AXL Is a Putative Tumor Suppressor and Dormancy Regulator ... · Cell Cycle and Senescence AXL Is a Putative Tumor Suppressor and Dormancy Regulator in Prostate Cancer Haley D. Axelrod1,2,

Cell Cycle and Senescence

AXL Is a Putative Tumor Suppressor andDormancy Regulator in Prostate CancerHaley D. Axelrod1,2, Kenneth C. Valkenburg2, Sarah R. Amend2, Jessica L. Hicks3,Princy Parsana4, Gonzalo Torga2, Angelo M. DeMarzo3,5, and Kenneth J. Pienta2

Abstract

Prostate cancer bone metastasis remains lethal and incur-able, and often arises years after elimination of the primarytumor. It is unclear what underlies the decades-long clinicallatency before recurrence, but evidence points to the existenceof dormant residual tumor cells that disseminated before theprimary tumor was eliminated. To design therapies to preventprogression of disseminated tumor cells (DTC) into lethalmetastases, it is crucial to understand the mechanism(s)underlying this dormancy. The current study functionallyvalidated our previous observation that implicated theGAS6/AXL axis in mediating DTC dormancy in the bonemarrow. AXL-null and AXL-overexpressing prostate cancer celllines were generated to determine if AXL was necessary and/orsufficient for dormancy. Characterization of these cells in vitroand using in vivomouse models of DTC growth demonstrated

that AXL was indeed sufficient to induce dormancy, but wasunable to maintain it long-term and was not absolutelyrequired for a dormancy period. Clinically, AXL expressioncorrelated with longer survival in prostate cancer patients,and AXL was not expressed by cancer cells in primary ormetastatic tissue. These data point to a tumor-suppressiverole for AXL in prostate cancer, and future work is requiredto determine if AXL is expressed on human bone marrowDTCs.

Implications: The ability of AXL to initiate but not maintaindormancy, coupled with its dispensability, suggests thattargeting AXL alone will not prevent lethal metastatic out-growth, and likely a cooperative network of factors exists tomediate long-term cellular dormancy.

IntroductionAlmost 165,000 men are diagnosed with prostate cancer every

year in the United States, making it the second-most commoncancer among men (1). Although approximately 70% of patientsare cured with primary local treatment, the remaining 30%experience biochemical recurrence [BCR; detection of circulatingprostate-specific antigen (PSA)], and approximately 29,000 menwill die each year from progression to fully metastatic disease(1, 2). Themost common site for prostate cancer metastasis is thebone, and virtually all men who die frommetastatic disease havesome level of bone involvement (3). There is currently no cure formetastatic prostate cancer, and the 5-year survival rate foradvanced cases with bone involvement is 1% compared with56% for those without (4).

Themajority ofmetastatic prostate cancer cases occur years afterelimination of the primary tumor followed by a long clinical

"disease-free" period. It is widely accepted that cancer cells dis-seminate early during primary tumor development, and it isthought that upon entering the secondary site (e.g., bone mar-row), disseminated tumor cells (DTC) can undergo a period ofdormancy, allowing them to evade therapy and clinical detectionfor years to decades (5, 6). The concept of tumor dormancy hasexpanded to include multiple definitions but is not well studieddue to the limitations of robust in vitro models. Tumor massdormancy, referring to both angiogenic and immunologic controlof growth by a balance of cellular proliferation and death, is likelynot able to be sustained long term (6, 7). Cellular dormancy, onthe other hand, the reversible quiescence of individual cells, likelyunderlies the observed clinical latency observed over a decade ormore (8–10). Because dormant bonemarrowDTCs are thought tobe the seeds for lethal prostate cancer metastasis, understandingthe mechanism(s) that govern cellular dormancy would providean opportunity for therapeutic intervention to prevent recurrentdisease altogether (11).

It has been demonstrated that prostate cancer DTCs preferen-tially home to the hematopoietic stem cell (HSC) endosteal nichewithin the bone marrow and bind to osteoblasts, displacingresident HSCs and co-opting their microenvironment (12).Because the role of the HSC niche is to support the quiescenceand self-renewal properties of HSCs, niche factors may similarlysupport the quiescence of DTCs (13, 14). We previously showedthat one of these niche factors, growth arrest–specific 6 (GAS6), issecreted by osteoblasts and restricts the growth of prostate cancercells (13). GAS6 is a ligand for the TAM (TYRO3, AXL, MER)family of receptor tyrosine kinases and plays various roles tosupport normal physiologic functions, such as platelet aggrega-tion, apoptotic cell clearance, and, importantly, the self-renewalpotential of HSCs (15). In many types of cancer, the TAM

1The Cellular and Molecular Medicine Program, Johns Hopkins University,Baltimore, Maryland. 2The James Buchanan Brady Urological Institute,Johns Hopkins University, Baltimore, Maryland. 3The Sidney KimmelComprehensive Cancer Center, Johns Hopkins University, Baltimore, Maryland.4Department of Computer Science, Johns Hopkins University, Baltimore,Maryland. 5Departments of Pathology, Urology, and Oncology, The JohnsHopkins School of Medicine, Johns Hopkins University, Baltimore, Maryland.

Note: Supplementary data for this article are available at Molecular CancerResearch Online (http://mcr.aacrjournals.org/).

Corresponding Author: Haley D. Axelrod, The Johns Hopkins School ofMedicine, 600 North Wolfe Street, Marburg Room 105, Baltimore, MD 21287.Phone: 410-504-4974; E-mail: [email protected]

doi: 10.1158/1541-7786.MCR-18-0718

�2018 American Association for Cancer Research.

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receptors become upregulated and contribute to various hall-marks of cancer (15). The functional consequences of GAS6binding to each of its receptors are context-dependent andwide-ranging. We previously showed that two of the receptors,AXL and TYRO3, play opposing roles in prostate cancer. In ametastatic prostate cancer mouse model, AXL expression is asso-ciated with dormant DTCs, while TYRO3 expression is associatedwith proliferating tumors (16).

In this study, we functionally validated the role of GAS6/AXLin mediating dormancy of DTCs with the overall goal ofdetermining its clinical utility in preventing lethal metastases.We generated AXL-overexpression and AXL-null prostate cancercell lines and characterized their growth in vitro and in vivo. Wefound that AXL alone was not necessary for a dormant phaseand is therefore likely not a suitable stand-alone target toprevent metastatic outgrowth. Importantly, however, AXL wassufficient to restrict proliferation of prostate cancer cells. Fur-ther, we found that cancer cells in outgrown human primaryprostate tumors and metastatic lesions are AXL-negative. Ourfindings highlight a potential tumor suppressor role for AXL asan inducer of dormancy in prostate cancer.

