R. Datasets were

normalized at a transcript level for core transcripts with the R/Bioconductor package oligo and annotated with the NetAffxBiological Annotations. Artifacts from technical batches wereremoved with ComBat (43) implemented in the R/Bioconductorpackage SVA (44). Differential expression analyses were per-formed on ComBat adjusted data using the R/Bioconductorpackage LIMMA (45). Genes with FDR-adjusted P values below0.05 and absolute log fold change greater than or equal to twowere called statistically significant. Gene set analysis was per-formed on the Kyoto Encyclopedia of Genes and Genomes(KEGG) cell-cycle pathway and the GeneMAPP TGFb familysignaling pathway obtained from the NetAffx Biological Anno-tations to confirm hypothesized functional mechanisms with aone-sided Wilcoxon gene set test implemented in LIMMA.

Quantification and statistical analysisUnless otherwise indicated, statistical comparisons between

two groups were performed using a two-tailed unpaired Student ttest. Statistical comparisons between multiple groups were per-formed using one-way ANOVA with Tukey or Newman–Keulsmultiple comparisons, as indicated in the figure legends. Statis-tical significance was defined as a P < 0.05, and P values wereindicatedwith asterisks in thefigures as follows: �,P�0.05; ��,P�0.01; ��, P � 0.001; ��, P � 0.0001. All statistical comparisonswere performed using GraphPad Prism software (v5.0).

ResultsASPN is highly expressed in MSCs

ASPN expression was examined in primary human MSCsisolated from fetal prostate, benign prostate, prostate cancer, andbone marrow. Primary heterogeneous cultures were expandedand shown to be enriched for MSCs by flow cytometry expressionprofile (CD73þ, CD90þ, CD105þ, CD14�, CD20�, CD34�,CD45�, and HLA-DR�) and by multi-lineage (osteoblast, adipo-cyte, and chondrocyte) differentiation potential (5). Comparedwith human primary benign prostate epithelial cells (PreC) and ahuman benign prostate epithelial cell line (RWPE-1), MSCs fromall sites had significantly elevated ASPN expression (Fig. 1A).Similar to human MSCs (20), Aspn mRNA and protein weredistinctly expressed in urogenital sinus mesenchyme (UGM)compared with urogenital sinus epithelium (UGE) of the devel-oping mouse prostate (Fig. 1B and C). MSCs were isolated fromboth fetal and adult mouse prostate and adult mouse bonemarrow, expanded in culture, and then examined for Aspn expres-sion. Mouse MSCs showed increased Aspn expression comparedwith UGE (Fig. 1B). In contrast to MSCs and fetal prostatemesenchyme, ASPN expression was markedly reduced in adultmouse benign prostate stromal cells as determined by IHC(Fig. 1C). These data suggest that ASPN is highly expressed inboth bone marrow–derived and tissue-resident human andmouse MSCs. However, the role of ASPN in regulating keyproperties of MSCs such as self-renewal, differentiation, andmigration has not been fully established.

ASPN regulates MSC self-renewalTo determine the role of ASPN in MSCs, we generated Aspn�/-

mice (B6J-Aspntm1Lex/Mmucd), which were then fully backcrossedat least nine generations to the C57BL/6J background. The firstcoding exon of Aspn, exon 2, was targeted for deletion by homol-ogous recombination (ref. 46; Supplementary Fig. S1A and S1B),

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Figure 1.

ASPN is expressed in MSCs and regulates MSC self-renewal.A, Relative ASPN expression in human primary prostate epithelial cells (PrEC), a benign humanprostate epithelial cell line (RWPE-1), a human prostate stromal cell line (WPMY-1), and primary human MSCs as measured by qRT-PCR. Statistical analysesperformed usingWelch t test (n� 3). B, Relative Aspn expression in mouse fetal prostate mesenchyme (UGM), mouse fetal prostate epithelium (UGE), andmouse MSCs as measured by qRT-PCR. Statistical analyses performed using one-way ANOVAwith Tukeymultiple comparison (n� 3). C, ASPN expression asmeasured by IHC in the urogenital sinus (UGS) and in adult mouse prostate. D and E, Aspnþ/þ and Aspn�/� bone marrow (D) and prostates (E) were analyzed byflow cytometry for MSCs (CD45�, CD105þ, CD29þ, and Sca-1þ). Statistical analyses performed using Student t test (n� 7). F–H, Aspnþ/þ and Aspn�/� adult bonemarrow–derived MSCswere isolated and then plated at equal densities for CFU assays. Displayed are the average number of colonies formed per 2.5� 103 cellsplated. Statistical analyses performed using Student t test (n�6). I–K, Aspnþ/þ and Aspn�/� adult prostate MSCs were isolated and then plated at equal densitiesfor CFU assays. Statistical analyses performed using Student t test (n� 3). L–N, Aspnþ/þ and Aspn�/� fetal MSCs were isolated and then plated at equal densitiesfor CFU assays. Statistical analyses performed using Student t test (n� 3). Graphs shown as mean� SEM. � , P� 0.05; �� P� 0.01; �� , P� 0.001; NS, notsignificant. Black bars, 100 mm.






which resulted in the loss of full-lengthAspn transcript andprotein(Supplementary Fig. S1C and S1D). Aspn�/- mice were viable,fertile, had comparable prostate size and histology, and hadsimilar expression of other SLRP family members includingDecorin (Dcn), Biglycan (Bgn), and Extracellular matrix protein 2(Ecm2) as Aspnþ/þ mice (Supplementary Fig. S1E–S1K).

To determine how ASPN regulates MSCs in vivo, we comparedbone marrow–derived MSCs from Aspnþ/þ and Aspn�/� malemice at 24 weeks of age by flow cytometry for MSC markers(CD45�, CD105þ, CD29þ, and Sca-1þ). By these markers, bothAspnþ/þ and Aspn�/� mice had a single population of bonemarrow–derived MSCs; however, Aspn�/� bone marrow hadsignificantly fewer MSCs than bone marrow from Aspnþ/þ mice(Fig. 1D; Supplementary Fig. S2A and S2B). We next comparedprostateMSCs fromAspnþ/þ andAspn�/�malemice at 24weeks ofage. In contrast to the singleMSCpopulation detected in the bonemarrow, both Aspnþ/þ and Aspn�/� prostates had two distinctpopulations of MSCs that were delineated by the level of Sca-1expression (Sca-1hi vs. Sca-1med; Supplementary Fig. S2C andS2D). Prior studies have shown that MSC stem progenitors withthe capacity for both self-renewal and differentiation are Sca-1hi,while more differentiated intermediate MSC progenitors withproliferative and differentiation capacity, but with reduced self-renewal ability, are Sca-1med (47). Interestingly, Aspn�/� prostateswere slightly enriched for Sca-1med

–intermediateMSCs comparedwith Aspnþ/þ prostates (Fig. 1E; Supplementary Fig. S2C andS2D), suggesting that in the absence of ASPN, prostate-derivedMSCs are more likely to differentiate and may thereby have adecreased potential for self-renewal.

