hypoxiastabilizesgas6/axlsignalinginmetastaticprostate …...extracellular domain containing 2...

Angiogenesis, Metastasis, and the Cellular Microenvironment

Hypoxia Stabilizes GAS6/Axl Signaling inMetastatic ProstateCancer

Anjali Mishra1,2, Jingcheng Wang1, Yusuke Shiozawa1, Samantha McGee1, Jinkoo Kim1, Younghun Jung1,Jeena Joseph1, Janice E. Berry1, Aaron Havens1, Kenneth J. Pienta3, and Russell S. Taichman1

AbstractThe receptor tyrosine kinase Axl is overexpressed in a variety of cancers and is known to play a role in proliferation

and invasion. Previous data fromour laboratory indicate that Axl and its ligand growth arrest–specific 6 (GAS6)mayplay a role in establishing metastatic dormancy in the bone marrow microenvironment. In the current study, wefound that Axl is highly expressed inmetastatic prostate cancer cell lines PC3 andDU145 and has negligible levels ofexpression in a nonmetastatic cancer cell line LNCaP. Knockdown of Axl in PC3 and DU145 cells resulted indecreased expression of several mesenchymal markers including Snail, Slug, and N-cadherin, and enhancedexpression of the epithelial marker E-cadherin, suggesting that Axl is involved in the epithelial–mesenchymaltransition in prostate cancer cells. The Axl-knockdown PC3 and DU145 cells also displayed decreased in vitromigration and invasion. Interestingly, when PC3 andDU145 cells were treated with GAS6, Axl protein levels weredownregulated.Moreover,CoCl2, a hypoxiamimicking agent, preventedGAS6-mediated downregulation of Axl inthese cell lines. Immunochemical staining of human prostate cancer tissuemicroarrays showed that Axl, GAS6, andhypoxia-inducible factor-1a (Hif-1a; indicator of hypoxia) were all coexpressed in prostate cancer and in bonemetastases compared with normal tissues. Together, our studies indicate that Axl plays a crucial role in prostatecancer metastasis and that GAS6 regulates the expression of Axl. Importantly, in a hypoxic tumor microenviron-ment Axl expression is maintained leading to enhanced signaling.Mol Cancer Res; 10(6); 703–12.�2012 AACR.

IntroductionCancer metastasis remains one of the most challenging

clinical problems. In the United States alone, more than350,000 people die each year due to skeletal complicationsassociated with bone metastasis common in many cancerssuch as prostate and breast cancer (1). The 5-year survivalrate of primary prostate cancer is nearly 100%, however afterthe detection of bone metastasis the 5-year survivalrate declines to 33% (2). In order for cancer cells tometastasize theymust at first detach from the primary tumor,invade the surrounding extracellular matrix (ECM), andcross the endothelium to enter the blood stream (3). Cancercells must then move across the endothelium and invade theECM before migrating and taking up residence in distantorgans such as bone. Understanding this multistep processis therefore crucial for elucidating the molecular events thatproduce a lethal phenotype.

Axl is one member of a tyrosine kinase receptor family(receptor tyrosine kinase, RTK), which also includes Sky(Tyro3) and Mer. Together, Axl, Sky, and Mer or the TAM(Tyro3-Axl-Mer) family of RTKs are characterized by anextracellular domain containing 2 immunoglobulin-likedomains and 2 fibronectin type 3–like domains. Axl encodesa 140 kD protein and was originally identified as a trans-forming gene in patients with chronic myelogenous leuke-mia (4, 5) and in chronic myeloproliferative disorders (6).Growth arrest–specific 6 (GAS6), is a vitamin K–dependentligand for Axl, Sky, and Mer (7–9), so named because it wasinitially discovered as a protein which is overexpressed ingrowth-arrested cells (10). Binding of GAS6 leads to Axlreceptor dimerization and autophosphorylation on its tyro-sine resides, which triggers a cascade of intracellular signaling(11). Several studies have indicated that Axl plays a role intumor invasion and metastasis in a number of cancers,including breast, pancreatic, glioblastoma, hepatocellular,and renal carcinomas (12–18). The expression level of Axl isdirectly proportional to tumor grade and predicts poorpatient survival rate (19–25).At present, less is known as to the biologic role played by