Materials and MethodsGeneration of AXL knockout cells using CRISPR/Cas9

A gRNA targeting the second exon of human AXL (50-AACCTG-GAGCTGACACCGAA-30), designed using CHOPCHOP (https://chopchop.rc.fas.harvard.edu/), was generated by annealing andamplifying two overlapping oligos (designed using http://crispr.technology/; ref. 17). The gRNA was cloned into the pH1v1 byGibson assembly40 (NEB) with AvrII digestion (18). PC3luc cellswere transfected with the gRNA plasmid and Cas9-P2A-GFPplasmid. GFPþ cells were sorted and seeded at a low density toisolate and pick colonies. Clones were screened by PCR (primersforward/reverse: 50-CTGTTTCTCTCTCTTTCACAGTCTC-30/50-TA-GAGGTTCCATCACATGCTCAAAG-30) followed by Sangersequencing (primers forward/reverse: 50-TCTCTCTCTCTCTTCT-CAGCCTC-30/50-ACAAGTGGTCAAACTGGGGT-30) of the gRNAtarget site (Johns Hopkins Synthesis and Sequencing Facility).Sequencing reads were aligned to PC3luc (SnapGene). Clonalityof the alterations was confirmed using TIDE analysis (19).The Cntl KO clone was isolated from clonal expansion andwas confirmed to have no genetic alterations around the gRNAtarget region by sequencing and TIDE analysis. The pH1v1 andCas9-P2A-GFP plasmids were a gift fromDonald Zack (The JohnsHopkins University School of Medicine, Baltimore, MA).

Generation of AXL overexpression cellsThe Gateway pENTR221 vector containing the AXL CDS from

the UltimateORF Clones collection (Invitrogen, clone IDIOH22600) was obtained through the Johns Hopkins High-Throughput Biology Center. Sanger sequencing confirmed aknown SNP at position 2456 of the corresponding mRNAsequence (BC032229). Site-directed mutagenesis was used tocorrect the SNP to match the gDNA sequence of human AXL(primers forward/reverse: 50-CTATCTGCGCCAGGGAAATCGCC-TGAAG-30/50-CTTCAGGCGATTTCCCTGGCGCAGATAG-30) asdescribed previously, and validated by Sanger sequencing (pri-mers: 50-CAGGAAACAGCTATGACC-30, 50-TACTACCGCCAGG-GACGTAT-30; ref. 20). The corrected AXL CDS was subclonedinto a pLenti CMV Neo DEST (705-1) vector gifted from Eric

Campeau and Paul Kaufman (Addgene plasmid #17392) andconfirmed by Sanger sequencing (primers: 50-ACGGGTCTGTG-TCCAATCTG-30, 50-CTTATCCCCACTTGCAGCCC-30, 50-CGCAA-ATGGGCGGTAGGCGTG-30; ref. 21).

For the Tet-On vector, PCR-based cloning of the correctedpENTR221-AXL CDS vector via restriction sites EcoRI and AgeIwas performed (primers forward/reverse: 50-TAAGCAGAAT-TCATGGCGTGGCGGTGCCCCAGGAT-30/50-TGCTTAACCGGTT-CAGGCACCATCCTCCTGCCCT-30) according to https://www.addgene.org/protocols/pcr-cloning/ (Addgene) and validated bySanger sequencing (primers: 50-TACTACCGCCAGGGACGTAT-30,50-CAGGAAACAGCTATGACC-30). The amplified EcoRI-AXLCDS-AgeI amplicon was cloned into the pLVX-Tet-One-Puro vector(Clontech, #631847) and confirmed by Sanger sequencing (pri-mers: 50-GCTTGGCAGCTCAGGTTGAA-30, 50-TACTACCGCCAG-GGACGTAT-30, 50-AACGGACGTGAAGAATGTG-30).

Lentivirus was made by cotransfection of HEK293T cells withthe appropriate expressionplasmid, an envelopeplasmid (pMD2.G, Addgene plasmid #12259), and a packaging plasmid (psPAX2,Addgene plasmid #12260). pMD2.G and psPAX2 plasmids weregifts from Didier Trono. For viral transduction, virus-containingmedia was removed from the HEK293T cells and applied to theC42Bluc target cells with polybrene (Sigma-Aldrich, #H9268).Target cells were selected: pDEST plasmids with Neomycin(0.5 mg/mL, Thermo Fisher, #10131035), and Tet-One plasmidswith Puromycin (1 mg/mL, Sigma-Aldrich, #P8833). Cellsremained under selection for 5 passages.

Cell cultureCells were cultured at 37�C and 5% CO2. HEK293T cells

(ATCC) were maintained in DMEM with 10% FBS and 1%penicillin/streptomycin. PC3luc cells, generated as described pre-viously, and C42Bluc cells (gift from Evan Keller, University ofMichigan, Ann Arbor, MI) were maintained in RPMI with 10%FBS and 1% penicillin/streptomycin (22). All cell lines wereauthenticated and tested for mycoplasma (Genetica) by passage20 and were not used past passage 40. Tet-On cell lines weremaintained in media containing Tet-free FBS (Clontech,#631107) and in the absence of doxycycline. When necessary,doxycycline (Sigma-Aldrich, #D9891) was administered at thetime of seeding at 100 ng/mL unless otherwise indicated. ForGAS6 treatment, rhGAS6 (R&D Systems, #885-GS-050) wasadministered at 100 ng/mL (unless otherwise indicated) for 30minutes. Each rhGAS6 lot was tested for activity using pAKT as areadout in H1299 cells (gift from Kolltan Pharmaceuticals Inc.;not authenticated or tested for mycoplasma since received), whichhave high AXL expression. Prior to all experiments, cells weresynchronized by serumdeprivation (0.1%FBS) overnight prior toseeding.

Western blottingLysates were collected in Frackelton lysis buffer with

Halt Protease and Phosphatase Inhibitor Cocktail (Thermo Fish-er, #78442; ref. 23). Proteinwas fractionatedon a4% to20%SDS-PAGE gel (Bio-Rad, #456-1093) and transferred to a nitrocellu-lose membrane (Bio-Rad, #1704158; Trans-Blot Turbo TransferSystem). After blocking, membranes were incubated with AXL(Cell Signaling Technology, #4939) and ACTIN (Sigma-Aldrich,#A5441) antibodies or TYRO3 (Cell Signaling Technology,#5585) and ACTIN antibodies (diluted 1:1,000). Anti-rabbitand anti-mouse secondary antibodies (LI-COR, #926-32211 and

AXL Plays a Tumor-Suppressive Role in Prostate Cancer

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https://chopchop.rc.fas.harvard.edu/

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http://crispr.technology/

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https://www.addgene.org/protocols/pcr-cloning/


#926-68070) were combined for secondary detection. Blots wereimaged using the LI-COROdyssey scanner (LI-COR Biosciences).PC3 samples were prepared for two simultaneous immuno-blots for AXL/ACTIN and TYRO3/ACTIN.

Reverse transcription and quantitative PCRRNA was extracted using the RNeasy Mini Kit (Qiagen), and

RNA was reverse transcribed with the iScript cDNA Synthesis Kit(Bio-Rad, #170-8891). qRT-PCR was performed in triplicate withSsoFast EvaGreen Supermix (Bio-Rad, #1725201). qRT-PCR anal-ysis was done using the delta-delta Ct method normalizing toACTIN. Primer sequences are listed in Supplementary File 1.

