Therapeutic Targeting of Tumor-Derived R-Spondin … › content › canres › 76 › 3 › ...R-Spondin Attenuates b-Catenin Signaling and Tumorigenesis in Multiple Cancer Types

Tumor and Stem Cell Biology

Therapeutic Targeting of Tumor-DerivedR-Spondin Attenuates b-Catenin Signaling andTumorigenesis in Multiple Cancer TypesCecile Chartier, Janak Raval, Fumiko Axelrod, Chris Bond, Jennifer Cain,Cristina Dee-Hoskins, Shirley Ma, Marcus M. Fischer, Jalpa Shah, Jie Wei, May Ji,Andrew Lam, Michelle Stroud,Wan-Ching Yen, Pete Yeung, Belinda Cancilla,Gilbert O'Young, Min Wang, Ann M. Kapoun, John Lewicki, Timothy Hoey, andAustin Gurney

Abstract

Deregulation of the b-catenin signaling has long been associ-ated with cancer. Intracellular components of this pathway,including axin, APC, and b-catenin, are frequently mutated in arange of human tumors, but the contribution of specific extra-cellular ligands that promote cancer development through thissignaling axis remains unclear. We conducted a reporter-basedscreen in a panel of human tumors to identify secreted factors thatstimulate b-catenin signaling. Through this screen and furthermolecular characterization, we found that R-spondin (RSPO)proteins collaborate with Wnt proteins to activate b-catenin.RSPO family members were expressed in several human tumorsrepresenting multiple malignancies, including ovarian, pancreat-ic, colon, breast, and lung cancer. We generated specific mono-

clonal antibody antagonists of RSPO family members and foundthat anti-RSPO treatment markedly inhibited tumor growth inhuman patient-derived tumor xenograft models, either as singleagents or in combination with chemotherapy. Furthermore,blocking RSPO signaling reduced the tumorigenicity of cancercells based on serial transplantation studies. Moreover, gene-expression analyses revealed that anti-RSPO treatment in respon-sive tumors strongly inhibited b-catenin target genes known to beassociated with cancer and normal stem cells. Collectively, ourresults suggest that the RSPO family is an important stimulator ofb-catenin activity in many human tumors and highlight a neweffective approach for therapeuticallymodulating this fundamen-tal signaling axis. Cancer Res; 76(3); 713–23. �2015 AACR.

Introductionb-catenin signaling is essential to developmental processes

and is deregulated in a wide range of diseases, including cancer(1–3). WNT ligands are able to activate b-catenin–dependentsignaling by binding to frizzled (FZD) receptor and LRP5/6coreceptor complex, inducing the release of unphosphorylatedb-catenin from a cytosolic destruction complex comprised ofaxin, APC, CKI, and GSK3 and its translocation to the nucleuswhere it associates with the TCF/LEF family of transcriptionfactors and induces canonical gene transcription. Intracellularcomponents of the signaling pathway such as APC, axin, andb-catenin itself are mutated in a number of human cancers.

However, no WNT ligand overexpression has yet been directlyassociated to the disease in humans (3).

The R-spondin (RSPO) family of four proteins representsanother group of secreted factors that enhance b-catenin signal-ing. RSPO1-4 have a similar structure and harbor two furindomains, which are necessary and sufficient for b-catenin activa-tion, and one thrombospondin type I domain, which promotesnon-canonical Wnt signaling (4, 5). Three classes of transmem-brane proteins have been discovered that interact with RSPO:syndecans, Leu-rich repeat-containing G protein-coupled recep-tors (LGR), and E3 ubiquitin ligases (5–10). Of note, one of thesereceptors, LGR5, is a robust marker of stem cells (11–18). Amechanism of action for RSPO has been elucidated; RSPOsprotect frizzled receptors from internalization and degradationand recruit IQGAP1 into the Wnt signaling complex, leadingto both an increase in b-catenin and Rho GTPase signalingactivities (9, 19). The LGR receptors involved in this process aremembers of the superfamily of seven-transmembrane receptors,suggesting the potential for additional signaling mechanismselicited by RPSO.

Like WNTs, RSPOs have important roles in development andact as powerful stem cell growth factors (20). RSPO1 is used as anindispensable agent for in vitropropagation of intestinal stem cellsand its activity cannot be replaced by exogenous WNT protein(21). In vivo, RSPO1 controls the phenotypic sex of humans andmice, RSPO2 is necessary for development of numerous mouseorgans, RSPO3 is involved in angiogenesis during placenta

OncoMed Pharmaceuticals Inc., Redwood City, California.

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

Current address for C. Bond: Center for Drug Research and Development,Vancouver, BC; current address for J. Shah: ZS Associates, San Mateo, CA; andcurrent address for J. Wei: Rinat Laboratories, Pfizer Inc., South San Francisco,CA.

Corresponding Author: Cecile Chartier, OncoMed Pharmaceuticals, 800 Che-sapeake Drive, Redwood City, CA 94063. Phone: 650-995-8216; Fax: 650-298-8600; E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-15-0561

�2015 American Association for Cancer Research.

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development inmice, andRSPO4 is the genedisrupted inhumanswith congenital anonychia (22). Inmost cases, RSPOs function byenhancing Wnt/b-catenin signaling. However, RSPO2 or 3 werealso shown to signal through cascades that are separate from thecanonical pathway (5, 8, 19, 23–25).