Axl in prostate cancer progression. Previous findings fromour laboratory have shown that Axl is highly expressed inmetastatic prostate cancer and suggested that the interactionof GAS6 and TAM RTKs may play a role in establishingtumor dormancy in the bone marrow microenvironment(26). In this present report, we show that Axl, which is highly

Authors' Affiliations: Departments of 1Periodontics and Oral Medicine,2Biologic and Materials Sciences, and 3Urology and Internal Medicine,University of Michigan School of Dentistry, Ann Arbor, Michigan

Note: K.J. Pienta and R.S. Taichman contributed equally to the work.

Corresponding Author: Russell S. Taichman, Periodontics & Oral Med-icine, University of Michigan School of Dentistry, 1011 North UniversityAve., Ann Arbor, MI 48109. Phone: 734-764-9952; Fax: 734-763-5503;E-mail: [email protected]

doi: 10.1158/1541-7786.MCR-11-0569

�2012 American Association for Cancer Research.

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expressed in metastatic prostate cancer cells, promotesmigration and invasion of prostate cancer cells in vitro andplays an important role in regulating expression of genesinvolved in epithelial–mesenchymal transition (EMT) ofcancer cells. Our studies also reveal that Axl expression levelsare negatively regulated by its ligand GAS6. However, inhypoxic environments such as in a tumor or in bonemetastases, downregulation of Axl expression is prevented.Together, these studies show that Axl plays a crucial role inprostate cancer metastasis, and its expression is regulated byboth GAS6 and hypoxia.

Materials and MethodsCell culturePC3 (CRL-1435), DU145 (HTB-81), and LNCaP

(CRL-1740) prostate cancer cell lines were obtained fromthe American Type Culture Collection. All prostate cancercell lines were cultured in RPMI-1640 (Invitrogen) supple-mented with 10% (v/v) FBS (Invitrogen) and 1% (v/v)penicillin-streptomycin (Invitrogen) and maintained at37�C, 5% CO2, and 100% humidity.

Antibodies and reagentsAnti-Axl antibody (catalog no. 4939), anti-Slug antibody

(catalog no. 9585), anti-Snail antibody (catalog no. 3879),anti-E-cadherin antibody (catalog no. 3195), anti-N-cad-herin antibody (catalog no. 4061), and anti-b-actin antibody(catalog no. 4970) were purchased from Cell SignalingTechnology. For immunostaining, anti-human GAS6 poly-clonal antibody (catalog no. AF885; R&D Systems) wasdetected using a ZenonAlexa Fluor 555 goat IgG labeling kit(Invitrogen). Staining with an anti-Axl antibody (catalogno. AF154; R&D Systems) and anti-human hypoxia-induc-ible factor-1a (Hif-1a;catalog no. 610958; BDBiosciences)were detected using a Zenon Alexa Fluor 488 mouse IgG1labeling kit (Invitrogen). Human recombinant GAS6 waspurchased from R&D Systems. CoCl2 (catalog no. C8861)was purchased from Sigma-Aldrich.

Axl knockdown by lentivirusesGIPZ Lentiviral shRNAmir vectors containing either Axl

shRNA or nonsilencing (scrambled) shRNAwere purchasedfromOpen Biosystems. Lentiviruses were constructed at theVector Core in University of Michigan (Ann Arbor, MI).Stable Axl knockdowns in DU145 and PC3 cells weregenerated by lentiviral infection followed by 1 week ofselection with 1 mg/mL of puromycin. Viral infectionefficiency was assessed by determining the percentage ofGFP-positive cells. Axl knockdown in these cells was con-firmed by both real-time PCR and Western blot analysis.

RNA extraction and real-time PCR analysisReal-time PCRwas carried out using standard techniques.