Flow-cytometric analysisGeneral processing for flow cytometry analysis was as follows:

cells were dissociated using enzyme-free Cell Dissociation Buffer(Thermo Fisher, #13151014). Cells were stained with AXL-488antibody (R&D Systems, #FAB154G). Data were collected using aBio-Rad S3 Cell Sorter, and analysis was performed using FlowJo.When indicated, 7-AAD (BioLegend, #420404) was added toidentify dead cells. For EdU incorporation analysis, seeded cellswere cultured � doxycycline, � R428 for 4 days, and EdU wasadded to cultures 2 hours prior to sample collection and Click-iTEdUdetection (ThermoFisher, #C10425)was followed accordingto themanufacturer's protocol except for a further 1:10 dilution ofthe azide. EdU was detected with an AF488 azide and AXL wasdetected with AXL-PE (R&D Systems, #FAB154P).

Immunofluorescence EdU proliferation assayCells were seeded on chamber slides (Corning, #354114) �

GAS6. On day 3, GAS6 was respiked and EdU added (finalconcentration 10 mmol/L). Twenty-four hours later, cells werestained by IF using the Click-iT EdU Alexa Fluor 555 Imaging Kit(Thermo Fisher, #C10338) and stained for DAPI. Stained slideswere imaged manually as described previously (24). Slides fromthe same experiment were imaged using the same settings. Threefields of view (FOV)were captured per sample, and the percentageof EdUþ cells was manually counted for each FOV and averaged.

Plate-based BrdUrd proliferation assayBrdUrd incorporation over 24 hours was detected using a

colorimetric ELISA (Sigma-Aldrich, #11647229001). GAS6 wasadded at time of seeding and respiked on day 4. Five technicalreplicates were averaged, and fold changes were calculated basedon day 2 signal.

Soft-agar assayThe soft-agar assay was performed according to ref. 25. Cells

were seeded (10,000 cells per well PC3 and constitutive AXLoverexpression; 5,000 cells per well for Tet-On cells), and doxy-cycline or GAS6 was included in the top layer of media and wasadded twice a week. After incubation of cultures in nitrobluetetrazolium chloride (Sigma-Aldrich, #N5514) overnight at 37�C,colonies were imaged. FIJI software (http://fiji.sc, particle analysisfunction using a pixel size range of 10-infinity and a circularityrange of 0.8–1.0) was used to quantify the number and size ofcolonies.

Human platelet isolationBlood from a healthy volunteer was drawn into a Vacutainer

containing ACD buffer (BD Biosciences, #364606) and processedaccording to http://www.abcam.com/protocols/isolation-of-

human-platelets-from-whole-blood. All recommended stepsincluded Prostaglandin E1 (Sigma-Aldrich, #P7527) to preventpremature platelet aggregation. Platelets were labeled with DiI(Thermo Fisher, #V22885) and filtered prior to coculture. Cellswere exposed � doxycycline for 3 days, dissociated usingenzyme-free buffer, and labeled with NucBlue Live ReadyP-robes Reagent (Thermo Fisher, #R37605) prior to plating.Coculture (1,000 C42B cells: 20 � 106 platelets) was platedin technical duplicates � doxycycline, � R428 (50 nmol/L orDMSO vehicle control; Selleckchem.com, #S2841) as indicated.Images (phase, DAPI, DsRed) were taken daily (EVOS FL AutoImaging System). Colocalization was quantified using the FIJIColocalization Threshold feature (percentage of DAPI volumecovered by DsRed signal). Results were averaged betweentechnical replicates, and treated samples were normalized tountreated.

Characterization of aggregates with R428Cells were seeded in duplicate � doxycycline, � R428 and

images taken daily. On day 3 NucLight Rapid Red Reagent(1:500; IncuCyte, #4717) was added. CellTiter96 (Promega,#G3580) was used to assess viability on day 5. Aggregates werequantified using FIJI 3D Object Counter (threshold: 3,140;min/max: 10/122880). Numbers of aggregate size were com-bined for technical duplicates, and skewness was measured(GraphPad Prism 6).

Mouse model of intracardiac injection and tissue harvestingThe Johns Hopkins Institutional Animal Care and Use Com-

mittee approved all experiments involving mice (protocol#M016M41). Four- to 8-week-old male NOD/SCID/gammanull (NSG) mice were obtained from an immunodeficientmouse colony housed at Johns Hopkins. 200,000 PC3, 1 �106 C42B, or DPBS (negative control) cells were injected intothe left ventricle of the heart. For C42B AXL overexpressionexperiments, half of the mice received 0.1 mg doxycyclineintraperitoneal (i.p.) 1 to 2 hours prior to intracardiac injec-tion, and doxycycline was administered in the drinking water(2 mg/mL þ 1% sucrose) for the duration of the experiment.Mice were imaged weekly to assess tumor burden (IVIS Spec-trum In Vivo Imaging System; PerkinElmer). Whole body totalflux (photons/sec) was quantified (Living Image 4.4). At 109

flux, mice were injected i.p. with EdU (1 mg; Thermo Fisher,#A10044) and 24 hours later euthanized. Soft tissues were fixedin 10%NBF and stored in 70% ethanol.Hind-limbbonemarrow(femur and tibia) was collected/processed/stored as describedpreviously (26, 27).

Immunofluorescent DTC detection in miceBone marrow slides were stained as previously detailed for

human bone marrow (24). Instead of human, mouse Fc block(BioLegend, #101320) was used. A cocktail of human/epithelial-specific antibodies was combined to detect DTCs (HLA-A: Abcam,#ab52922; pan-cytokeratin: Abcam, #ab9377; human nucleolin-488: Abcam, #ab154028) and detected by Alexa Fluor (AF) 488 F(ab) fragment goat anti-rabbit secondary antibody (JacksonImmunoResearch Laboratories, #111-547-003). A cocktail ofWBC markers (CD45: BioLegend #103102; CD11b: BioLegend#101201) was detected by an AF555 goat anti-rat secondaryantibody (Thermo Fisher, #A-21434). EdU was detected usingthe Click-iT EdU Alexa Fluor 647 Imaging Kit (Thermo Fisher,

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#C10340). DTCs were counted using theMetafer5 (MetaSystems,V3.11.8) automated scanning system as described previously(24). A 4-color classifier was used (minimum/maximum expo-sure times, seconds: DAPI 0.0012/0.0092, AF488 0.0092/0.16,AF555 0.0092/0.08, and AF647 0.0092/0.16).

IHC staining of mouse samplesTissue was paraffin-embedded and sectioned by the Johns

Hopkins Reference Histology Laboratory. Sections were stainedaccording to https://www.cellsignal.com/contents/resources-protocols/immunohistochemistry-protocol-(paraffin)/ihc-paraffinfor chromogenic staining with slight modification: deparaffiniza-tion with Citrisolv (Fisher Scientific, #22-143-975); antigenunmasking with steaming in Target Retrieval Solution (Dako,#S1699); AXL antibody (1:300; Cell Signaling Technology,#8661); goat anti-rabbit biotinylated secondary antibody(1:200; Vector Labs, #BA-1000) detected by ABC HRP reagent(Vector Labs, #NC9313719).