To examine MSC self-renewal in vitro, total MSCs were isolatedfrom Aspnþ/þ and Aspn�/� bone marrow, compact bone, andprostates at 24 weeks of age and then plated at equal numbersin vitro for colony-forming unit (CFU) assays. Compared withAspnþ/þ, Aspn�/� bone marrow-, compact bone-, and prostate-derived MSCs formed significantly fewer CFUs (Fig. 1F–K; Sup-plementary Fig. S3A and S3B). To determine further the role ofASPN in self-renewal, fetal MSCs were isolated from Aspnþ/þ andAspn�/�mice and examined at early (P2) and late (P7) passage forCFU ability. CFU number was comparable between early passageAspn�/� and Aspnþ/þ fetal MSCs; however, late passage Aspn�/�

fetalMSCs formed significantly fewer colonies comparedwith latepassage Aspnþ/þ fetal MSCs (Fig. 1L–N; Supplementary Fig. S3Cand S3D). When added to late passage Aspn�/� fetal MSCs,recombinant mouse ASPN did not increase CFU number. Col-lectively, these data suggest that ASPN is necessary to sustain asubpopulation ofMSCswith self-renewal capacity, but exogenousASPN is not able to rescue self-renewal once MSCs have lost thiscapacity.

ASPN restricts MSC proliferation and differentiationThe loss of bone marrow MSCs in Aspn�/� mice suggests that

ASPN may regulate MSC differentiation. Consistent with amore quiescent phenotype, Aspnþ/þ fetal MSCs had a signifi-cantly decreased proliferation rate compared with Aspn�/� fetalMSCs (Fig. 2A). Microarray analyses of gene expression changesbetween Aspnþ/þ and Aspn�/� fetal MSCs demonstrated thatgenes annotated to the cell cycle by KEGG were significantlydownregulated in Aspnþ/þ compared with Aspn�/� fetal MSCs(Fig. 2B). Consistent with a more proliferative phenotype,Aspn�/� fetal MSCs had elevated expression of several DNAsynthesis–regulatory genes including Cdc45, Mcm2, Mcm3,

Mcm5, and Mcm6. In contrast, Aspnþ/þ fetal MSCs showedincreased expression of genes that restrict cell-cycle progressionincluding Cdkn2a. Interestingly, two genes with reported germ-line SNPs associated with aggressive prostate cancer, Cdkn1aand Cdkn1b (48), were differentially elevated between Aspnþ/þ

and Aspn�/� MSCs.To more clearly define the role of ASPN in regulating MSC

differentiation, early passage (P3) fetal MSCs from Aspnþ/þ andAspn�/� mice were cultured in osteogenic, adipogenic, andchondrogenic differentiation–inducing media and then ana-lyzed for in vitro lineage differentiation. Differentiation to allthree lineages (osteoblast, adipocyte, and chondrocyte) wassignificantly greater in Aspn�/� fetal MSCs compared withAspnþ/þ fetal MSCs as measured by Alizarin Red, Oil Red O,and Alcian Blue staining, respectively (Fig. 2C–H). In addition,lineage-specific differentiation markers such as Runx2, Adipoq,and Col10a1 were significantly higher in Aspn�/� fetal MSCscompared with Aspnþ/þ fetal MSCs when cultured in osteogen-ic, adipogenic, and chondrogenic differentiation-inducingmedia, respectively (Fig. 2I–K). Compared with control media,Aspn expression in Aspnþ/þ fetal MSCs significantly decreased indifferentiation-inducing media, indicating that Aspn is down-regulated during differentiation (Fig. 2L–N). Consistent withthese findings, recombinant mouse ASPN decreased osteogenicdifferentiation of Aspn�/� fetal MSCs (Supplementary Fig. S3Eand S3F). Interestingly, late passage (P8) Aspn�/� fetal MSCsshowed increased differentiation in the absence of differenti-ation-inducing media (Supplementary Fig. S3G–S3K). Collec-tively, these data suggest that ASPN restricts MSC proliferationand inhibits early MSC differentiation prior to lineagespecification.

ASPN binds to BMP-4 and restricts BMP-4–induced signalingand differentiation

How ASPN regulates MSC differentiation is not fully known.ASPN has been reported to regulate many extracellular signalingmolecules (27, 28, 49–52), including multiple TGFb familymembers (26, 27, 49, 51–53). Most TGFb family members havepleiotropic and lineage-specific roles during differentiation; how-ever, BMP-4 distinctly induces progenitor differentiation to oste-oblast, adipocyte, and chondrocyte lineages (54). Microarrayanalysis of gene expression changes between Aspnþ/þ andAspn�/� fetal mouse MSCs demonstrated that TGFb family sig-naling pathway genes annotated in GeneMAPP, including genesrestricted to BMP signaling, were significantly downregulated inAspnþ/þ compared with Aspn�/� fetal MSCs (Fig. 3A). Consistentwith this, BMP-4–induced SMAD1/5/9 phosphorylation wassignificantly decreased in Aspnþ/þ compared with Aspn�/� fetalmouse MSCs (Fig. 3B and C). Similarly, recombinant mouseASPN also restricted BMP-4–induced signaling in Aspn�/� fetalmouse MSCs (Fig. 3D and E).