Briefly, total RNA was isolated by RNeasy Mini Kit (Qia-gen), and first-strand cDNA was synthesized in a 20 mLreaction volume using 0.4 mg of total RNA. Reverse tran-scription products were analyzed by real-time PCR inTaqMan Gene Expression Assays of several target genes

including GAS6, Axl, Sky, Mer, Slug, Snail, N-cadherin,E-cadherin, and b-actin (Applied Biosystems). Real-timePCR analysis was conducted with 15.0 mL of TaqManUniversal PCR Master Mix (Applied Biosystems), 1.5 mLof TaqMan Gene Expression Assay, 1 mL of cDNA, andRNAse/DNAse-free water in a total volume of 30 mL. Allsample concentrations were standardized in each reaction toexclude false-positive results. Reactions without templateand/or enzyme were used as negative controls. The second-step PCR (95�C for 30 seconds and 60�C for 1 minute) wasrun for 40 cycles after an initial single cycle of 95�C for 15minutes to activate the Taq polymerase. The PCR productwas detected as an increase in fluorescence using an ABIPRISM 7700 sequence detection system (Applied Biosys-tems). RNA quantity (CR) was normalized to the house-keeping gene b-actin control by using the formula:CR ¼ 2ð40�Ct sampleÞ�ð40�Ct controlÞ. The threshold cycle (Ct)is the cycle at which a significant increase in fluorescenceoccurs.

Western blot analysisProstate cancer cells (1� 106 cells per well) were cultured

in 6-well plates in RPMI medium. For Axl knockdown andEMT studies, whole-cell lysates were made from cells grownin media containing serum. For GAS6 regulation studies,prostate cancer cells were serum starved overnight, afterwhich they were either left untreated or treated with 100ng/mL of GAS6 for 6 hours and overnight, or with 100mmol/L of cobalt chloride (CoCl2) for 6, 7 hours, andovernight. Whole-cell lysates were prepared from cells,separated on 10% SDS-PAGE and transferred to a poly-vinylidene difluoride membrane. The membranes wereincubated with 5% milk for 1 hour and incubated withprimary antibodies overnight at 4�C. Primary antibody wasused a 1:1,000 ratio with 5% dry milk. Blots were incubatedwith peroxidase-coupled secondary antibodies (Promega) for1 hour at a ratio of 1:10,000, and protein expression wasdetected with SuperSignal West Pico ChemiluminescentSubstrate (Thermo Scientific). Membranes were reprobedwith polyclonal anti-b-actin antibody (1:1,000; Cell Signal-ing) to control for equal loading.

In vitro migration and invasion assaysIn vitromigration and invasion assays were done using the

Corning Cell migration, chemotaxis, and invasion assayprotocol. PC3 and DU145 cells were serum starved over-night. A total of 2 � 105 cells were plated in the upperchamber of a 24-well transmembrane permeable supportwith 8 mm pore size (catalog no. 3422; Corning). For theinvasion assay only, 100 mL of basement membrane extract(BME)-coating solution (catalog no. 3455-096-02; Trevi-gen) was placed in the top chamber before cell plating. GAS6(100 ng/mL) in serum-free media or serum-containingmedia were used as chemoattractants in the bottom cham-ber. After 24 hours, the cells which had migrated or invadedwere dissociated using a cell dissociation buffer (catalog no.3455-096-05; Trevigen). Cells were then stained with

Mishra et al.

Mol Cancer Res; 10(6) June 2012 Molecular Cancer Research704




Calcein AM fluorescent dye (catalog no. C3100MP;Molec-ular Probes; Invitrogen) for 1 hour and placed in a 96-wellsolid black microplate (catalog no. 3916; Corning). At theend of the experiments, the fluorescent intensities of bottomchambers were read by a fluorescent plate reader at 485 nmexcitation and 520 nm emission. A standard curve was madeby staining known numbers of cells with Calcein AM.Fluorescent data from migration and invasion assay experi-ments were converted to cell numbers based on this standardcurve.