Several lines of evidence suggest a role for RSPOs in cancerdevelopment. The identification of Rspo2 and Rspo3 as sites ofintegration for MMTV-induced mammary tumors in mice sup-ported a role as breast cancer oncogenes (26–28). Ectopic expres-sion of Rspo2 was shown to increase tumorigenic and invasiveproperties of mouse breast cell lines (25). Genomic rearrange-ments and transcriptional activation in human tumors that resultin elevated RSPO expression have been identified, suggesting thatRSPO may be functionally important for tumor development(29–32). However, no direct demonstration of a functional rolefor RSPO in cancer development and maintenance has beenreported to date.

Here, we report an analysis of patient-derived xenograft (PDX)tumor samples for RSPO expression and activity. FunctionalRSPO activity was found to be produced by human tumors ofmultiple types. The importance of RSPO in driving the growth ofoverexpressing tumors was tested in vivo with anti-RSPO mono-clonal antibodies (mAb) directed against various members of thefamily. Efficacy was observed in PDX models established withminimally passaged human tumors of multiple types. Gene-expression analyses revealed that RSPO promoted expression ofgenes associated with theWnt signaling pathway and normal andcancer stem cells (CSC). These data provide the first demonstra-tion that RSPO familymembers play amajor role in the growth ofhuman cancer and validate RSPO1-3 as therapeutic targets.

Materials and MethodsXenograft models and tissue processing

NOD/SCID mice were purchased from Harlan Laboratoriesand maintained under specific pathogen-free conditions andprovided with sterile food and water ad libitum. Animals werehoused in a U.S. Department of Agriculture-registered facility inaccordance with NIH guidelines for the care and use of laboratoryanimals. The mice were allowed to acclimate for several daysbefore the studies.

The establishment of OMP PDX models was described previ-ously (33). Patient-derived tumorswere passaged inmicewithoutany intervening cell culture. Tumor cells were stored in liquidnitrogen. Experiments for obtaining fresh tumor cells or testingantibody therapeutic efficacy were initiated from frozen cellstocks. CR3150, CR1560, CR2506, and CR2513 models wereestablished at Crown BioScience (Beijing, China).

For in vitro assays and in vivo tumorigenicity assessments, single-cell suspensions were obtained from freshly dissociated tumorsaccording to a previously described procedure (34). Cells werethen incubated with biotinylated antibodies (a-mouse CD45-biotin 1:200 dilution and a-mouse H2Kd-biotin 1:100 dilution;BioLegend) on ice for 30 minutes followed by addition of 50 mLMagnaBind streptavadin-labeled magnetic beads (Thermo Scien-tific) per 10E6 cells/mL. Mouse cells were removed on amagneticstand.

Cell linesThe HEK-293 STF cell line was a kind gift from Dr. Jeremy

Nathans (Johns Hopkins School of Medicine, Baltimore, MD)

received in 2005. It contains the TOP-FLASH reporter systemcomprised of 8 copies of a TCF-binding site (AGATCAAAGG)upstream of a minimal promoter and firefly luciferase. Cells werecultured in DMEM, 10% FBS, 500 mg/mL Geneticin (Life Tech-nologies) and incubated at 37�C, 5% CO2. HEK-293T (CRL-3216) and WNT3A-L cells (CRL-2647) were obtained in 2005from the ATCC and cultured according to the ATCC guidelines.

STF reporter assayTumor cells were cultured in DMEM, 10% FBS (Life Technol-

ogies) for 24 hours at 37�C and 5% CO2. One volume ofconditioned medium and cells was added on top of a monolayerof HEK-293 STF cells grown in 1 volume of DMEM, 10% FBS.When used, 5 ng/mL of recombinant human RSPO (R&D Sys-tems) were added to the reporter cells. WNT3A-conditionedmedium prepared from WNT3A-L cells was added to the tumorcell conditioned medium or purified RSPO at a final concen-tration of 25% v/v. The activity of purified soluble receptors(10 mg/mL) and mAbs (40 mg/mL) was compared with thecontrol immunoadhesin JAG1-Fc and control antibody LZ1.Reporter cells were incubated for 16 hours before luciferaseactivity measurement using Steady-Glo according to the man-ufacturer's instructions (Promega).

Real-time RT-PCRFor RSPO gene-expression analyses, RNA was extracted from

xenograft tumors using an RNeasy Fibrous Tissue mini kit(Qiagen) according to the manufacturer's instructions. Thirtyng RNA were submitted to a one-step RT-PCR reaction usingApplied Biosystems 7900HT instrument and buffer systemaccording to the manufacturer's instructions (Life Technolo-gies). GUSBwas used as endogenous control against which DCtswere calculated and the lowest RSPO expressing sample asbaseline for the DDCt calculations. Relative expressions weredetermined by raising 2 to the power of the negative value ofDDCt for each sample. All gene-expression assays were pur-chased from ABI and verified for species specificity beforescreening the OMP PDXs.

– GUSB: Hs00939627_m1– RSPO1: Hs01045335_mH– RSPO2: Hs00379983_m1– RSPO3: Hs00262176_m1– RSPO4: Hs01382765_m1

For select target gene-expression analyses, frozen tumortissues were pulverized in liquid nitrogen with a mortar andpestle. RNA was extracted as above. All RNAs were verified tocontain intact ribosomal bands by Bioanalyzer (Agilent).cDNA template was generated from 50 ng RNA using the CellsDirect One-step qRT-PCR Kit (Life Technologies). PCR wasperformed on ABI 7900HT (Life Technologies) using the rel-ative standard curve method. Gene-expression normalizationwas performed with GAPDH. Statistical calculations wereperformed using the t test with Excel software (Microsoft).Primer (forward and reverse) and probe sequences wereas follows. TDGF1: 50-CCAGAGTGCTGAAGGAATGGA-30,50-CCTCTCTCTTCTATTTGCTTCCTCTT-30, and 50-CCTACC-CAGTCTCCCTGCACACACG-30; ASCL2: 50-CCCCTCCCCA-CAGCTTCT-30, 50-AAATGGATTCTCTGTGCCCTTAGA-30, and

Chartier et al.