Sections stained by multiplex IF were blocked with TrueBlack(Chemometech, #23007) and stained with the Click-iT EdUAlexa Fluor 647 Imaging Kit. Reagents: Human nucleolinAF488 (1:200), AXL antibody (1:300; Cell Signaling Technology,#8661), and Cy3 F(ab) fragment goat anti-rabbit secondaryantibody (Jackson ImmunoResearch Laboratories, #111-167-003).

Metastatic patient tissue IHCThe rapid autopsy program was approved by the Institutional

Review Board (IRB numberNA_00036610) at The JohnsHopkinsUniversity School of Medicine. Metastatic castrate-resistant pros-tate cancer samples were obtained from different patients. Tissueswere isolated 0 to 24 hours postmortem and fixed in 10%neutral-buffered formalin for 48 hours and paraffin-embedded andsectioned by the Johns Hopkins Reference Histology Laboratory.Sections were stained as above for mouse tissues with AXLantibody.

Human prostate tissue microarray (TMA) stainingThis study was approved by the Johns Hopkins institutional

internal review board and followed the U.S. Common Rule.Prostate TMAs were prepared as in ref. 28. AXL staining (1:400,Cell Signaling Technology, #8661) was performed on four TMAsusing the Discovery anti-Rabbit HQ kit (Roche). One TMAincluded 52 cases of matched normal/benign, tumor, and lymphnode metastases from prostatectomies of untreated patients. The3 other TMAs each contained 40 cases of normal/benign andtumor tissue from prostatectomies.

Microarray sample collection and analysisRNA was extracted from Tet-ø, Tet-Axl 1, and Tet-Axl 2 cells

seeded � doxycycline for 4 days. Quality assessment, cDNAconversion, and the Agilent Human Gene Expression 4 � 44KMicroarray were conducted at The Sidney Kimmel CancerCenter Microarray Core Facility at Johns Hopkins University.Samples were background-corrected and normalized betweenarrays. Probe sets without gene names were removed, andbatch effects were corrected before transforming the data tolog fold change. While accounting for batch, limma package inR tested for differential expression. Differentially expressedgenes had logFC � 1.5 and BH adjusted P value � 0.05. Rawdata can be found on the NCBI Gene-Expression Omnibus

database with the accession number GSE119003. Relevantcode is available at Code Ocean: https://codeocean.com/2018/07/02/axl-putative-tumor-supressor-and-dormancy-regulator-in-prostate-cancer/.

AXL expression analysis in patient tumor data setsThe expression profiles (n ¼ 1,405) of retrospective patients

were extracted from the Decipher GRID registry (NCT02609269).The retrospective GRID cohort was pooled from five publishedmicroarray studies: Cleveland Clinic (PMID: 25466945), JohnsHopkins (PMID: 26058959), Mayo Clinic (PMID: 23826159 and23770138), and Thomas JeffersonUniversity (PMID: 25667284).IRB approval was obtained from the participating institutionsprior to initiating the current study.

Survival curves: recurrence-free survival was determined forpatients with high (above median) and low (below median)AXL expression. P value was calculated using the log-rank test(reanalysis of GSE21032, Taylor and colleagues 2010 (33);visualized using Project Betastasis http://www.betastasis.com).

Statistical analysisAll statistical analyses were performed using GraphPad

Prism 6. For comparisons of fold change to control samples,analysis of test samples was performed using a one-samplet test against the null hypothesis, log fold change ¼ 0. Formouse experiments, mice were randomized into groups.Fifteen mice from each group were injected intracardiac toobtain 10 successfully injected mice. Successful injections wereclassified as having no bioluminescent imaging (BLI) signal inthe heart. BLI imaging and analysis and bone marrow DTCcounting were all performed blinded to sample type. Allcomparisons are not considered significant (ns, P > 0.05)unless otherwise indicated. Significance: �, P � 0.05; ��, P �0.01; ��, P � 0.001; ��, P � 0.0001.

ResultsAXL is not necessary for prostate cancer cell dormancy

We initially observed an increase in AXL expression in PC3prostate cancer cells when grown at low density as individualcells ("early DTCs") relative to full confluence ("proliferatingtumor mass"; Fig. 1A). We tested if loss of AXL in these cellswould prevent dormancy and/or increase proliferation bygenerating AXL-null PC3 cells using CRISPR. We isolated twoAXL knockout clones (Axl KO 1 and Axl KO 2) and a controlclone from the same population (Cntl KO) and confirmedloss of expression by Western blot, qPCR, and Sangersequencing (Fig. 1B; Supplementary Fig. S1A–S1B). Interest-ingly, AXL knockout increased expression of TYRO3, but onlyat the protein level (Supplementary Fig. S1A and S1C). Lossof AXL did not affect the genes involved in the epithelial-to-mesenchymal transition (EMT) or stemness, two commonphenotypes known to be influenced by AXL (SupplementaryFig. S1D–S1E).

There was no change in the proliferation rate of Axl KOcompared with controls as assessed by BrdUrd incorporation,and upon rhGAS6 treatment, only one Axl KO clone actuallyincreased proliferation (Fig. 1C). When grown in 3D culture insoft agar, Axl KO clones did not display any difference in theirability tooutgrow into large colonies comparedwithPC3parentalor Cntl KO cells, independent of GAS6 treatment (Fig. 1D;Supplementary Fig. S1F).





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AXL knockout does not prevent dormancy.A,Western blot showing AXL expression (left) in PC3 cells seeded at various densities (right, phase images; n¼ 1). Arrowrepresents 125 kDa. Scale bar, 500 mm. B, AXL expression by Western blot in PC3 cells (Par), a control knockout clone (Cntl KO), and two AXL knockoutclones (Axl KO1 andAxl KO2; n¼ 1). Arrow represents 125 kDa.C,BrdUrd incorporation over 24 hours in control and knockout cell lines�GAS6. Results represent foldchange between the incorporation readout on days 6 to 2 (n ¼ 3). Error bars, mean � SEM; P values calculated using the t test between Axl KO and Cntl KO,andwithin each cell line between treatments.D,Quantification of the number of large colonies (number of colonies in the top 65% of the range of colony size) in softagar � GAS6, relative to reference sample Par -GAS6 (n ¼ 3). Error bars, mean � SEM; P values calculated using multiple t-tests to assess differences due totreatment. E, Number and proliferation status of bone marrow DTCs detected in mice at 1 week after intracardiac injection. Each bar represents 1 mouse (n¼ 7 pergroup). F, Time in days to tumorigenesis after intracardiac injection. Tumorigenesis was defined as BLI signal over control PBS-injected mice (Cntl KO, n ¼ 9;Axl KO, n¼ 12; each dot represents onemouse;mean� SD).G,Duration of tumor growth of each group after intracardiac injection. Duration of growthwas defined asthe time between the onset of tumorigenesis and time to lethal tumor burden (Cntl KO, n ¼ 9; Axl KO, n ¼ 12; each dot represents one mouse; mean � SD).H, Survival proportions for each group of mice over time (Cntl KO, n¼ 9mice; Axl KO, n¼ 12 mice). P value calculated by the log-rank test. For relevant experiments,GAS6 was added at 100 ng/mL at the time of seeding. For mouse tumor growth P values were calculated by the t test. I, Number and proliferation statusof bone marrow DTCs detected in mice at the time of death. Counting stopped after the first 500 BM-DTCs (n ¼ 5 for each group).