Tumor tissue microarrays and immunostainingHuman tissue microarrays were provided by the Prostate

SPORE Tissue Core Laboratory, Urology Department,University of Michigan, for immunofluorescence staining.Tissue microarray (TMA) sections were unmasked withpepsin solution (Lab Vision) at 37�C for 15 minutes andpH balanced with PBT (PBS plus 0.2% Triton X-100) atroom temperature for 5minutes. Sections were blocked withImage-iT FX signal enhancer (Invitrogen) for 30 minutesbefore incubating with fluorescence-labeled primary anti-bodies at room temperature for 2 hours in the dark. Sectionswere washed with PBS, fixed with 10% formalin (Sigma-Aldrich) for 10 minutes, mounted with ProLong Goldantifade reagent with 40,6-diamidino-2-phenylindole(DAPI; Invitrogen), and covered with cover glass. Imageswere taken with Olympus FV-500 confocal microscope. Fordata analysis, Software NIS Elements BR3.2 64-bit (Nikon)was used in conjunction with 20�DICM (0.19 um/px) forall 20� images using an automated mass field measurement.For single color, GAS6 or Axl 555 mono wavelengthchannels were used, 488 and 555 wavelengths were usedto quantify colocalization withHif-1a. Data are presented aspercent positive of each 20� field.

Statistical analysisAll in vitro experiments were carried out at least 3 times

with similar results. Results from representative assays areshown. Numerical data are expressed as mean � SD.Significance of the difference between 2 measurements wasdetermined by unpaired Student t test. Values of P < 0.05were considered significant.

ResultsMetastatic prostate cancer cells express high levels of AxlOur earlier study indicated that Axl was highly expressed

in prostate cancer cell lines (26). In that study, immunos-taining of Axl in TMA samples revealed that Axl expressionhad a positive correlation with tumor grade. To confirm ourearlier observations, real-time PCR was carried out to detectmRNA expression levels of theGAS6 receptors Axl, Sky, andMer in the metastatic prostate cancer cell lines PC3 andDU145 and in a nonmetastatic cell line LNCaP. Asexpected, Axl was highly expressed in metastatic cancer cellsPC3 and DU145, whereas very low levels of Axl weredetected in the nonmetastatic cell line LNCaP (Fig. 1A).Little or no expression of Sky andMer was observed in all celllines (Fig. 1A). These results corroborate our earlier data and

strongly indicate that Axl plays a role in metastasis inprostate cancer cells. To further explore the biology of Axlexpression in prostate cancer cells, Axl expression wasdownregulated using a lentivirus vector harboring eitherscrambled shRNA (sc.shRNA) or Axl shRNA. Real-timePCR analysis confirmed Axl shRNA-induced knockdown ofAxl mRNA in PC3 and DU145 compared with controls(Fig. 1B and C).

Axl regulates EMT in prostate cancer cellsThe process of EMT allows a cancer cell to undergo

biochemical changes to assume a mesenchymal phenotype.This results in cancer cells which have enhanced migratorycapacity, increased invasiveness, enhanced resistance to apo-ptosis, and which exhibit an ability to degrade the ECMcomponents (27). To determine whether Axl plays a role inregulating the process of EMT, Western blot analysis wasconducted to compare the protein levels of molecules

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Figure 1. Axl is highly expressed in metastatic prostate cancer cells. A,Axl, Sky, and Mer mRNA expression was analyzed by real-time PCR inPC3, DU145, and LNCaP cells. B andC, following infectionwith lentivirusbearing Axl shRNA or scrambled shRNA (sc.shRNA), Axl mRNA levelswere determined by real-time PCR in PC3 and DU145 cells, respectively.Gene expression shown is relative to b-actin levels.