Cancer Res; 76(3) February 1, 2016 Cancer Research714




50-CACCAACACTTGGAGATTTTTCCGGAGG-30; CLDN2: 50-CCT-AAGTCCCCAACCCTCAAC-30, 50-GGGCAAAGGGATCCTCTGA-30, and 50-TGAAACCCCATTCCCTTAAGCCAGGA-30; PTPRO: 50-GAACTCTGTTGCTGTCTGAGCAA-30, 50-GCTCTCACATGTATG-CAGGTCAA-30, and 50-CGTGGTGCCTAGACTTTGCATTCCTTG-30; RPRD1A: 50-CACCTGAGGCAGATCATGCA-30, 50-ACGGCAC-CATGCTTTTCTCT-30, and 50-CTTTGAGTGCAGTTTGGTCTGAC-CCCTC-30; LGR5: 50-TGGACTCAAGAGACTCAGTAACGTATTAT-30, 50-TAAGCAGAGAAGTAATGTTCCTAACATCA-30, and 50-ATT-TAGCTTGGTTTTAGCTGTGTTCTCTCTGGATAACC-30; ZNRF3: 50-CCATGTCTTATGTTGAGAGTGTGACA-30, 50-TGCAAATATG-TAAAATCTGTGTGCAA-30, and 50-TGGAATAATCATTGAAAATG-ACTAACACAAGACCCTGTAA-30; AXIN2: 50-CCTTTCCTCCACA-CACCTTCA-30, 50-TGTATAGTACAAGTAACAATGGCAAACAG-30,and 50-ATGTACAGATTAACTTAACACAAAAACCCGAACATCAA-A-30; GAPDH: 50-CCACCCATGGCAAATTCC-30, 50-TGGGATTTC-CATTGATGACAAG-30, and 50-TGGCACCGTCAAGGCTGAGAA-CG-30.

AntibodiesAnti-RSPO antibodies were generated by immunizing mice

with purified recombinant human RSPO1, RSPO2, or RSPO3(R&D Systems) followed by hybridoma generation and char-acterization. The antibodies LZ1, an anti-lysozyme monoclonalgenerated by panning a phage display library obtained fromMorphoSys (Martinsried/Planegg) and 1B711, a murine mono-clonal directed against dinitrophenol (anti-hapten) andobtained from the ATCC were used as negative controls in thein vitro and in vivo experiments, respectively (35). Anti-RPSO3mAb #2 was isolated by a mammalian cell antibody displaytechnique, MAbTrap, in which antibody sequences derivedfrom the immunized mice were transiently expressed and thenretained on the cell surface by interaction with a membrane-anchored CH2-CH3 antibody fragment. FACS was used toisolate cells expressing the antibody of interest and correspond-ing cDNA isolated.

The binding of anti-RSPO antibodies to RSPO was assessedby flow cytometry. First, HEK-293T cells were transiently trans-fected with cDNA expression vectors for RSPO extracellulardomain fused to human CD4 transmembrane domain fusedto GFP using Fugene 6 (Promega). Transfected cells were thenincubated with 20 mg/mL antibody and washed with PBS.Antibody binding was detected with a PE-conjugated anti-mouse Fc antibody and light signals measured for PE and GFP,using a Canto II instrument (BD Biosciences).

To assess ability of anti-RSPO antibodies to block interac-tion with LGR, binding studies were conducted. RSPO pro-teins were expressed as Fc fusion proteins containing RSPON-terminal furin domains and human IgG1 Fc. HEK-293Tcells were transiently transfected with cDNA expression vectorencoding the N-terminal extracellular domain of human LGRfused to human CD4 transmembrane domain fused to GFP.After 24 hours, cells were incubated with RSPO–Fc fusionproteins and anti-RSPO antibodies at 10 mg/mL. RSPO bind-ing was detected by flow cytometry with a PE-conjugated anti-human Fc antibody and light signals measured for PE andGFP.

In vivo efficacy experimentsAbout 50,000 freshly thawed tumor cells re-suspended in

100 mL 50% Matrigel in FACS buffer (OMP PDXs) or tumor

fragments (Crown BioScience PDXs) were injected s.c. into theleft flank region of 6- to 8-week-old NOD/SCID mice with a25-gauge needle. Tumor-bearing animals were randomizedand treatment started when the mean tumor volumes reached100 to 150 mm3. Chemotherapeutic agents were given onceweekly at various doses ranging from 10 to 100 mg/kg asspecified in the figure legends. Antibodies were dosed onceweekly or biweekly at a dose ranging from 10 to 25 mg/kg. Allagents were administered i.p.

For the tumorigenicity study, single tumor cell suspensionsprepared as described from control and treated tumors werecounted and diluted to 50 cells in 100 mL 50%Matrigel in FACSbuffer. Ten NOD/SCID mice were injected s.c. per group.Tumor growth was monitored for 59 days without any furthertreatment.

Microarray analysisRNA was isolated and microarray analyses were conducted

as detailed in Supplementary Data. Data are available throughGEO database, accession GSE73906.