Prior studies have shown that polymorphisms in the asparticacid (D)-repeat length of human ASPN differentially regulatesignaling (27) and that germline D-repeat variants are differen-tially associated with prostate oncologic outcomes (21). Thegermline variant of human ASPN containing 14-D repeats (ASPND14) was shown to be significantly associated with metastaticprostate cancer recurrence after surgery while homozygosity forthe ASPN D13 variant was significantly associated with a reducedrisk of metastatic recurrence (21). To determine whether ASPND14 and ASPN D13 differentially regulate BMP-4 signaling in

Hughes et al.





human prostate stromal cells, we generated independent clones(A and B) of the prostate fibroblast cell line, WPMY-1, over-expressing either ASPN D13 or ASPN D14. Independent clonesof WPMY-1 expressing the neomycin empty vector alone (Neo)were also generated as control lines. WPMY-1, which are germlineASPN D13/13, did not express ASPN under standard conditions(Fig. 3F; ref. 21). Similar to mouse ASPN, ASPND14 significantlyrestricted BMP-4–induced signaling in WPMY-1 cells comparedwith WPMY-1-Neo and WPMY-1-ASPN D13 (Fig. 3F and G).BMP-4–induced signaling was slightly decreased in WPMY-1-ASPN D13 compared with WPMY-1-Neo, but was not significant(Fig. 3F and G).

To determine whether ASPN directly binds to BMP-4, mouseASPN was overexpressed in HEK293T cells and then incubatedwith recombinant mouse BMP-4. Immunoprecipitation ofsecreted ASPN from the media demonstrated binding toBMP-4 (Fig. 3H). To determine whether BMP-induced signalingis necessary for differentiation in the absence of ASPN, weinterrogated the ability of a BMP inhibitor to rescue differen-tiation in Aspn�/� MSCs. To do this, we compared osteogenic-induced differentiation in Aspnþ/þ and Aspn�/� fetal MSCs inthe absence of BMP-induced signaling by treating MSCs withthe BMP inhibitor LDN-193189 or vehicle. Inhibition of BMP-induced signaling significantly restricted osteogenic-induceddifferentiation in Aspn�/� fetal MSCs as assayed by AlizarinRed staining and by expression of osteogenic-induced genes

(Fig. 3I and J). To determine whether BMP-4 could rescuedifferentiation in Aspnþ/þ fetal MSCs, we compared osteogen-ic-induced differentiation in Aspnþ/þ and Aspn�/� fetal MSCs inthe presence of exogenous recombinant mouse BMP-4. BMP-4strongly induced differentiation in Aspnþ/þ fetal MSCs (Fig. 3Iand J). Taken together, these findings support a model by whichmouse ASPN and human ASPN D14 restrict BMP-4–inducedsignaling and differentiation of MSCs.

ASPN expression pattern is distinctly conserved between MSCsand reactive stromal cells in the tumor microenvironment

MSCs also give rise, in part, to pathologic cell lineagesincluding CAFs and injury or inflammation-associated fibro-blasts (2, 3, 7, 8, 39, 47, 55, 56). We and others have previouslyreported that ASPN is highly expressed by reactive stromal cellsin the microenvironment of primary tumors, with minimal toundetectable expression by primary adenocarcinoma cells oradenocarcinoma-derived cell lines (refs. 21–25; Fig. 4A and B;Supplementary Fig. S4A). In fact, ASPN expression in cancer celllines negatively correlated with the epithelial marker EPCAM(Pearson r: �0.2478; P < 0.0001) and positively correlated withMSC markers including THY1 (Pearson r: 0.4064; P < 0.0001),ENG (Pearson r: 0.3607; P < 0.0001), VCAM (Pearson r: 0.1237;P < 0.0001), and ALCAM (Pearson r: 0.0952; P < 0.0019;Supplementary Fig. S4A–S4G; ref. 57). Elevated ASPN in pri-mary prostate cancer from the TCGA was significantly

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ASPN restricts MSC proliferation and differentiation. A, Cell growth of Aspnþ/þ and Aspn�/� fetal MSCs. B, Gene set enrichment analysis of cell-cyclegenes in Aspnþ/þ and Aspn�/� fetal MSCs as determined from microarray data (n ¼ 3). C–H, Aspnþ/þ and Aspn�/- fetal MSCs were cultured inosteogenic (C and D), adipogenic (E and F), and chondrogenic differentiation-inducing media (G and H) and then stained for Alizarin Red, Oil Red O,and Alcian Blue, respectively. Staining was quantified using ImageJ (n ¼ 3). Expression of osteogenic (I), adipogenic (J), and chondrogenicdifferentiation-induced (K) genes in Aspnþ/þ and Aspn�/� fetal MSCs as determined by qRT-PCR (n � 3). Aspn expression in Aspnþ/þ fetal MSCscultured in osteogenic (L), adipogenic (M), and chondrogenic differentiation-inducing (N) media (n ¼ 3). Statistical analyses in A and C–N performedusing Student t test (mean � SEM). � , P � 0.05; �� , P � 0.01; �� , P � 0.001; n � 3. Black bars, 100 mm.






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Figure 3.

ASPN binds to BMP-4 and restricts BMP-4–induced signaling.A, Gene set enrichment analysis of TGFb family pathway genes in Aspnþ/þ and Aspn�/� fetal MSCsas determined frommicroarray (n¼ 3). B and C, BMP-4–induced signaling in Aspnþ/þ and Aspn�/� fetal MSCs (A and B represent independently derived MSCs)as measured by immunoblotting and quantified using ImageJ. Aspnþ/þ and Aspn�/� fetal MSCs were serum-depleted and then incubated with recombinantmouse BMP-4 (5 ng/mL) for 30minutes. Statistical analyses performed using Student t test (n� 3). D and E, Recombinant mouse ASPN restricts BMP-4–inducedsignaling in Aspn�/� fetal MSCs as measured by immunoblotting and quantified using ImageJ. Aspnþ/þ and Aspn�/� fetal MSCs were serum-depleted and thenincubated with recombinant mouse BMP-4 (5 ng/mL) with vehicle or recombinant mouse ASPN (100 ng/mL) for 30 minutes. Statistical analyses performedusing Student t test (n� 2). F andG, BMP-4–induced signaling inWPMY-1-Neo, WPMY-1-ASPN D13, andWPMY-1ASPN D14 (A and B represent independentlyderived clones) as measured by immunoblotting and quantified using ImageJ. WPMY-1-Neo, WPMY-1-ASPN D13, andWPMY-1ASPN D14 were serum-depletedand then incubated with recombinant human BMP-4 (5 ng/mL) for 30minutes. Statistical analyses performed using one-way ANOVAwith Tukey multiplecomparison (n� 3). H, Coimmunoprecipitation of ASPN and BMP-4. FLAG-tagged mouse ASPN was transfected in HEK293T cells and then incubated withrecombinant mouse BMP-4. ASPNwas immunoprecipitated with anti-FLAG beads and then examined by immunoblotting for ASPN and BMP-4 (n¼ 2).I, Aspnþ/þ and Aspn�/� fetal MSCs were cultured in osteogenic-inducing media with either vehicle, 250 nmol/L LDN-193189, or recombinant mouse BMP4(5ng/mL) and then stained for Alizarin Red. J, Aspnþ/þ and Aspn�/� fetal MSCs were cultured in osteogenic-inducing media with either vehicle, 250 nmol/LLDN-193189, or recombinant mouse BMP4 (5 ng/mL) and then examined for osteogenic-induced genes by qRT-PCR. Statistical analyses performed usingone-way ANOVAwith Tukeymultiple comparison (n� 3). Graphs shown as mean� SEM. � , P� 0.05; �� , P� 0.01; �� , P� 0.001. Black bars, 100 mm.