Role of Axl in Prostate Cancer Bone Metastasis

www.aacrjournals.org Mol Cancer Res; 10(6) June 2012 705




involved in the process of EMT. As shown in Fig. 2A, B, andD, PC3 and DU145 cells in which Axl expression is down-regulated expressed diminished levels of the mesenchymalmarkers Snail, Slug, and N-cadherin and enhanced levels ofthe epithelialmarker E-cadherin.Whereas there were changesin the protein levels of these EMT markers, no changes inmRNA levels were seen following Axl knockdown in PC3cells (Fig. 2C) and DU145 (not shown), suggesting that Axlregulates EMT marker expression at the protein level.

Knockdown of Axl in prostate cancer cells leads toinhibition of migration and invasion in vitroTo determine whether expression of Axl plays a functional

role in prostate cancer metastasis, in vitro migration andinvasion assays were conducted. Following overnight serumstarvation, migration across a Boyden chamber towardsserum or GAS6 decreased significantly for PC3 and DU145cells in which Axl was knocked down, as compared controlcells (Fig. 3A and C). Similarly, in vitro invasion assays werealso conducted to determine the invasion potential of Axlknockdown PC3 and DU145 cells. Similar to the migrationassay, PC3 and DU145 cell invasion across a Boydenchamber was suppressed when Axl expression was reduced(Fig. 3B andD). Together these in vitro data suggest that Axlplays a role in promoting migration and invasion of prostatecancer cells.

Exogenous treatment with GAS6 reduces Axl proteinexpression in prostate cancer cellsTo determine whether GAS6 regulates the expression of

its receptor Axl, PC3 and DU145 cells were treated withGAS6 at different time points following overnight serumstarvation. Axl protein levels in both cell lines were decreasedat 6 hours followingGAS6 treatment.Moreover, Axl proteinlevels remained low even after overnight treatment withGAS6 (Fig. 4A and B). While Axl protein levels went downupon GAS6 treatment, there were no significant changes inAxl mRNA levels (Fig. 4C andD). This indicates that GAS6regulation of Axl expression is mediated at the protein ratherthan the mRNA level.

Hypoxia prevents GAS6-mediated downregulation ofAxl in prostate cancer cellsSeveral studies have indicated that the tumor microenvi-

ronment is hypoxic in nature (28). CoCl2, a reagent whichmimics a hypoxic environment when added in vitro (29), wasused to create a hypoxic environment for PC3 and DU145cells in culture. Interestingly, CoCl2 treatment preventedGAS6 downregulation of Axl protein expression in bothPC3 and DU145 cells (Fig. 5A and B). To verify CoCl2'srole as a hypoxia mimicking agent, Hifl-a levels wereanalyzed in PC3 and DU145 cells after CoCl2 treatment.It was observed that Hifl-a levels increased within 6 hours of

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Figure 2. Axl regulates EMT inprostate cancer cells. A and B,Western blot analysis was used todetermine protein levels of Axl,Slug, Snail, N-cadherin, E-cadherin, and b-actin in PC3 andDU145 with and without AxlshRNA. C, mRNA levels of Axl,Slug, Snail, N-cadherin, andE-cadherin relative to b-actin weredetermined by real-time PCR inPC3 cells with and without AxlshRNA. Values displayed here arefold differences in mRNAexpression between controland Axl knockdown cells. sc.shRNA, scrambled shRNA.D, immunohistochemicalevaluation of Axl, N-caderhin, andE-cadherin expression followingAxl shRNA knockdown.

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treatment with CoCl2 treatment of both PC3 and DU145cells (Fig. 5C). Our results indicate that hypoxia stabilizesAxl protein and prevents its degradation via GAS6.

GAS6 and Axl colocalize with Hif-1a in both primarytumors and bone metastasis in human prostate TMAsamplesOur in vitro studies indicate that GAS6 negatively reg-