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Figure 2.

AXL overexpression decreases proliferation in vitro.A,AXL expression byWestern blot in C42B control Neomycin-resistant cells (Neo), and twoAXL overexpressionclones (Axl 1 and Axl 2; n ¼ 1). Arrow represents 125 kDa. B, EdU incorporation over 24 hours in C42B Axl 1 and 2 cells compared with Neo control � GAS6 by IF.Individual cells were counted manually in 3 FOV per sample (n ¼ 2). P values calculated using the t test between cell lines and their control, and within eachcell line between treatments. C, Quantification of the number of large colonies (number of colonies in the top 16% of the range of colony size) in soft agar� GAS6, relative to reference sample Neo -GAS6 (n ¼ 2). D, AXL expression after dead cell exclusion by flow cytometry in an AXL overexpression clone overincreasing passage number. Cells were reselected with Neomycin over 2 passages between passages 16 and 19 (n ¼ 1). E, AXL expression by Western blot inconditionally overexpressing AXL clones (Tet-Axl 1 and Tet-Axl 2) compared with empty vector control (Tet-ø) � doxycycline (n ¼ 1). Arrow represents125 kDa. F, EdU incorporation over 2 hours in Tet-Axl 1 and 2 cells compared with Tet-ø control � doxycycline by flow cytometry (n ¼ 3). P values calculated usingone-sample t test against the null hypothesis (log fold change ¼ 0) was performed between treated samples. G, EdU incorporation over 2 hours in all celllines� doxycycline and AXL inhibitor R428 by flow cytometry; fold change is relative to untreated (Tet-ø and Tet-Axl 2, n¼ 3; Tet-Axl 1, n¼ 2). P values calculatedusing the Dunnett multiple comparison test against þDox samples for each cell line, excluding �Dox samples. H, Quantification of the number of largecolonies (number of colonies in the top 13% of the range of colony size) in soft agar � doxycycline, relative to reference sample Tet-ø �Dox (n ¼ 2). For eachexperiment, treatments were initiated at the time of cell seeding; GAS6: 100 ng/mL, doxycycline: 100 ng/mL, R428: 50 nmol/L. Error bars, mean � SEM.For soft-agar assays, P values were calculated using multiple t tests to assess differences due to treatment.






Because in vitro studies are a limited model to study dor-mancy and are only an approximation of the dormancy phasein vivo, we next directly tested whether loss of AXL wouldprevent the dormant phase of bone marrow DTCs in a mousemodel of DTC growth. Cntl KO or Axl KO 2 cells wereinoculated intracardiac in NSG mice. Mice were injected withEdU 1 day prior to sacrifice to detect proliferating cells. At the 1-week time point, there was no difference in the number orproliferation status of DTCs that reached the bone marrow. Thepresence of a similar number of EdU� DTCs in each groupindicated the dormancy period was not bypassed by the loss ofAXL (Fig. 1E). A separate cohort of mice from each group wasmonitored over time by weekly BLI until whole body tumorsignal reached a lethal threshold. There was no difference

between the two groups in terms of either the time it tookto reach first detectable tumor burden or the duration of thetumor growth from first detectable signal to lethal threshold(Fig. 1F and G). However, there was a statistically significantdifference indicating better survival overall for mice with AXLKO tumors; this is likely due to the greater number of mice inthe KO group with a successful intracardiac injection despiterandomizing the groups, indicating this may not be biologi-cally significant (Fig. 1H). Focusing on bone-specific signal,there was not a statistically significant increase in the number ofmice with hind leg and/or jaw signal in the Axl KO group, andthere was no difference in the number or proliferation status ofbone marrow DTCs at the time of death (SupplementaryFig. S1G; Fig. 1I).

(73.2%)

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Figure 3.

HighAXL expression leads to cell aggregation.A,Overlay of phase and nuclear labeling (NucLight Rapid RedReagent) images of conditional AXL overexpression celllines seeded � doxycycline for 4 days (n ¼ 1). B, Tet-Axl cells (pooled population prior to Tet-Axl 1 and 2 cloning) seeded with increasing concentrations ofdoxycycline as indicated (ng/mL) were grown for 3 days and were imaged or collected for protein expression analysis byWestern blot (n¼ 1). Arrow represents 125kDa. C, Phase images of Tet-Axl 1 cells seeded without doxycycline (�) over 4 days (top row), with 100 ng/mL doxycycline (þ) over 4 days (middle row),and after doxycycline withdrawal on day 2 (bottom row). Numbers indicate the percentage of AXL-positive cells in each condition by flow cytometry (n¼ 1). Imageswere taken on indicated days, and contrast was enhanced to the same degree for each image to more easily visualize cells. (Continued on the following page.)

Axelrod et al.





AXL overexpression decreases proliferation in vitroTo determine if AXL is sufficient to induce dormancy, we

generated C42B prostate cancer cells with constitutive overexpres-sion of AXL. C42B cells expressing a control Neomycin-resistancevector (Neo) did not express endogenous AXL, and AXL expres-sion was observed only in two overexpression clones (Axl 1 andAxl 2; Fig. 2A; Supplementary Fig. S2A–S2B). EMT and stemnessgenes were largely unaffected by AXL overexpression (Supple-mentary Fig. S2C–S2D).

To determine if AXL overexpression decreased the rate ofproliferation, we performed EdU labeling. We observed a slightdecrease in the number of EdUþ cells by immunofluorescent (IF)detection in AXL-overexpressing cell lines relative to Neo control(Fig. 2B). This was also confirmed by detection of BrdUrd incor-poration by a plate-based ELISA assay; however, this population-based method was not as sensitive as individual cell counting(Supplementary Fig. S2E). Surprisingly, when grown in soft agar,AXL-overexpressing cells did not show significantly reduced out-growth compared with Neo control cells, regardless of GAS6(Fig. 2C; Supplementary Fig. S2F).

We hypothesized that the modest proliferation changesobserved may have been due to the loss of AXL expressionover time in the population, wherein AXLhi cells do not pro-

liferate, leading to the overrepresentation of AXLneg or AXLlow

cells. We did in fact observe a decrease in the percentage of AXL-positive cells with increasing passage of an AXL overexpressionclone that was unaffected by reselection of the cells withNeomycin (Fig. 2D). Therefore, to improve our model of AXLoverexpression, we cloned the AXL ORF into a tetracycline-Onsystem (Tet-Axl 1 and Tet-Axl 2) in which AXL was expressedonly in the presence of doxycycline ("AXL-on"), and cells weremaintained without doxycycline ("AXL-off"; Fig. 2E; Supple-mentary Fig. S2G–S2H). Again, AXL overexpression didnot alter EMT or stemness gene expression (SupplementaryFig. S2I–S2J).