Hughes et al.





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ASPN is expressed in primary andmetastatic prostate cancer tumor microenvironments, but not in the microenvironment of prostate inflammation. A,ASPNexpression in human prostate cancer as measured by IHC. B, ASPN (red), pancytokeratin (green), and DAPI (blue) expression in human prostate cancer asmeasured by immunofluorescence. C, Elevated ASPN (amplification, gain, expression >2) was associated with worse disease/progression-free survival in TCGAdata as determined by Kaplan–Meier survival curves and visualized on cBioPortal (n¼ 491; log-rank test). D–F, ASPN expression in mouse adult prostate (D),murine prostatic intraepithelial neoplasia (mPIN) in the TRAMPmodel (E), and mouse prostate adenocarcinoma (F) in the TRAMPmodel as measured by IHC andqRT-PCR (G).H and I,Mean ASPN H-score in prostate cancer metastases from the PELICAN rapid autopsy study of prostate cancer (n¼ 15 patients with anaverage of 4 metastases per patient), benign adjacent prostate stroma (n¼ 11), benign bone (n¼ 2), benign lymph node (n¼ 3), benign lung (n¼ 4), and benignliver (n¼ 4) as measured by IHC. Historical controls of stroma adjacent to benign prostate and stroma adjacent to Gleason grade� 6 prostate cancer (G� 6)were measured and calculated using the samemethodology and included for comparison. Statistical analyses performed using one-way ANOVAwithNewman–Keuls multiple comparison. J, ASPN expression in human prostate inflammation and human prostate cancer as measured by IHC on radicalprostatectomy sections that contained regions of both chronic inflammation and prostate cancer in nonoverlapping locations. K,Quantification of ASPNexpression by IHC (H-score) in stroma adjacent to inflammation and stroma adjacent to cancer on single sections from radical prostatectomy sections. Statisticalanalyses performed using Student t test (n¼ 13 patients). L–N, ASPN (red), vimentin (green), and DAPI (blue) expression in mouse adult prostate (L),CP1 E. coli–induced mouse prostate inflammation (M), and mouse prostate adenocarcinoma in the TRAMPmodel (N) as measured by immunofluorescence.Graphs shown asmean� SEM. � , P� 0.05; �� , P� 0.001. Black and white bars, 100 mm.






associated with a worse disease/progression-free survival(Fig. 4C; refs. 58, 59). Similar to human prostate cancer, bothAspn mRNA and protein expression were elevated in the tumormicroenvironment of mouse models of prostate adenocarci-noma compared with benign adjacent prostate stroma andstroma associated with mouse prostatic intraepithelial neopla-sia (mPIN; Fig. 4D–G; Supplementary Fig. S4H).

To determine whether ASPN is also expressed in the meta-static tumor microenvironment, prostate cancer metastasesfrom a Johns Hopkins School of Medicine rapid autopsy cohort(n ¼ 15 patients; 60 metastases; ref. 37) were analyzed forASPN expression by IHC and quantified by H-score. ElevatedASPN expression was detected in reactive stromal cells atvarious metastatic sites including lymph node, bone, and softtissue such as lung and liver (Fig. 4H; Supplementary Fig. S4I).ASPN expression in metastatic prostate cancer adjacent stromawas elevated compared with benign prostate, lymph node,lung, and liver stroma, and it was comparable with ASPNexpression in primary prostate cancer (Gleason sum �6) adja-cent stroma (Fig. 4I). Thus, we demonstrate that ASPN is highlyexpressed in MSCs isolated from multiple sites and in reactivestromal cells associated with both primary and metastaticprostate cancers.

To determine whether ASPN expression is selective to reac-tive stroma in the tumor microenvironment or is generallyconserved among reactive stroma, we next examined ASPNexpression in human and mouse reactive stroma associatedwith prostate inflammation. Human prostate sections obtainedafter radical prostatectomy for prostate cancer that containedregions of both benign chronic inflammation and prostatecancer, but in distinct locations (�2mm apart), were analyzedfor ASPN expression by IHC. Compared with reactive stromaassociated with human prostate cancer, stroma associated withdistinct areas of chronic prostate inflammation expressed sig-nificantly lower levels of ASPN that were comparable withbenign adjacent stroma (Fig. 4J and K). Similar to humanprostate inflammation, CP1 E. coli–induced prostate inflam-mation, which is characterized by a marked vimentin-positivestromal response (38), was largely negative for ASPN expres-sion (Fig. 4L–N). ASPN expression in inflammation adjacentstroma was comparable with benign prostate stroma and wasonly detected in perivascular regions. Collectively, these datasuggest that ASPN expression is selectively conserved betweenMSCs and cancer-associated reactive stromal cells.

ASPN induces MSC and cancer cell migrationA fundamental property of MSCs is their ability to migrate

to both local and distant areas of tissue injury or damage.Migration was significantly elevated in Aspnþ/þmouse fetal MSCscompared with Aspn�/� mouse fetal MSCs (Fig. 5A and B).Consistent with elevated motility, Aspnþ/þ fetal MSCs cells hadincreased cytoskeletal remodeling compared with Aspn�/� MSCsas assayed by spontaneousmotions of Arg-Gly-Asp (RGD)-coatedmicrobeads that bind to the cytoskeletal network through cellsurface integrin receptors (Fig. 5C and D). Similar dynamics ofincreased migration and cytoskeletal remodeling were alsoobserved in WPMY-1-ASPN D14–overexpressing cells comparedwithWPMY-1-ASPND13–overexpressing cells andWPMY-1-Neocontrol cells (Fig. 5E–H).