ResultsIdentification of RSPO as a tumor-derived b-cateninstimulating activity

To identify possible b-catenin–stimulating activities pro-duced by human tumor cells, a functional screen was devel-oped. Tumor cells freshly obtained from minimally passagedPDXs (referred to as OMP-xxxx tumors hereafter, where xxxxrefers to our internal nomenclature for that tumor PDX line)were cocultured with an HEK-293 cell-based reporter cellline containing a TCF-luciferase reporter (STF). This reportercell line responds to WNT and RSPO signals through b-catenin–driven transcription. Forty eight OMP tumors (SupplementaryTable S1) were screened in this assay, of which, three (6.25%)induced reporter activity more than 2-fold relative to control(Fig. 1A and Supplementary Table S2). OMP-LU2, OMP-LU25,and OMP-OV38 induced luciferase activity 6.7-, 4.7-, and 3.1-fold over medium alone, respectively. In this survey, WNT3Awas also tested, alone as a positive control and added tothe coculture assay. Interestingly, the three tumors that werepositive for b-catenin signaling activity displayed further ele-vated activation upon addition of WNT3A (Fig. 1A). OMP-LU2,OMP-LU25, and OMP-OV38 boosted WNT3A activity 4.3-, 9.6-,and 1.6-fold over WNT3A alone. Such potentiation of WNThas been noted as a hallmark of RSPO activity, suggesting thatRSPO ligands might be responsible for the activation of theb-catenin reporter in the assay (4). Of note, additional OMPtumors were shown to score in the assay only in the presence ofWNT3A, possibly reflecting lower levels of the b-catenin acti-vating factor(s). OMP-B37, OMP-B39, and OMP-LU102increased WNT3A activity 2.7-, 9-, and 1.8-fold, respectively(Fig. 1A and Supplementary Table S2). To examine the tumor-derived activity further, FZD8-Fc and LGR5-Fc decoy receptorswere tested in the assay. FZD8-Fc acts as an inhibitor of Wntsignaling by binding WNT ligands, whereas LGR5-Fc binds toRSPO and antagonizes RSPO potentiation of Wnt signaling(Fig. 1B and C). In conditions in which FZD8-Fc abolishedWNT3A-induced reporter activity, it partially inhibited theactivity induced by the tumor cells alone and dramaticallydecreased the activity induced by the tumor cells in presence

RSPO Signaling in Cancer

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of exogenously added WNT3A. Conversely, although LGR5-Fcremained mostly ineffective at inhibiting WNT3A alone, itefficiently decreased the activity of the tumor cells alone andsupplemented with WNT3A. LGR5-Fc reduced the activity ofthe cocultures containing added WNT3A to a level comparablewith that observed with WNT3A alone (Fig. 1D). These resultssuggest that the activation of the b-catenin reporter observed inthe tumor cell culture assay reflects the presence of RSPO activitypotentiating low levels of endogenous WNT ligands. The mod-est inhibition of the tumor-induced activation by FZD8-Fcsuggests the presence of a distinct (WNT or non-WNT) ligandnot bound by FZD8-Fc.

Interestingly, the tumors previously found to exhibitb-catenin activity possessed markedly elevated levels of par-ticular RSPO family members. Generally, only a single RPSO

family member was highly expressed within a tumor as deter-mined by quantitative real-time polymerase chain reaction(qPCR; Fig. 2 and Supplementary Table S2). Thus, OMP-LU2,OMP-LU25 and OMP-OV38 expressed high levels of RSPO2or RSPO3. Additional RSPO mRNA-positive tumors, for exam-ple, OMP-OV19 and OMP-PN7 (Fig. 1A and SupplementaryTable S2), had not been identified by the in vitro functionalassay, maybe due to the low sensitivity of the assay. It hasbeen reported that RSPO2 and RSPO3 genes are targets ofrearrangements in colon cancer and that resulting fusions in-crease gene expression (29, 31). On the basis of these reports,we performed a PCR-based screen and also mRNAseq analy-sis. These analyses did not find fusions in either RSPO2or RSPO3 in our tumor bank, including RSPO2-high OMP-C28, suggesting that other mechanisms are involved in the

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Figure 1.Detection of OMP tumor sample-derived b-catenin activation. The ability of human tumor-derived soluble factors to activate b-catenin was assessed in theSTF assay. A, fold inductions are shown for a subset of the 48 OMP tumor samples tested. They represent the ratio of tumor (red bars), WNT3A (green bars),and tumor þ WNT3A (purple bars)-induced luciferase activity relative to the medium alone. B, FZD8.Fc inhibitory activity was demonstrated inthe STF reporter assay. STF cells were exposed to WNT3A or non-WNT3A (solid triangle) conditioned medium supplemented with the indicatedconcentrations of FZD8.Fc (hollow dots). Mouse JAG1-Fc (solid dots) was used as negative control. Results are displayed as luciferase activity levels (RLU,relative light units) as a function of the test agent's concentration (mg/mL). C, serial dilutions of LGR5.Fc were used to inhibit the induction of luciferase byRSPO1 (squares), RSPO2 (circles), RSPO3 (triangles), or RSPO4 (diamonds) added to WNT3A-conditioned medium. Results are shown as the measuredRLUs as a function of the decoy protein concentration. D, both FZD8.Fc and LGR5.Fc inhibited the luciferase reporter activity (RLUs) of STF cells stimulatedwith tumor cell cultures supplemented (purple bars) or not (red bars) with WNT3A. WNT3A activity was only inhibited by FZD8.Fc (green bars).

Chartier et al.





overexpression of RSPO in these tumors. RSPO3-high humanCRC tumors possessing the reported PTPRK–RSPO3 genefusion were identified at an external service provider (Sup-plementary Fig. S1).