ulates Axl protein levels, yet in hypoxic conditions Axlprotein levels do not change upon in response to GAS6stimulation. It was therefore critical to explore whether thisin vitro result is also true in the hypoxic regions of tumors (asdefined by Hif-1a staining; ref. 30). Consequently, humanprostate cancer TMAs were probed with antibodies thattarget GAS6 and Hif-la or Axl and Hif-1a to determinetheir colocalization. Overall, Hif-1a levels were higher inprostate tumors tumor than in the normal prostate (Fig. 6).It was also observed that GAS6 is highly expressed in regionspositive for Hif-1a in both primary tumor and in bonemetastasis (Fig. 6). Similarly it was seen that Axl wasexpressed in regions positive for Hif-1a in both primarytumors and significantly in bone metastasis (Fig. 7). Thus,bothGAS6 and Axl are expressed within hypoxic regions of atumor and significantly in metastases. Together these resultsshow that GAS6 negatively regulates Axl under normoxic

conditions (Figs. 4 and 5), but not under hypoxic conditionsin vitro, and within a hypoxic tumor Axl expression remainshigh (Fig. 7) despite the presence of abundant GAS6expression (Fig. 6). These data suggest that the continuousGAS6-Axl signal in regions of hypoxia may contributesignificantly to migration, invasion, and metastasis.

DiscussionIn this study, we evaluated the role of Axl in prostate

cancer progression. We observed that reducing the expres-sion of Axl in prostate cancer cell lines significantly decreasedboth the in vitro migration and the invasion of cancer cellsacross a Boyden chamber toward either GAS6 or serum.Reducing Axl expression also resulted in the downregulationof several mesenchymal markers including Snail, Slug, andN-cadherin whereas at the same time enhanced expression ofthe epithelial marker E-cadherin. Not surprisingly, theexpression of Axl is negatively regulated by the presence ofits ligand. However under hypoxic conditions, such as thosein the bone marrow or in a tumor setting, GAS6 does notdownregulate Axl expression, which may result in tumorprogression toward an EMT state.The physiologic role of Axl and its ligand GAS6 has been

extensively studied in vascular smooth muscle cells and in

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Figure 3. Knockdown of Axl in prostate cancer cells leads to inhibition of migration and invasion in vitro. Following infection with lentiviruses bearingAxlshRNA or scrambled shRNA (sc.shRNA), migration (A, C) or invasion (B, D) assays were conducted in Boyden chambers in either serum-containingmedia,serum-free media, or serum-free media containing 100 ng/mL GAS6 as a chemoattractant. PC3 (A, B). DU145 (C, D). Data are presented as mean � SD fortriplicate determinations.






neurons. GAS6 stimulation of Axl plays a role in chemotaxisin these cells (31, 32). Recently, the role of Axl andGAS6 hasbeen studied in a variety of cancers (12). Enhanced Axlexpression correlates with increased lymph node metastasisand poor clinical outcomes in oral squamous cell carcinoma(19). Downregulation of Axl in hepatocellular carcinomaattenuates proliferation andmigration in vitro and peripherallymph node metastasis in vivo (15). Axl was found to beoverexpressed in 70% of stage II pancreatic ductal adeno-carcinoma (PDA) samples. GAS6 and Axl overexpression inPDA samples are associated with poor prognosis in patientswith stage II PDA (12), and Axl expression in pancreaticcancer is significantly associated with lymph node metastasis(12). Axl knockdown prevents migration and invasion inpancreatic cancer cell lines and results in decreased amountsof activated (GTP-bound) GTPase proteins Rho and Rac;significant downregulation in transcript levels of the EMT-associated transcription factors Slug, Snail, and Twist; andsignificant decrease in matrix metalloproteinase (MMP)-9mRNA levels (21). The presence of Axl in primary breastcancers predicts reduced overall survival. In mammary epi-thelial cells Axl is induced by EMT and Axl knockdownprevents the spread of highly metastatic breast carcinomacells to lymph nodes and several major organs (17). Over-expression of Axl in lung adenocarcinomas and gliomas alsopredicts a less than favorable outcome (24, 25). Severalstrategies are being developed to inhibit GAS6 signalingin cancer (33). Using mouse models it has been shownthat a small-molecule inhibitor of Axl kinase, R428,significantly reduces metastasis of breast cancer andextends survival (34).Little is known about how Axl expression is regulated in

cancer cells. In breast cancer cells, the intermediate filamentand mesenchymal marker vimentin positively regulates Axlexpression (13). In colorectal and cervical carcinoma celllines, myeloid zinc finger protein 1 (MZF1) binds to the Axlpromoter, transactivates promoter activity, and enhances