As a secreted protein in the tumor microenvironment, ASPNmay also induce cancer cell migration. We examined cancer cell

migration in response to conditioned media from Aspnþ/þ andAspn�/� fetal MSCs. Conditioned media from Aspnþ/þ fetalMSCs increased B6MycCaP (60) and TRAMP-C2 mouse pros-tate cancer cell migration compared with Aspn�/� fetal MSC-conditioned media (Fig. 5I and J; Supplementary Fig. S5Aand S5B). Conditioned media from WPMY-1-ASPN D14 cellsalso increased PC-3 and DU-145 migration compared withconditioned media from WPMY-1-ASPN D13 cells and condi-tioned media from WPMY-1- Neo cells (Fig. 5K and L; Sup-plementary Fig. S5C and S5D). Interestingly, a 1:1 mix ofconditioned media from WPMY-1-ASPN D14 and WPMY-1-ASPN D13 cells also increased PC-3 migration similar toconditioned media from WPMY-1-ASPN D14 alone, therebysuggesting a dominant function of ASPN D14 relative to ASPND13 (Fig. 5K and L).

ASPN-induced migration is calcium-dependentHowASPN regulates prostate stromal and cancer cellmigration

is not fully understood. Migration in Aspn�/� fetal MSCs wasneither rescued by the BMP inhibitor LDN-193189 nor furtherrestricted by BMP-4, suggesting that ASPN regulates migration byan alternative mechanism (Supplementary Fig. S5E). To deter-mine whether ASPN is able to directly enhance migration asopposed to functioning indirectly through the expression of othersecreted factors, migration was examined in Aspn�/� cells in thepresence of exogenous recombinant mouse ASPN. Elevatedmigration was observed in Aspn�/� cells when cultured withexogenous ASPN (Fig. 6A), suggesting that ASPN can, at least inpart, directly enhance migration. To better determine how ASPNregulates migration, Aspnþ/þ MSCs, Aspn�/� MSCs, WPMY-1-ASPN D14, WPMY-1-ASPN D13, and WPMY-1-ASPN Neo werecompared by microarray for gene expression. Differentiallyexpressed genes in Aspnþ/þ MSCs compared with Aspn�/� MSCsas well as in WPMY-1-ASPN D14 compared with WPMY-1-ASPNNeo and in WPMY-1-ASPN D13 compared with WPMY-1-ASPNNeo were examined for KEGG Pathway enrichment. Genes anno-tated to cell adhesion, actin cytoskeleton, and cytokine–cytokinereceptor interaction by KEGG were altered in cells expressingASPN (Aspnþ/þ MSCs, WPMY-1-ASPN D14, WPMY-1-ASPND13) compared with cells deficient for ASPN (Aspn�/� MSCs andWPMY-1-ASPN Neo; Supplementary Fig. S5F). Because mouseASPNandhumanASPND14 enhancedmigration comparedwithASPN-null and ASPN D13 cells, KEGG pathways selective toAspnþ/þ MSCs and WPMY-1-ASPN D14 were highlighted as ameans to determine pathways potentially involved in ASPN-mediated migration. Interestingly, genes annotated to calciumsignaling, Hedgehog signaling, chemokine signaling, WNT sig-naling, and TGFb signaling by KEGG were altered in Aspnþ/þ

MSCs and WPMY-1-ASPN D14 when compared with cells defi-cient for ASPN (Fig. 6B and C). ASPN has been reported to bindcalcium, likely through its poly-aspartate domain (61). Consis-tent with this, hierarchical clustering showed differential expres-sion of several Gene Ontology (GO) calcium-related genesbetween Aspnþ/þ and Aspn�/� fetal MSCs (Fig. 6D). Similarly,differential expression of calcium-related genes was also observedin WPMY-1-ASPN D14 compared with WPMY-1-ASPN D13 andWPMY-1-Neo (Fig. 6E). To determine whether ASPN-mediatedmigration depends on calcium, Aspn�/�MSCs were examined formigration in the presence of exogenous recombinant mouseASPN and an extracellular calcium chelator, BAPTA. BAPTArestricted ASPN-induced migration, suggesting that extracellular

Hughes et al.





calcium is necessary for ASPN's promigratory function (Fig. 6Fand G).

ASPN alters the primary tumor microenvironmentTo determine the role of ASPN in the tumor microenviron-

ment, we utilized a novel allograft generated from a C57BL/6JHi-Myc mouse metastatic lesion (B6CaP). This allograft is notto be confused with the B6MycCaP cell line used for migrationstudies that was generated on a C57BL/6N background (60).B6CaP allografts consisted of both cancer and tumor-infiltrating host cells including reactive stromal cells. Becauseof this stromal infiltration, B6CaP allografts highly expressedAspn (Supplementary Fig. S6A). B6CaP organoids, which wereenriched for cancer cells, had undetectable Aspn expressionfurther establishing that Aspn expression was restricted toinfiltrating stromal cells (Supplementary Fig. S6A). Subcutane-ous growth of B6CaP allografts was similar in Aspnþ/þ, Aspnþ/�,and Aspn�/� mice as average tumor volume at resection andaverage time to resection were not significantly differentbetween mice (Fig. 7A and B). Despite comparable Aspn expres-sion at tumor inoculation, B6CaP allografts in Aspn�/� mice

had significantly reduced levels of Aspn at resection (Supple-mentary Fig. S6B).

B6CaP allografts were examined for MSCs and CAF-mediatedproperties including cancer stem cells, immune infiltration, andvascular development. Consistent with our in vivo and in vitrofindings pertaining to the roles of ASPN in MSC self-renewal,differentiation, and migration, flow cytometry demonstratedthat B6CaP tumors in Aspn�/� mice had significantly fewerMSCs (CD45�, CD105þ, CD29þ, and Sca-1þ) compared withB6CaP tumors in Aspnþ/þ mice (Fig. 7C). B6CaP tumors inAspn�/� mice also had significantly fewer CD44þ cancer stemcells (CD45�, CD105�, CD31�, CD29þ, Sca-1þ, CD44þ) com-pared with B6CaP tumors in Aspnþ/þ mice (Fig. 7D). Flowcytometry for myeloid and lymphoid lineage cells demonstrat-ed that Aspn�/� tumors had an increased percentage of tumor-infiltrating CD8þ T cells compared with Aspnþ/þ cells (Fig. 7E;Supplementary Fig. S6C and S6D). IHC for tumor-associatedvasculature showed that Aspnþ/þ tumors were enriched forlarger blood vessels compared with Aspn�/� tumors (Fig. 7Fand G). These findings suggest that ASPN pleiotropically altersboth the tumor and the tumor microenvironment.

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Figure 5.