To investigate the functional consequence of inhibiting theactivity of specific RSPOs,mAbs were generated that bind RSPO1,RSPO2, or RSPO3 (Fig. 3A) and block the ability of RSPO topotentiate WNT stimulation of the b-catenin luciferase reporterthrough blocking binding to LGR (Fig. 3B and C). All antibodieswere shown to be specific for their targeted RSPO andnot to cross-react with the other RSPOs. Anti-RSPO antibodies were tested forimpact on the tumor-induced b-catenin activity observed in theluciferase reporter screen. Antibody to RSPO2 blocked the activityproduced by OMP-LU2 tumor cells and antibody to RSPO3blocked the activity observed in response to OMP-LU25, OMP-LU102, and OMP-OV38 tumor cells (Fig. 4). In each case, theantibodies inhibited the reporter activity back to control levels.None of the three antibodies affected the reporter activity whennon–b-catenin–inducing tumor cells such as OMP-PN25 andOMP-M6were used in the assay (Supplementary Fig. S2).Overall,the results parallel the relative RSPO gene-expression pattern(Fig. 2, Supplementary Table S2), strongly implicating the RSPOproteins as the inducers of the observed b-catenin signalingactivity produced by these human tumor cells. In particular, theabove data revealed RSPO2 or RSPO3 as the principal factorsresponsible for the b-catenin activity derived from several of ourpatient-derived tumors, making potential therapeutic targets ofRSPO2 and RSPO3.

RSPO antagonists inhibit the growth of human tumorxenografts

The impact of inhibiting RSPO signaling on tumor growthwas assessed using PDXs in mice. Tumor-bearing mice weretreated with antibodies to individual RSPO family members.Anti-RSPO treatment produced significant inhibition of tumorgrowth in several types of human tumors (Fig. 5A). Growth ofOMP-OV19, an ovarian tumor noted to express RSPO1, wasinhibited by antibody to RSPO1. OMP-C28 and OMP-PN7, acolon and a pancreatic tumor, respectively, noted to expressRSPO2, were inhibited by antibody to RSPO2. Anti-RSPO3displayed significant antitumor activity in several RSPO3-high tumors, including non–small cell lung cancer (NSCLC)models OMP-LU25 and OMP-LU102, colorectal cancer modelsCR3150, CR2513, and CR2506, and ovarian cancer OMP-OV38. Colon tumor CR3150 is noteworthy as a tumor posses-sing a RSPO3 chromosomal translocation identical to thosepreviously reported (29). Thus, anti-RSPO treatments showedsingle-agent therapeutic activity in most RSPO-high models.Combination with chemotherapy, including taxane treatmentin NSCLC and ovarian models, gemcitabine treatment in pan-creatic tumor, and irinotecan in colorectal cancer models,resulted in further significant inhibition of tumor growthbeyond the impact of chemotherapy alone (Fig. 5A). Themechanism of combination activity with paclitaxel was inves-tigated in OMP-OV38. Anti-RSPO3 treatment combined withpaclitaxel was found to increase mitotic cell death (Supple-mentary Fig. S3).

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Figure 2.RSPO gene-expression profile across multiple different tumor types. RSPO1, 2, 3, and 4 gene expression in breast, colon, lung, melanoma, ovary, andpancreas tumor models was assessed by real time RT-PCR and expressed as a relative quantity using the lowest positive sample as baseline and GUSBas endogenous reference. Each purple bar represents one patient-derived tumor sample.






Not all RSPO-high tumors were sensitive to RSPO inhibition.For example, the NSCLCOMP-LU2, despite high levels of RPSO2mRNA, and colorectal cancer CR1560, despite high levels ofRSPO3 mRNA, were not significantly inhibited by anti-RSPO2and anti-RSPO3, respectively, whether alone or combined withchemotherapy (Fig. 5A). Analysis of LGR4, 5, and 6 gene expres-sions revealed no correlation between receptor expression andresistance to antibody treatment (Supplementary Fig. S4). Tumorsthat did not express RSPO were unresponsive to antibody treat-ment (Supplementary Fig. S5).

The responsive NSCLC model OMP-LU25 was used to assessthe impact of RSPO3 inhibition on CSCs, also known as tumorinitiating cells. For this, tumors previously treated with antibodyand/or chemotherapy were harvested and processed to a single-

cell suspension and 50 cells were injected into recipient mice for asecond (treatment free) round of propagation. The ability of thislow cell number to re-grow a tumor provided a direct measure oftheir CSC content. In such a setting, anti-RSPO3 significantlyreduced the tumorigenicity of OMP-LU25 tumor cells both as asingle agent and in combination with paclitaxel (Fig. 5B).Although all 10 control mice and 8 of 9 paclitaxel group micedeveloped tumors, the anti-RSPO3 and combination-treatedtumor cells engrafted poorly (7:10 and 6:10, respectively) andgrew slowly. Similar reductions in tumorigenicity after anti-RSPOtreatment were observed in other tumors, including recentlyobtained pancreatic and lung tumors that were not included inour original panel (Supplementary Fig. S6). In addition, levels ofCD44, a well-known colon CSC marker, were significantly