Axl-mRNA and protein expression in a dose-dependentmanner (14). In Axl-transformed NIH-3T3 cells, Axl isposttranslationally regulated by proteolytic-mediated degra-dation, and half life of Axl is around 2 hours (4). Thisindicates that Axl protein is highly unstable and is rapidlydegraded under certain conditions. Studies in human lens

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Figure 4. Exogenous treatmentwithGAS6 reducesAxl protein expression in prostate cancer cells. Following overnight serumstarvation, PC3 andDU145 cellswere left untreatedor treatedwith100ng/mLofGAS6 for 6 hours or overnight (O/N). AandB, protein levels ofAxl andb-actin (usedascontrol)weredeterminedby Western blot analysis for PC3 and DU145. C and D, mRNA levels for Axl were determined by real-time PCR and normalized to b-actin levels.

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epithelial cells have indicated that GAS6 promotes down-regulation of Axl protein by inducing phosphorylation andubiquitination of Axl and the interaction of Axl with theubiquitin ligase c-Cbl (35).In this report, we used a hypoxia mimicking agent,

CoCl2, to show that GAS6 secretion is not able tonegatively regulate expression Axl in PC3 and DU145cells under hypoxic conditions. Quite likely Axl proteinexpression is stabilized in a hypoxic microenvironment aswe observed very little alteration in Axl mRNA expressionin response to GAS6 signaling. Regions of the tumormicroenvironment are known to be very hypoxic (28). In aprimary tumor, hypoxia induces an EMT, whereas in thebone hypoxia may increase the expression of transcriptionof factors which can further promote skeletal metastasis ofthe tumor (36). Hif-1a is a highly unstable protein thatunder normoxic conditions is degraded by proteolytic-mediated degradation. Hypoxia stabilizes Hif-1a proteinby preventing its degradation (37). We observed that inprimary prostate cancer lesions and in bone metastases Axlcolocalizes with GAS6 and Hif-1a. These findings suggestthat Axl expression under hypoxic conditions may nolonger be regulated by the GAS6 ligand, and thereforemay remain available for signaling or for the promotionof an EMT-like state which may drive progression ofmetastasis.Earlier, we showed that GAS6 is produced by primary

human osteoblasts (26, 38). We have shown that in acutelymphoblastic leukemia cells migrate toward the bone using

the GAS6/Mer axis. In prostate cancer we have shown thatGAS6 production by osteoblasts influences tumor dormancyand thereby protects tumor cells from chemotherapy-induced apoptosis. There have been other studies usingmelanoma, breast, pancreatic, and colon cancer cell linesin which cancer cells were shown to promote their owngrowth by educating infiltrating leukocytes to upregulate theproduction of GAS6 (39). These observations indicate thatGAS6 production is high in the bone marrow microenvi-ronment and that the presence of a tumor further promotesGAS6 production. Yet GAS6 can either be producedby osteoblasts (paracrine loop) or by the tumor cells them-selves (autocrine loop). The hypoxic microenvironment inthe bone may facilitate the high expression of Axl by thetumor cells despite high GAS6 expression. This results inincreased production of both the ligand and receptor,which boosts GAS6/Axl signaling within the bone micro-environment that in turn drives tumorigenesis (Modelin Fig. 8). A similar phenomenon can be imagined in aprimary prostate cancer microenvironment, which is alsohighly hypoxic in nature.It is well known that normal cells control the length and

intensity of RTK signaling by receptor downregulation. Thispathway involves ligand-induced internalization by endo-cytosis, followed by degradation in lysosomes, as seen inEGF receptors (EGFR) and in platelet-derived growth factorreceptors (PDGFR; refs. 40–42). In cancer cells, however,some of these deactivation pathways are compromised,leading to overexpression of RTKs (43, 44). It is highly

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Hif-1α

GAS6

DAPI

Bone metastasis (60x)

Merged

MergedMerged


Hif-1α

GAS6

DAPI

GAS6 / Hif-1αGAS6NTissue

Normal 0.2 ± 0.2122prostate 0. 2 ± 0.2

Localized 1.3 ± 0.6123PCa 0.9 ± 0.5

a

Bone 6.5 ± 4.112metastasis 4.8 ± 3.3a,b

aP < 0.01 from normal prostate. bP < 0.01 from localized PCa.