ASPN enhances MSC and cancer cell migration. A and B,Migration of Aspnþ/þ and Aspn�/� fetal MSCs across a membrane. Statistical analyses performed usingStudent t test (n¼ 3). C and D, Cytoskeletal remodeling as measured by mean square displacement (C) and analyzed at t¼ 300 s nm2 in Aspnþ/þ and Aspn�/�

fetal MSCs (D). Statistical analyses performed using one-way ANOVAwith Tukey multiple comparison (n� 3). E and F,Migration ofWPMY-1-Neo,WPMY-1-ASPN D13, andWPMY-1-ASPN D14 as determined by scratch assay. Statistical analyses performed using one-way ANOVAwith Tukeymultiple comparison (n�9). G and H, Cytoskeletal remodeling as measured by mean square displacement (G) and analyzed at t¼ 300 nm2 (H) inWPMY-1-Neo, WPMY-1-ASPN D13, andWPMY-1-ASPN D14 (n¼ 2 independent clones per experimental group). Statistical analyses performed using one-way ANOVAwith Tukeymultiple comparison(n� 3). I and J,Migration of B6MycCaP cells in conditioned media from Aspnþ/þ and Aspn�/� fetal MSCs as determined by scratch assay. Statistical analysesperformed using Student t test (n¼ 3). K and L,Migration of PC-3 cells in conditionedmedia fromWPMY-1-Neo, WPMY-1-ASPN D13, WPMY-1-ASPN D14, and a 1:1mix fromWPMY-1-ASPN D13:WPMY-1-ASPN D14 cells. Statistical analyses performed using one-way ANOVAwith Tukeymultiple comparison (n� 9). Graphsshown as mean� SEM. � , P� 0.05; �� , P� 0.01; �� , P� 0.001; �� , P� 0.0001. Black bars, 100 mm.






KEGG Pathways selective enriched in Aspn+/+ MSCs and WPMY-1-APSN D14 cells

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CALCIUM SIGNALINGBASAL CELL CARCINOMAHEDGEHOG SIGNALINGCHEMOKINE SIGNALING

WNT SIGNALINGTGF BETA SIGNALING

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CCL11LILRB3HRH4PKHD1FCRL5RAMP3PCDHB2SLC24A3CRPUTS2CCR8PKD2L1CACNG1CRHGRM6P2RX1TEX101PCDH12CDH17KCNK3ADRA1DCCL1NPY2RPKDREJNPPAPKD1L1NEUROD2SCGNCACNA1AGPRC6ACALCRSYTL1FCER1APLEKRXFP3SYT13CASQ2CCR1CACNA1IHCRTHCRTOXTSYT5BDKRB1KRT10PYGMP2RY12RIC3CXCL9ARVCFCCKARDCHS1CD8AUCNFGF14JPH3ANO3CACNA2D3DRD5HRH3KSR2SYT17CCL8KISS1CRACR2BTFF2SEMG1TRPV6AVPSYT3CACFD1DSG1GCM2CASQ1CCL7GNA15HRH3SYT17PCDHB16TGM2ATP2B3PTGDR2ATP13A5DPEP1HOMER3GNAT2PTGESSHPKJUNBSYT12AKAP5RCVRNTMEM37FFAR1TRPC3C5AR1PPAN-P2RY11FZD4KCTD17PPP1R9BTPCN2KCNMB4ORAI1SYT6HSPA2SLC30A1CRACR2AF2RL3RASA3ORAI2P2RX5-TAX1BP3MCTP1ORAI3SV2ACUL5FXYD5GNA13CPNE3CPNE3SDF4STXBP3TSNAXITPR3UBASH3B

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Figure 6.

ASPN-mediatedmigration is calcium-dependent. A,Migration of Aspn�/� cells in low calciummedia with vehicle or 100 ng/mL recombinant mouse ASPN asdetermined by transwell assay. B, Venn diagram of altered KEGG Pathways between differentially expressed genes in Aspnþ/þMSCs compared with Aspn�/�

MSCs as well as inWPMY-1-ASPN D14 compared withWPMY-1-ASPN Neo and inWPMY-1-ASPN D13 compared withWPMY-1-ASPN Neo. C, KEGG Pathwayenrichment selective to Aspnþ/þMSCs andWPMY-1-ASPN D14 cells compared with cells deficient for ASPN. D, Hierarchical clustering of GO calcium-relatedgenes in Aspnþ/þ and Aspn�/� fetal MSCs. E, Hierarchical clustering of GO calcium-related genes inWPMY-1-ASPN D14, WPMY-1-ASPN Neo, andWPMY-1-ASPND13 cells. F and G,Migration of Aspn�/� cells in low calciummedia with vehicle, 100 ng/mL recombinant mouse ASPN, 1 mmol/L BAPTA, or ASPN and BAPTA asdetermined by transwell assay. Quantification by ImageJ. Statistical analyses performed using one-way ANOVAwith Newman–Keuls multiple comparison (mean� SEM. � , P� 0.05; �� , P� 0.01; n� 3.

Hughes et al.





ASPN promotes metastatic progressionMSCs, cancer stem cells, angiogenesis, and immune infiltration

have all been shown to regulate metastatic development. Prioroverexpression studies indicate that ASPN (62) and specificallyASPN D14 (21) promotes invasion and metastasis; however, thishas not been determined in a genetic, immunocompetent mousemodel. Thus, we examinedmetastatic progression in Aspnþ/þ andAspn�/�micewith B6CaP allografts.When grown subcutaneouslyand then resected, B6CaP allografts form lungmetastases in 60%–

70% of C57BL/6J mice. While primary tumor size was similarbetween Aspnþ/þ and Aspn�/� mice, the development of lungmetastases was strikingly different. B6CaP subcutaneous allo-grafts formed lung metastases in 67% (4/6) of Aspnþ/þ mice andin 50% (3/6) of Aspnþ/� mice, but not in any of the Aspn�/� (0/6) mice (Fig. 7H–J). Collectively, these findings indicate thatASPN in the tumor microenvironment broadly impacts multi-ple cell types, and ultimately influences metastatic progression(Fig. 7K).

DiscussionMSCs and CAFs reside in the tumor microenvironment and

impact cancer growth and progression (4, 5, 9–13). These studiesestablish ASPN as a novel MSC factor expressed in the tumormicroenvironment that regulates metastatic development. ASPNrestricts earlyMSCdifferentiation, shownhere by inhibiting BMP-4 signaling. While ASPN expression is reduced during differenti-ation tomostMSC lineages, ASPN is highly expressed inCAFs andbroadly impacts multiple cell types in the tumor microenviron-ment. Collectively, these findings underpin the role of ASPN as akey mediator of MSC multipotency and as a critical regulator ofmetastasis in the tumor microenvironment.