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Figure 3.Characterization of anti-RSPO1, RSPO2, and RSPO3 mAbs. RSPO-binding properties of the anti-RSPO mAbs were assessed by flow cytometry. Theability of anti-RSPO antibodies to inhibit RSPO signaling function was monitored in the cell-based b-catenin reporter assay. A, RSPO1, 2, 3, or 4 wasdisplayed at the surface of HEK-293 cells by means of a transiently transfected RSPO1, 2, 3, or 4.CD4TM.GFP fusion cDNA expression vector thattethers RSPO to the cell surface via a transmembrane anchor. Binding of the RSPO moieties by the anti-RSPO1, anti-RSPO2, and two anti-RSPO3 mAbswas detected using a PE-conjugated secondary antibody. The PE (y axis) and GFP/FITC (x axis) signals are plotted for each individual cell analyzed.The relative increase of both signals translates specific antibody binding to the antigen. B, anti-RSPO antibodies' inhibitory activity was confirmed inthe STF reporter assay. STF cells were exposed to WNT3A conditioned medium supplemented with RSPO1, RSPO2, or RSPO3. Anti-RSPO antibodieswere added. Results are displayed as luciferase activity levels (RLU, relative light units). C, LGR5 was displayed at the surface of HEK-293T cells bymeans of a transiently transfected LGR5.CD4TM.GFP fusion cDNA expression vector that expresses the N-terminal extracellular domain of LGR5 tetheredto the cell surface via a transmembrane anchor. Ability of anti-RSPO mAb to block the binding of the indicated RSPO was assessed by incubating cellswith RSPO-Fc fusion protein and mAb as indicated and followed by detection of bound RSPO protein with PE-conjugated anti-human Fc secondaryantibody. Specific RSPO-LGR5 binding is indicated by detection of cells within the inset box of the flow-cytometry plot.

Chartier et al.





reduced in anti–RSPO3-treated tumors (Supplementary Fig. S7;ref. 33). These results show that RSPO3 is important to maintainCSCs in multiple tumor types.

RSPO blockade inhibits stem cell signaling pathways intumors

RSPO1 functions as a potent regulator of stem cell growth incolon (21). The expression of a panel of genes associated withstem cell and/or RSPO signaling was, thus, monitored in anti–RSPO3-treated colorectal cancer xenografts. Control and anti-body-treated tumor RNAs from the in vivo efficacy experimentsconducted with CR3150, CR2506, CR2513, and CR1560 weretested for the expression of ASCL2, AXIN2, CLDN2, LGR5,PTPRO, RPRD1A, TDGF1, and ZNRF3 genes, using qPCR. Strik-ingly, 7 of the 8 genes were downregulated in response to anti-RSPO3 in all three sensitive colorectal cancer models (Fig. 6).The downregulation was highly significant in at least two of themodels for these genes. For example, ASCL2 expression couldno longer be detected in any of the anti–RSPO3-treated CR2506and CR2513 samples; LGR5 was downregulated 7.24- and5.75-fold in treated CR2506 and CR3150 tumors, respectively,and TDGF1 mRNA levels were reduced in all three models, upto 234-fold. All 7 downregulated genes are reported canonicalWnt signaling target genes (36, 37). Several of these genes,ASCL2, LGR5, and TDGF1, have also been directly associatedwith CSCs in various tumor types, supporting a role for theRSPO pathway in CSC biology and consistent with the in vivotumorigenicity results presented herein (38–40). Downregula-tion of Wnt and stem cell genes by anti-RSPO treatment couldbe involved in the sensitization of tumor cells to chemother-apeutic agents (41, 42). Two of the 8 studied genes, AXIN2 andZNRF3, were downregulated by anti-RSPO3 in the nonrespond-ing CR1560 model. The two genes are the only Wnt target genesof the panel that were highly expressed in this model, possiblyreflecting a lower state of Wnt signaling activation. Thus, iso-latedWnt targets could be regulated by anti-RSPO3 therapy and

not be associated with growth inhibition of non-RSPO/Wnt-driven tumors.

To further study themechanismof action of RSPO3,microarrayanalysis was performed on anti–RSPO3-treated colon tumors.Findings were consistent with the qPCR results (Fig. 6) and alsorevealed new insights into the RSPO3 mechanism of action(Supplementary Table S3). In the three responsive models, mod-ulation ofWnt signaling was confirmed by the downregulation ofnumerous Wnt genes that included CLDN2, EPHB2, CD44, LRP4,ASCL2, AXIN2, LGR5, PTPRO, TDGF1, and ZNRF3. In addition,effects onNotch signalingwere revealed by the downregulation ofgenes such as NOTCH1 and PSEN1. Both Wnt and Notch path-ways are associated with CSCs (43–45). Consistently, numerousstem cell genes were inhibited by anti-RSPO3 in the three respon-ders. Interestingly, differentiation genes, including BAMBI,CADM1, POU2F2, and RGS2were upregulated. Most of the genesdescribed above were not significantly modulated by anti-RSPO3in the nonresponsive CR1560. Together, these results show thatRSPO3 blockade inhibited CSC signaling pathways in colorectalcancer, and suggest that the tumor cells may adopt a moredifferentiated cell fate. Microarray data for anti-RSPO3 plusirinotecan-treated CR2506 further support this mechanism byshowing that most Wnt and stem cell genes targeted by anti-RSPO3 remained modulated upon addition of chemotherapeu-tics, with some of them responding to a greater extent to thecombination than to anti-RSPO3 alone (Supplementary TableS3). Overall, the gene-expression data support the inhibition ofself-renewal pathways as the main mechanism of anti-RSPO3'santitumor activity.