Whenwe assessed the percentage of cells that incorporated EdUin these cell lines, Tet-Axl clones proliferated at a slower rate whentreated with doxycycline, compared with Tet-ø control cells thatwere unaffected by doxycycline treatment (Fig. 2F). The decreasein proliferation was more evident in the Tet-Axl 2 clone that hadhigher AXL expression. Inhibition of AXL signaling with R428, anAXL-specific kinase inhibitor, increased proliferation rates of Tet-Axl cells with doxycycline back to baseline levels (Fig. 2G).Surprisingly, these cells did not exhibit any differences in out-growth ability when grown in soft agar (Fig. 2H; SupplementaryFig. S2K).

D Tet-Axl 2Tet-ø0

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Figure 3.

(Continued. ) D, Representative phase and nuclear labeling (NucLight Rapid Red Reagent) overlay images of Tet-ø and Tet-Axl 2 cells seeded with doxycycline(100 ng/mL) for 5 days � AXL inhibitor R428 (left) and quantification of skewness of aggregate size with increasing concentrations of R428 (right; n ¼ 3).P values calculated using the t test betweenR428-treated and untreated. E, Left, representative images of Tet-ø and Tet-Axl 1 cells (phase, NucBlue Live-Cell Stain)�doxycycline (100 ng/mL) and cocultured with human platelets (red). Right, fold change of quantified colocalization of nuclear area (blue) and platelet signal(red) relative to �Dox (n ¼ 2 for samples without R428 treatment, n ¼ 1 for samples with R428 treatment; 50 nmol/L). P values calculated using the t test tocompare Tet-Axl to Tet-ø for þDox samples only. Error bars, mean � SEM. Scale bars, 1 mm.






High AXL expression leads to cell aggregationUpon doxycycline treatment of Tet-Axl cells, we consistently

observed a clear aggregation phenotype. This wasmore evident inthe Tet-Axl 2 clone that had higher AXL expression (Fig. 3A). Theaggregation occurred only in Tet-Axl cells and in the presence ofdoxycycline. Weak aggregation became apparent with 25 ng/mLof doxycycline and reached full ability by 100 ng/mL (the con-centration used for all other experiments), correlating with AXLexpression (Fig. 3B). Removal of doxycycline after 2 days ofexposure and aggregation formation led to the rapid dissociationof aggregates by the following day but did not correlate with asignificant decrease in AXL expression (Fig. 3C). The formation ofaggregates by the addition of doxycycline in Tet-Axl cells wasblocked with simultaneous AXL kinase inhibition, indicating therequirement of AXL signaling (Fig. 3D). Treatment did not affectviability (Supplementary Fig. S3A).

As AXL signaling plays a critical role in platelet aggregation, andcancer cell clustering with platelets has been demonstrated topromote their survival in the blood of prostate cancer patients, wenext investigated the ability of AXL-overexpressing cells to aggre-gate with platelets (29–31). Upon coculture, human platelets

preferentially coated the cell surface of doxycycline-pretreatedAXL-on cells, compared with coculture with AXL-off cells (Fig.3E). Interestingly, microarray analysis comparing genes upregu-lated in doxycycline-treated Tet-Axl 2 cells (robust aggregateformation) relative to Tet-Axl 1 cells (weak aggregate formation)revealed an upregulation of less than 30 genes, two of which(AGR1 and ELF3) were also found to be upregulated in breastcancer patient circulating tumor cell (CTC) clusters comparedwith single CTCs (Supplementary Fig. S3B; Supplementary TableS1; ref. 32).

AXL expression delays but is not sufficient to preventtumorigenesis in vivo

To determine if AXL overexpression could induce dormancyin vivo, we injected either Tet-ø or Tet-Axl 2 cells intracardiacinto NSG mice that were administered doxycycline in theirdrinking water ("AXL-on," refers only to Tet-Axl 2 cells exposedto doxycycline) or received normal water ("AXL-off"). After 3weeks, a cohort with representative mice from each group wasinjected with EdU to label proliferating cells and sacrificed aday later to capture differences in bone marrow–specific

0

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Figure 4.

AXL overexpression delays but does not prevent tumorigenesis in a mouse model of disseminated tumor cell growth. A, Number and proliferation status of bonemarrow DTCs detected in mice at 3 weeks after intracardiac injection. Each bar represents 1 mouse; positive BLI signal is indicated below (AXL-off groups,n¼ 7; AXL-on group, n¼ 6). B, Time in days to tumorigenesis of each group after intracardiac injection at the 6week (Tet-ø�Dox, n¼ 10; Tet-øþDox, n¼ 8; Tet-Axl�Dox, n ¼ 9; Tet-Axl þDox, n ¼ 6) and 14 week (Tet-ø �Dox, n ¼ 12; Tet-ø þDox, n ¼ 11; Tet-Axl �Dox, n ¼ 11; Tet-Axl þDox, n ¼ 12) time points. Tumorigenesiswas defined as BLI signal over control PBS-injected mice; each dot represents one mouse. C, Duration of tumor growth after intracardiac injection. Durationof growth was defined as the time between the onset of tumorigenesis and time to lethal tumor burden; each dot represents one mouse (Tet-ø �Dox, n ¼ 9;Tet-ø þDox, n ¼ 8; Tet-Axl �Dox, n ¼ 7; Tet-Axl þDox, n ¼ 9). D, Survival proportions for each group of mice over time (left) with tabulated log-rank testresults for each group compared with Tet-AxlþDox group at the 8- or 12-week time point (right). Vertical dashed line indicates 8 weeks (Tet-ø�Dox, n¼ 14; Tet-øþDox, n ¼ 13; Tet-Axl �Dox, n ¼ 12; Tet-Axl þDox, n ¼ 14). Error bars, mean � SD. For mouse tumor growth, P values were calculated by the t test.

Axelrod et al.





dormancy periods that occur prior to tumorigenesis. There werea greater number of total and proliferating bone marrow DTCsdetected in AXL-off mice, compared with almost no DTCs andno proliferating DTCs found in AXL-on mice (Fig. 4A). OneAXL-on mouse was not representative of the AXL-on group,harboring a high number of total and proliferating DTCs.Notably, only AXL-off mice developed tumor signal either inthe jaw or liver at the 3-week time point. We monitored a largercohort of mice over time by BLI and observed that at 6 weeksthere was a significant delay in tumorigenesis in the AXL-onmice. By 14 weeks, however, these data were no longer signif-icant, indicating that almost all mice eventually developedtumors (Fig. 4B). AXL had no effect on the duration of tumorgrowth once tumors were initiated (Fig. 4C). This translated tothe overall survival differences mimicking those of the onset oftumorigenesis, with a statistically significant increased survivalrate for AXL-on mice compared AXL-off groups at 8 weeks afterinjection, but by 12 weeks they had reached a lethal tumorburden (Fig. 4D). At the time of death, there was no differencein bone marrow DTC number between AXL-on or AXL-offgroups. The increased bone marrow DTC number in the Tet-øgroups compared with Tet-Axl groups is likely due to uninten-tional selection for intrinsic phenotypes during the clonalexpansion of these cell lines and is unrelated to AXL, especiallyas these data were not recapitulated in the AXL-on groupcompared with the AXL-off controls (Supplementary Fig.S4A). Overt bone-specific metastasis was observed at low fre-quency across all groups, and therefore no conclusions aboutAXL preventing bone metastasis could be made (Supplemen-tary Fig. S4B).