MSCs are a heterogeneous population of cells that include cellsof mesenchymal origin including mesenchymal stem cells, mes-enchymal stromal cells, and fibroblasts. We demonstrate thatASPN is expressed in MSCs isolated from distinct sites includingbone marrow, adult prostate, and fetal prostate in humans andmice. MSCs give rise to physiologic connective tissue lineages,

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Figure 7.

ASPN regulates the tumor microenvironment and promotes metastatic development. A and B, Tumor volume (A) and time to resection (B) of B6CaPsubcutaneous allografts in Aspnþ/þ (n¼ 6), Aspnþ/� (n¼ 6), and Aspn�/- (n¼ 6) mice. Statistical analyses performed using one-way ANOVAwith Tukeymultiple comparison. C, Percent tumor-associated MSCs (CD45�, CD105þ, CD29þ, and Sca-1þ) in B6CaP subcutaneous allografts from Aspnþ/þ and Aspn�/�

mice as measured by flow cytometry. Statistical analyses performed using Student t test (n� 13). D, Percent cancer stem cells (CD45�, CD105�, CD31�, CD29þ,Sca-1þ, CD44þ) in B6CaP subcutaneous allografts from Aspnþ/þ and Aspn�/- mice as measured by flow cytometry. Statistical analyses performed using Studentt test (n� 8). E, CD8þ T cells as a percent of CD3þ cells in B6CaP subcutaneous allografts from Aspnþ/þ and Aspn�/�mice as measured by flow cytometry.Statistical analyses performed using Student t test (n� 4). F, SMAa-positive vasculature detected by IHC of B6CaP subcutaneous allografts from Aspnþ/þ andAspn�/�mice.G,Quantification of vasculature length in B6CaP subcutaneous allografts from Aspnþ/þ and Aspn�/�mice. Statistical analyses performed usingStudent t test (n� 4). H and I, Photograph (H) and hematoxylin and eosin staining (I) of lungs from Aspnþ/þ and Aspn�/�mice with B6CaP allograft.J, Percentage of Aspnþ/þ (4/6), Aspnþ/� (3/6), and Aspn�/� (0/6) mice with lung metastases from B6CaP subcutaneous allografts as determined byhematoxylin and eosin staining. Statistical analyses performed using x2 test. K, Schematic of the role of ASPN in MSCs andmetastasis. Graphs shown asmean� SEM. � , P� 0.05; �� , P� 0.01; �� , P� 0.001; NS, not significant. Black bars, 100 mm.






and differentiation is regulated by both conserved and lineage-specific factors (6). ASPN expression is diminished duringMSC differentiation to multiple lineages, and our findingsindicate that ASPN functions prior to MSC lineage commitmentto suppress the differentiation of a stem populationof multipotent MSCs capable of self-renewal. In the absenceof ASPN, fewer MSCs reside in the bone marrow and anintermediate population of multipotent MSCs with diminishedself-renewal potential predominates in the prostate. This isevidenced by our flow cytometry data demonstrating that incontrast to Aspnþ/þ MSCs in the prostate, MSCs from Aspn�/�

prostates were enriched for intermediate MSCs (Sca-1med) asopposed to stem MSCs (Sca-1hi). Consistent with these in vivofindings, Aspn�/� bone marrow–derived, compact bone-derived, prostate-derived, and late passage fetal MSCs had adecreased capacity for self-renewal in vitro compared withAspnþ/þMSCs. Studies using ASPN-deficient cells demonstratedthat ASPN restricts MSC differentiation in vitro along multiplelineages. Taken together, these data indicate that ASPN func-tions as a novel MSC factor that maintains MSC multipotency.

MSC lineage commitment and progression through differen-tiation is regulated bymanyTGFb familymembers (54). ASPN is asecreted extracellular protein that has been shown to antagonizeseveral members of the TGFb family including BMP-2 (49), BMP-4 (51), and TGFb1 (27). Most TGFb family members havepleiotropic roles in regulating MSC differentiation to connectivetissue lineages (54). In contrast to other TGFb family members,BMP-4 distinctly induces early MSC differentiation along severallineages including osteoblasts, adipocytes, and chondro-cytes (54). Our data demonstrate that ASPN binds to BMP-4 andrestricts BMP-4–induced signaling in MSCs and support a modelby which MSC-secreted ASPN locally restricts BMP-4–inducedMSC differentiation.

Similar to MSCs, reactive stroma cells in multiple cancer types,including prostate (20–22), breast (23), scirrhous gastric (24),and pancreatic (25), also highly expresses ASPN while benign-associated stroma is largely negative for ASPN expression. Inaddition to localized cancer, we show that ASPN is widelyexpressed in prostate cancer metastases. In contrast to the tumormicroenvironment, ASPNwas not expressed in reactive stroma inthe inflammatory microenvironment. This suggests that ASPNexpression is not conserved across all types of reactive stroma, buthas some specificity to reactive stroma in the tumor microenvi-ronment. Tumor-associated reactive stroma is a heterogeneouspopulation of cells that includes MSCs and CAFs. Studies supportthat CAFs are, in part, derived from MSC precursors (47, 55, 56).Accordingly, CAFs may be more similar to MSCs than inflamma-tion-associated reactive stromal cells based on the data presentedherein. Collectively, these findings indicate that ASPN expressionis highly conserved betweenMSCs and the reactive stroma in bothprimary and metastatic tumor microenvironments.

These studies indicate that ASPN broadly impacts both thetumor and the tumormicroenvironment. In the absence of ASPN,B6CaP allografts had fewer tumor-associated MSCs supportingthat ASPN regulates the number of multipotent MSCs in thetumor microenvironment. In addition, the immune infiltrationand vasculature was significantly altered in Aspn�/� mice. Effectson other cell types in the tumormicroenvironment and on cancerstem cells may be directly mediated by ASPN or indirectly regu-lated through MSC deficiencies. In addition to impacting thetumor microenvironment, ASPN may promote tumor progres-

sion by directly enhancing migration of both stromal and cancercells. ASPN has been implicated in the activation of multiplemigratory pathways including FGF-2 (50), IGFR (51), RAC1 (24),CD44 (24, 25), and EGFR (28, 62). Our data support thatexogenous ASPN is able to enhance migration, likely, by amechanism independent of its ability to restrict BMP4-inducedsignaling. Our findings support that ASPN-mediated migration isdependent on extracellular calcium and that genes annotated tothe KEGG calcium signaling pathway are differentially expressedin ASPN-expressing cells compared with ASPN-null and ASPND13 cells. Future studies will be needed to determine how ASPNmechanistically regulatesMSC and prostate cancer cell migration.