DiscussionBecause the initial discovery of WNT1 as a gene product

capable of promoting breast cancer thirty years ago, therehas been a recognition that this signaling pathway may con-tribute to human cancer (46). This recognition has been greatly

ND ND ND

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Figure 4.Inhibition of tumor-derived b-cateninactivity by the anti-RSPO antibodies. Theuse of specific anti-RSPO antibodies in thetumor-STF cell coculture reporter assayidentified RSPO ligands as the tumor-derived factors responsible for inducingb-catenin transcriptional activity.OMP-LU2, -LU25, -LU102, and -OV38 cellswere tested for their ability to induceluciferase activity of a TCF-respondingelement in absence (1st series) andpresence (2nd series) of WNT3A whencombined with a control mAb (red bars),anti-RSPO1 (tan bars), anti-RSPO2 (purplebars), or anti-RSPO3 mAb1 (blue bars)antibody. Effect of the anti-RSPOantibodies could not be assessed (ND) onOV38 in absence of WNT3A. Relativeluciferase activity (RLU) was plotted foreach condition next to a no tumor cellcontrol (black bars).






strengthened by the understanding that intracellular compo-nents of the signaling cascade, APC, axin, and b-catenin, aremutated in approximately 90% of human colon tumors (47).Mutations in Wnt pathway components have also now beenobserved in a broad range of human tumors (3). Recently,therapeutic agents have been developed that target the Wntpathway, and these agents show efficacy in a range of preclinicaltumor models (43, 48). OMP-18R5 (vantictumab), an antibodythat functions by blocking WNT binding to 5 of the 10 humanfrizzled receptors, demonstrates activity in inhibiting the growthof a range of human tumor xenograft models, indicating thatextracellular Wnt pathway signals play an important role in thegrowth of many human tumors. Despite this progress, a majorunanswered question remains the specific contribution of var-ious members of the large family of WNT ligands in humantumors, and other signaling proteins that might contribute tob-catenin activation, particularly in tumors that do not possessknown mutations impacting intracellular signaling compo-nents. For instance, despite the demonstration that murineWNT1 can drive the formation of breast tumors when over-

expressed due to retroviral MMTV integration, there has notbeen clear evidence that WNT1 is frequently overexpressedin human tumors. To address this question of which factormight be responsible for driving b-catenin signaling in humantumors, we surveyed a series of human tumors for productionof b-catenin signaling activity using a functional reporterassay. This effort has identified RSPO family members as impor-tant stimulators of b-catenin signaling activity in a numberof human tumors. Patient-derived lung and ovarian tumorcells were shown to induce b-catenin activation in an in vitrocell-based assay and the activity was directly dependent onRSPO2 or RSPO3 expression as demonstrated using specificblocking anti-RSPO antibodies. Interestingly, although RSPO1-or RSPO4-high tumors were identified, none of them displayedany detectable activity in the STF coculture assay. This couldreflect the assay detection limit or/and the induction of differ-ential b-catenin activation levels by different RSPO proteins.

Subsequently, the growth of several of the RSPO-producingtumors was shown to be driven by the RSPO activity whenimplanted in NOD/SCID mice. Using anti-RSPO antibodies

1,000A

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Figure 5.Inhibition of RSPOhigh tumor growth in vivo using specific anti-RSPO antibodies. Ovarian (OMP-OV19 and -OV38), pancreatic (OMP-PN7), lung (OMP-LU2,-LU25, and -LU102), and colorectal (OMP-C28, CR3150, CR2513, CR2506, and CR1560) xenograft models were treated with anti-RSPO1 (green symbols),anti-RSPO2 (blue symbols), or anti-RSPO3 mAb2 (red symbols) antibody alone (up-pointing triangles) or in combination with chemo (down-pointingtriangles) to assess the role of RSPO in driving the in vivo growth of these models. Anti-RSPO1 was used at 10 mg/kg once weekly; anti-RSPO2 was used at10 (OMP-PN7) or 15 (OMP-LU2) mg/kg once weekly or 25 mg/kg every other week (OMP-C28); anti-RSPO3 mAb2 was used at 25 mg/kg once weekly.The chemo agents and regimens were as follows: 15 mg/kg taxol once weekly for OMP-OV19, -OV38, and –LU25, 10 mg/kg taxol once weekly for OMP-LU2, 100mg/kg gemcitabine once weekly for OMP-PN7, and 10 mg/kg irinotecan once weekly for OMP-C28, CR3150, CR2513, CR2506, and CR1560. A, tumor volumeswere measured weekly, averaged for each treatment group (n¼ 10) and graphed as a function of time to obtain growth curves relative to the control antibody(open black circles) and chemo (black circles) groups. Tumor models were deemed responsive when P values calculated using a two-way ANOVA analysisand Bonferroni post-tests were <0.05 (�), <0.01 (��), or <0.001 (��) at termination. Antibody alone was compared with control antibody and chemocombination was compared with control antibody þ chemo agent as depicted by black vertical lines on the graphs. B, the tumorigenicity of anti–RSPO3-treated OMP-LU25 tumors was assessed upon passaging low cell doses in na€�ve NOD/SCID mice. All 10 individual tumor volumes were plotted for each groupafter 100% take rate was observed in the control group.