Tumors fromTet-Axlmiceondoxycycline areheterogeneous forAXL expression and EdU incorporation

One explanation for the development of tumors in AXL-onmice could simply be that some of the DTCs lost AXL expres-sion and that the delay in tumorigenesis represented the time ittook for those cells to outgrow, similar to the loss of AXL-positive cells in the constitutively driven AXL model (Fig. 2D).To determine if this was true, we sectioned and stained livertumors (the most common tumor site and the site with thegreatest tumor burden, regardless of group) from each groupfor AXL expression. Unexpectedly, there was robust AXLexpression in tumors from AXL-on mice (Fig. 5A, top). Torule out that AXL was being inactivated by extracellular cleav-age, we stained the same tumors with an antibody recognizingthe N-terminal extracellular region and still observed strongAXL staining (Fig. 5A, bottom). To determine if AXL-positivecells were perhaps not proliferating, or were only a fraction ofthe entire tumor, we used multiplex IF to stain for AXL (C-termAb), human nucleolin as a cancer cell marker, and EdU toindicate proliferating cells. AXL was not expressed on everycancer cell yet did not show any correlation with EdU status(Fig. 5B).

AXL is not expressed in human prostate tumorsIf AXL is sufficient to induce dormancy in human prostate

cancer, we would hypothesize that it would have low or noexpression in primary and metastatic tumors and wouldcorrelate with better disease outcome. Indeed, prostate cancerpatients who did not have a BCR had higher AXL expressionin their primary tumors compared with those who did recur

(GenomeDx Decipher GRID database; Fig. 6A). Furthermore,higher AXL expression also correlated with better progression-free survival (Taylor and colleagues 2010 data set; ref. 33; Fig.6B). We stained a prostate cancer TMA comprised of healthyprostate tissue, primary cancer tissue of varying Gleasonscores, and metastatic tissue for AXL expression. We foundno AXL staining in any cancer cells of any correspondingtissue; all positive AXL expression is localized to macrophagesand/or endothelial cells (Fig. 6C). To query additional met-astatic tumors, we stained tissue sections from liver, lymphnode, and tibia metastases and again found no AXL expres-sion (Fig. 6D).

DiscussionDormancy of bone marrow DTCs is thought to underlie the

emergence of lethal prostate cancer metastases years afterelimination of the primary tumor (34–36). Treatment strategiesaimed at eliminating the primary tumor are not able to preventmetastasis because cancer cells escape the primary tumor earlyduring its development and dormant DTCs are not susceptibleto chemotherapy. Therefore, in order to prevent the outgrowthof lethal metastases, we must understand the mechanism(s) bywhich their DTC "seeds" persist in a dormant state. Previously,we have shown that the bone marrow HSC niche factor GAS6restricts prostate cancer cell growth, and that dormant bonemarrow DTCs express AXL, a receptor for GAS6 (13, 16). Inmost cancer types, AXL and the other GAS6 receptors are knownto play protumorigenic roles by promoting EMT phenotype,migration, stemness, angiogenesis, therapeutic resistance, andproliferation (15). Interestingly, the TAM receptors have beenwidely characterized to promote cancer progression, but noactivating mutations were identified, unlike those in cancer-driving oncogenes (37). Early investigation of the role of AXL incancer described the transforming potential of AXL as being cellline–specific (38). There is increasing evidence that the func-tional output of GAS6/TAM receptor signaling is heavily con-text-dependent (15). In the work presented here, we provideevidence that AXL signaling mediates prostate DTC dormancyin the bone marrow.

Our initial evidence demonstrated that AXL was not necessaryfor prostate cancer cell dormancy. AXL loss did not have asignificant effect on tumor initiation and growth, or on overallsurvival in our in vivo model. Importantly, loss of AXL did notaffect the number or proliferation status of bone marrow DTCsafter intracardiac injection in mice, indicating that the dormancyphase in this model was not bypassed. In other processes, includ-ing platelet aggregation, EMT, and therapeutic resistance, AXL andthe other TAM receptors are redundant with, and often potentiate,other signaling pathways (15, 39, 40). Indeed, mice null for allthree TAM receptors or forGAS6 alone are viable, in contrast withthe lethal phenotype of mice null for other essential RTKs (41–43). Similarly, it is likely that other dormancymechanisms exist tocompensate for the loss of AXL in this setting. Our data supportthe paradigm that the TAM receptors are dispensable in manyprocesses and participate as actively signaling passengers andcollaborators rather than as drivers.

AlthoughAXL is nonnecessary, we found that overexpression ofAXL was sufficient to induce, though not to maintain, dormancyinourmodels. AXLoverexpressiondecreased proliferation in vitro,but only at high levels, and delayed tumorigenesis in vivo. High






AXL overexpression alone was sufficient to drive the proliferationphenotype independent of GAS6. The reversal of this phenotypeby AXL kinase inhibition suggests it was autoactivated by over-expression, as has previously been shown with high expressionlevels of AXL and other RTKs (44, 45). Because activation of AXLwas thus cell-intrinsic, assessment of AXL-specific effects in vivo isindependent of GAS6 availability at a particular organ site. AXLwas able to prolong the dormancy phase of cells across the entirebody, including in the bone. The more evident in vivo phenotypecomparedwith effects of AXLmodulation in vitro is not surprising,as this has been reported for another dormancy regulator (46).Also consistent with these results, AXL was shown to be signif-icantly upregulated in dormant myeloma cells in mouse bonemarrow compared with proliferating cells (10).

That AXL-on cells eventually outgrew into overt tumors in theliver while retaining full-length AXL expression indicated that AXLis not able to maintain dormancy in the long term, and that otherfactors are required to maintain dormancy. Perhaps these addi-tional dormancy factors are present in the bone marrow microen-vironment. In our experience, intracardiac injection of C42B cellsdoes not frequently yield bone metastases before mice die fromtumor burden at other sites, yet we were able to detect individualviable bone marrow DTCs. This suggests there are indeed othersignaling axes that can regulate dormancy. The growth behavior ofindividual AXL-on cells with andwithout additional factors can beassessed by comparing the in vivo bonemarrow DTC data with the3Dgrowthof the cells in soft agar. Althoughweobserved adecreasein cell number in the bonemarrow, this was not replicated in vitro,

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Figure 5.