Mouse andhumanASPNarehighly homologous, including theD-repeat domain. While C57BL/6 mice have eight D-repeatsinterrupted by a single asparagine (N), the D-repeat length inhumans is variable. Recent reports suggest that the D-repeatdomain lengthmay regulate cancer progression (21, 29). Patientsharboring germline allele(s) of ASPN D14 were more likely todevelop metastatic prostate cancer, while homozygous germlineASPN D13 was associated with a reduced risk of metastaticprostate cancer (21). Animal models have shown that ASPND14 promotes metastatic prostate cancer progression (21), whileASPN D13 restricts breast (63) and prostate (21) cancer progres-sion. Our data show that ASPND14 increased stromal and cancercell migration compared with ASPN D13. Intriguingly, prostatecancer patient–based data suggest that ASPN D14 may be dom-inant over ASPN D13. Consistent with this, mixed conditionedmedia fromASPND13- andASPND14–expressing cells increasedcancer cell migration similar to conditioned media from ASPND14–expressing cells alone while conditioned media from ASPND13 did not increase cancer cell migration. Collectively, ourfindings reveal that ASPN D14 and ASPN D13 may differentiallyregulate molecular and cellular pathways that are fundamental tometastasis.

In conclusion, our data identify ASPN as a novel MSC factorthat is conserved in the tumor microenvironment and pro-motes metastatic progression. Further studies are warranted todetermine the efficacy and feasibility of therapeutically target-ing ASPN in prostate cancer, and potentially other solid tumortypes.

Disclosure of Potential Conflicts of InterestT.L. Lotan reports receiving other commercial research support from

GenomeDx, Ventana/Roche, Myriad Genomics, and is a consultant/advisoryboardmember for Janssen. E.Davicioni has ownership interest (including stock,patents, etc.) in GenomeDx. D.L.J. Thorek has ownership interest (includingstock, patents, etc.) in Diaprost AB and is a consultant/advisory board memberfor Diaprost AB. B.H. Park reports receiving a commercial research grant fromAbbvie, reports receiving other commercial research support from FoundationMedicine, has ownership interest (including stock, patents, etc.) in LoxoOncology, and is a consultant/advisory board member for Loxo Oncology. Nopotential conflicts of interest were disclosed by the other authors.

Authors' ContributionsConception and design: R.M. Hughes, B.H. Park, P.J. HurleyDevelopment of methodology: R.M. Hughes, W.N. Brennen, P.J. HurleyAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): R.M. Hughes, B.W. Simons, H. Khan, V. Kugler,S. Torquato, D. Theodros, M.C. Haffner, T.L. Lotan, J. Huang, E. Davicioni,S.S. An, R.C. Riddle, D.L.J. Thorek, I.P. Garraway, J.T. Isaacs, W.N. Brennen,P.J. HurleyAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): R.M. Hughes, B.W. Simons, H. Khan, V. Kugler,

Hughes et al.





S. Torquato, E. Davicioni, S.S. An, R.C. Riddle, D.L.J. Thorek, E.J. Fertig,W.N. Brennen, B.H. Park, P.J. HurleyWriting, review, and/or revision of the manuscript: R.M. Hughes, H. Khan,S. Torquato, D. Theodros, E. Davicioni, S.S. An, D.L.J. Thorek, I.P. Garraway,E.J. Fertig, W.N. Brennen, B.H. Park, P.J. HurleyAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): R.M. Hughes, H. Khan, R. Miller, S. Torquato,E. Davicioni, P.J. HurleyStudy supervision: P.J. Hurley

AcknowledgmentsWe thank Edward M. Schaeffer, Ashley E. Ross, Daniel Ardeljan, Leigh

Ellis, Daniele M. Gilkes, Ines Godet, Timothy E. Krueger, and Karen Craverofor thoughtful discussions and for sharing resources and experimentalprotocols. We thank the Johns Hopkins Sidney Kimmel ComprehensiveCancer Center Flow Cytometry and Cell Imaging Core Facilities supported bythe Cancer Center Core Grant P30 CA006973. We also thank the ProstateCancer Biorepository Network (PCBN), supported by the Department ofDefense Prostate Cancer Research Program, Department of Defense AwardNo. W81XWH-10-2-0056 and W81XWH-10-2-0046. We acknowledge theuse of tissues procured by the National Disease Research Interchange (NDRI)

with support from NIH grant 2 U42 OD011158. This work was supported byDepartment of Defense Prostate Cancer Research Program W81XWH-11-PCRP-IDA (to P.J. Hurley), The Patrick C. Walsh Prostate Cancer Fund(to P.J. Hurley), The Willowcroft Foundation (to P.J. Hurley), The JohnsHopkins Catalyst Award (to P.J. Hurley), The American Cancer Society131356-RSG-17-160-01-CSM (to P.J. Hurley), The National CancerInstitute/NIH RO1CA211695-01A1 (to P.J. Hurley, B.W. Simons, T.L. Lotan,W.N. Brennen, R.M. Hughes), R01CA194024 (to B.H. Park), andR01CA201035 (to D.L.J. Thorek), and the Commonwealth Foundation (toB.H. Park). W.N. Brennen acknowledges support from the Prostate CancerFoundation as the Clay and Lynn Hamlin Young Investigator. B.H. Parkacknowledges support from Susan G. Komen as a Komen Scholar.

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received September 18, 2018; revised April 17, 2019; acceptedMay 20, 2019;published first May 23, 2019.

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Hughes et al.




2019;79:3636-3650. Published OnlineFirst May 23, 2019.Cancer Res Robert M. Hughes, Brian W. Simons, Hamda Khan, et al. the Tumor Microenvironment, and Drives Metastatic ProgressionAsporin Restricts Mesenchymal Stromal Cell Differentiation, Alters

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Asporin Restricts Mesenchymal Stromal Cell Differentiation, … · Asporin Restricts Mesenchymal Stromal Cell Differentiation, Alters the Tumor Microenvironment, and Drives Metastatic