Chartier et al.





alone and in combination with standard of care chemothera-peutic agents, we were able to significantly decrease the growthrate of RSPO3-positive lung and ovarian tumors that wereidentified in our cell-based screen. In addition, RSPO-expres-sing tumors with no detectable ex vivo b-catenin–activatingproperties, were also shown to respond to antibody-mediatedanti-RSPO treatment. Anti-RSPO1 was shown to inhibit the invivo growth of an RSPO1-high ovarian tumor and anti-RSPO2that of RSPO2-high colon and pancreas tumors. This suggeststhat the reporter assay used to monitor tumor cell–derivedRSPO activity is not sensitive enough to allow for detecting lowlevels of functional RSPO or that a b-catenin–independentfunction of RSPO is at play in these models. Our results alsosuggest that gene-expression analysis may be sufficient toidentify tumors that respond to anti-RSPO therapy. According-ly, three xenograft models in which no RSPO gene expressionwas detected failed to respond to antibody-mediated RSPOinhibition. Testing additional models will be required to fur-ther defining minimal RSPO gene-expression levels associatedwith anti-RSPO treatment sensitivity. Finally, the identificationof an anti-RSPO2 antibody-resistant lung tumor and an anti–RSPO3-resistant colon tumor despite respective high RSPO2and RSPO3 expression indicates that not all instances of highRSPO expression may reflect tumor dependence upon thissignaling axis. Of note, RSPO4 was not tested as a therapeutictarget in this work. However, its relative prevalence across theOMP tumor bank supports evaluating RSPO4 in future studies.Overall, the comprehensive set of in vivo efficacy data presentedhere demonstrates the therapeutic value of RSPO blockade totreat RSPO-expressing tumors.

In anti–RSPO3-sensitive colorectal cancer models, the sig-nificant downregulation of target genes associated with bothnormal stem cells and CSCs supports a mechanism of action by

which the antibody would inhibit tumor growth by decreasingCSC frequency, a most desirable outcome for long-term cancertherapies. CSCs are defined as the most tumorigenic subsetof malignant cells that supports tumor growth by conferringstem cell properties, including self-renewal and multipotencyto tumor cells (49). The major impact of RSPO on stem cells isthought to be due to RSPO potentiation of the Wnt/b-cateninsignaling and thus, one hypothesis is that RSPO may havelimited signaling activity in absence of WNT ligands (4, 50).In this model, tumors may achieve enhanced b-catenin signal-ing by elevated RSPO, which functions to potentiate lowlevels of WNT that are present in the tumor microenvironment,rather than by substantial upregulation of the expression ofWNT family members. Thus, it appears that, in many tumors, asynergistic potentiation of b-catenin function is mediated bytumor-derived RSPO and blocking RSPO represents a mostefficient therapeutic approach in these tumors.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: C. Chartier, C. Bond, J. Cain, M.M. Fischer, W.-C. Yen,A.M. Kapoun, J. Lewicki, T. Hoey, A. GurneyDevelopment of methodology: C. Chartier, F. Axelrod, C. Bond, M.M. Fischer,B. CancillaAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.):C.Chartier, J. Raval, F. Axelrod, C. Bond,M.M. Fischer,J. Shah, J. Wei, M. Ji, A. Lam, M. Stroud, W.-C. Yen, P. Yeung, B. CancillaAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): C. Chartier, J. Raval, C. Bond, J. Cain, S. Ma,M.M. Fischer, J. Shah, J. Wei, M. Ji, W.-C. Yen, B. Cancilla, G. O'Young,M. Wang, A.M. Kapoun, T. Hoey, A. Gurney

CR3150 CR2506 CR2513 CR1560 CR3150 CR2506 CR2513 CR1560 CR3150 CR2506 CR2513

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Figure 6.Downregulation of select Wnt target genes in anti–RSPO3-treated colorectal cancer xenografts. ASCL2, AXIN2, CLDN2, LGR5, PTPRO, RPRD1A, TDGF1, andZNRF3 genes were analyzed for expression levels in colorectal cancer xenografts CR3150, CR2506, CR2513, and CR1560 treated with control antibody(small plaid pattern bars) or anti-RSPO3 (large plaid pattern bars). Only high quality RNA samples were included in the analysis, resulting in thefollowing sample sizes: n ¼ 15 for control antibody in CR3150 and CR2506, n ¼ 14 for control antibody in CR2513 and CR1560, n ¼ 3 for anti-RSPO3 in CR3150,n ¼ 10 for anti-RSPO3 in CR2506, n ¼ 8 for anti-RSPO3 in CR2513, and n ¼ 9 for anti-RSPO3 in CR1560. Results were expressed as relative quantityover GAPDH endogenous control gene. P values were calculated using a t test with Welch correction and anti–RSPO3-induced decrease considered significantwhen below � , 0.05, �� , 0.01, or �� , 0.001.






Writing, review, and/or revision of the manuscript: C. Chartier, J. Cain,M.M. Fischer, W.-C. Yen, A.M. Kapoun, J. Lewicki, T. Hoey, A. GurneyAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): C. Chartier, S. Ma, G. O'Young, T. HoeyStudy supervision: C. Chartier, C. Bond, P. Yeung, T. HoeyOther (as the lead in the in vivo experiment): C. Dee-Hoskins

AcknowledgmentsThe authors thank many people at OncoMed Pharmaceuticals for their

contributions to this work, including Peter Stathis, Ian Scott, Esohe Idu-sogie, Jim Evans, Xiaomei Song, Diane Pardi, and Michael Mulkerrin.The authors thank Zhun Wang, Jie Cai, and Henry Li from Crown Biosci-

ence for their assistance. Research funding was provided by OncoMedPharmaceuticals.

Grant SupportResearch funding was provided by OncoMed Pharmaceuticals.The costs of publication of this articlewere defrayed inpart by the payment of

page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received February 25, 2015; revisedOctober 12, 2015; acceptedNovember 5,2015; published OnlineFirst December 30, 2015.

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2016;76:713-723. Published OnlineFirst December 30, 2015.Cancer Res Cecile Chartier, Janak Raval, Fumiko Axelrod, et al. -Catenin Signaling and Tumorigenesis in Multiple Cancer Types

βTherapeutic Targeting of Tumor-Derived R-Spondin Attenuates

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