AXL-on tumors are heterogeneous for AXL expression and EdU incorporation.A,Representative IHC staining to detect either the C-terminus or N-terminus of AXL inliver tumors from each group. Dashed white lines depict the border between tumor and liver tissue; T, tumor region (n ¼ 3 per group; scale bar, 100 mm). B,Multiplex IF staining for DAPI, human nucleolin, AXL (C-terminus), and EdU incorporation in liver tumors from 3 different AXL-on mice; scale bar, 50 mm.

Axelrod et al.





supporting the idea that AXL requires additional bone marrowfactors for dormancy maintenance.

The striking aggregation phenotype we observed upon highAXLoverexpression is not unprecedented: AXLoverexpression hasbeen shown to cause aggregation via homophilic binding both inthe presence and the absence of GAS6 (44, 47). GAS6/AXLpromotes platelet aggregation and activation, and AXL�/� miceare protected against thrombosis (39, 48). The extracellulardomain of AXL contains two immunoglobulin and two fibro-nectin III domains, features shared by cell adhesion moleculessuch as NCAM. It is possible, therefore, that artificially high AXL

overexpression may simply cause cells to "stick" to each otherby virtue of their extracellular adhesion features, rather thandue to any AXL-specific signaling effects. However, when cellswere simultaneously treated, an AXL kinase inhibitor and withdoxycycline to induce AXL expression, aggregates failed toform, indicating a requirement for active AXL signaling. CTCclustering with platelets has been demonstrated to promotecancer cell survival in the circulation, allowing for successfulhoming to secondary sites (30, 49, 50). Although it remainsunknown whether AXL is expressed on prostate CTCs, wedemonstrated here that human platelets preferentially coated

NO BCR BCR

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Figure 6.

AXL is not expressed in human prostate tumors. A, AXL expression in prostate cancer patients who did and did not experience a biochemical recurrence (n¼ 1,405patients, GenomeDx GRID data set). P value was calculated using the Wilcoxon rank test. B, Recurrence-free survival of patients with high (above the median)and low (below the median) AXL expression (n ¼ 571 patients, Taylor et al. 2010; visualized and analyzed with Project Betastasis). C, Representative images ofAXL IHC staining of a prostate tissue microarray; LN, lymph node. C42B cells served as a negative control and DU145 as a positive control. All images atoriginal magnification �200. D, AXL IHC staining in metastatic patient tissue; top: scale bar, 100 mm; bottom: scale bar, 50 mm.






AXL-expressing cells. Initial results suggested the coating wasindependent of AXL kinase inhibition, implying this interac-tion was structurally mediated.

Inmany types of cancers, AXL expression becomes upregulatedin the primary tumor and/or metastases and correlates withadvanced disease and poor survival (15). If AXL plays a protumorrole regardless of cancer type, we would expect to see similarassociations in prostate cancer. We found, however, that AXLexpression correlates with decreased BCR and better recurrence-free survival. Moreover, gain or loss of AXL in our prostate cancercell lines did not consistently alter EMT or stemness gene expres-sion, unlike what has been reported for other cancer cell types.Furthermore, we show that AXL is not expressed by cancer cells inprimary tumors of any grade or in metastases. Interestingly, AXLexpression seems to localize to macrophages and/or endothelialcells. These cellsmay be the source ofAXL signal in the correlationdata that predict better prognosis, indicating a potential antitu-mor role for AXL-positive cells in the tumormicroenvironment. Itis worth considering that AXL may be regulated carefully at theprotein level, while its RNA expression does not change (as wasobserved for TYRO3 upon AXL KO; Supplementary Fig. S1A andS1C), in which case its transient protein expression would bedifficult to capture. The ability of the cell to quickly adapt throughregulation of protein stability would allow it to easily respond tomicroenvironmental signals that would induce the aggregation ordormant phenotypes we observed. Overall, these data supports atumor-suppressive role for AXL inhumanprostate cancer but doesnot determine if AXL induces dormancy of human bone marrowDTCs. It is currently not feasible to accurately detect and charac-terize bonemarrowDTCs, asmore sensitivemethods anddisease-specific markers are required.

Our study functionally validates a role for AXL in promotingprostate tumor cell dormancy. Although AXL is not necessaryfor dormancy, it is sufficient to induce short-term dormancy inprostate cancer models. The inability of AXL to maintaindormancy, coupled with the idea that there are likely otherdormancy-inducing factors, indicates that AXL alone is not asuitable target for preventing lethal metastases. More impor-tantly, this study demonstrates the pleotropic nature of AXLand further highlights the complexity of GAS6/TAM receptorsignaling and downstream functions. Careful observation

should be made when studying the roles of the TAM receptorsin various contexts, and caution should be taken when con-sidering targeting AXL for therapeutic purposes.

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

Authors' ContributionsConception anddesign:H.D. Axelrod, K.C. Valkenburg, S.R. Amend, K.J. PientaDevelopment of methodology: H.D. Axelrod, K.C. Valkenburg, G. Torga,A.M. DeMarzo, K.J. PientaAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.):H.D. Axelrod, K.C. Valkenburg, S.R. Amend, J.L.Hicks,A.M. DeMarzoAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): H.D. Axelrod, P. Parsana, G. Torga, A.M. DeMarzo,K.J. PientaWriting, review, and/or revision of the manuscript: H.D. Axelrod,K.C. Valkenburg, S.R. Amend, J.L. Hicks, G. Torga, K.J. PientaAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): H.D. AxelrodStudy supervision: H.D. Axelrod, K.C. Valkenburg, K.J. Pienta

AcknowledgmentsThe authors thank Dr. Russell Taichman for his guidance and thoughtful

discussion during this work and members of the Pienta Lab for helping toprocess mice. Thanks toWayne Yu at the SKCCCMicroarray Core andmembersof the Johns Hopkins Synthesis and Sequencing Facility and the Johns HopkinsReference Histology Laboratory.

This work was supported by the Prostate Cancer Foundation (Young Inves-tigator Award to S.R. Amend), the American Cancer Society (Postdoctoral Fel-lowship PF-16-025-01-CSMtoS.R.Amend), theNIH (grants F32-CA206394 toK.C. Valkenburg, P01-CA093900 to K.J. Pienta, and P30 CA006973 to The SidneyKimmel Cancer Center Microarray Core Facility at Johns Hopkins University),Department of Defense (Prostate Cancer Research Program AwardW81XWH-14-2-0182 toA.M.DeMarzo), andNCI/NIHProstate SPOREGrant P50CA58236andCancer Center Support Grant 5P30CA006973 to A.M. DeMarzo.

The costs of publication of this articlewere defrayed inpart by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received July 5, 2018; revised August 25, 2018; accepted September 26, 2018;published first October 5, 2018.

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2019;17:356-369. Published OnlineFirst October 5, 2018.Mol Cancer Res Haley D. Axelrod, Kenneth C. Valkenburg, Sarah R. Amend, et al. Prostate CancerAXL Is a Putative Tumor Suppressor and Dormancy Regulator in

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