% positive area

Figure 6. GAS6 colocalizes with Hif-1a in both primary tumors and bone metastasis in human prostate cancer. TMA samples were coimmunostained withDAPI, Hif-1a, and GAS6. Representative micrographs showing normal prostate, localized prostate cancer at 20� or 60�, or bone metastasis at 20� or 60�.Quantification of the TMA images including the number of samples evaluated and percentage of total area double positive and was carried out with SoftwareNIS Elements BR3.2 64-bit (Nikon). PCa, prostate cancer.






likely that the regulation of Axl expression is disrupted in asimilar manner in cancer, particularly in a hypoxic tumormicroenvironment, and our group is focusing on this in ourongoing studies.In conclusion, our data provide evidence for the role

played by Axl and GAS6 in prostate cancer tumorigenesisand also offers insights into how GAS6/Axl signaling isregulated in prostate cancer. While we still do not knowhow Axl expression is stabilized during hypoxia, a furtherunderstanding of this process may provide significant newtherapeutic possibilities, especially when coupled with theknowledge that GAS6 signalingmay regulate prostate cancerdormancy in themarrow (26). In fact, it will be interesting toexplore whether other RTK inhibitors which are beingconsidered for as cancer therapeutic testing cancer will alterthese pathways, andwhether theymay bemore effective withagents that prevent receptor stabilization (45, 46). Clearly, asevidence continues to mount that dormant microscopictumors are prevalent in healthy individuals, understandingthe molecular mechanisms that regulate dormancy willbecome increasingly important as our population ages.

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

Authors' ContributionsConception and design: A. Mishra, K.J. Pienta, R.S. TaichmanDevelopment of methodology: A. Mishra, J. Wang, K.J. Pienta

GAS6

Autocrine loop

Paracrine loop

Hypoxic bone microenvironment

Degradation of Axl after GAS6 binding

Axl

Osteoblasts

Y

Hypoxiaprevents

degradationof Axl

Figure 8. Amodel for Axl regulation in a hypoxic tumormicroenvironment.In a bone marrow microenvironment GAS6 is either produced byosteoblasts (paracrine loop) or by the tumor cells (autocrine loop). Thehypoxic microenvironment in the bone facilitates high expression of Axlby the tumor cells despite high GAS6 expression. This results inincreased production of both the ligand and receptor which boostsGAS6/Axl signaling within the bone microenvironment, which enhancestumorigenesis.

Hif-1α

Axl

Merged

Normal prostate (20x)

DAPI

Hif-1α

Axl

DAPI

Localized PCa (20x)

Merged

Hif-1α

Axl

DAPI

Localized PCa (60x)

Hif-1α

Axl

DAPI


Merged

MergedMerged


Hif-1α

Axl

DAPI

Axl / Hif-1αAxlNTissue

Normal

121prostate 0.1 ± 0.10.2 ± 0.2

Localized

120PCa 1.2 ± 0.7a

1.5 ± 1.0

Bone

10metastasis 5.8 ± 2.2 4.9 ± 2.0a,b

% positive area

aP < 0.01 from normal prostate. bP < 0.01 from localized PCa.

Figure 7. Axl colocalizes with Hif-1a in both primary tumors and bonemetastasis in human prostate TMA samples. TMA samples were coimmunostained withDAPI, Hif-1a, and Axl. Representativemicrographs showing normal prostate at 20�, localized prostate cancer at 20� or 60�, and bonemetastasis at 20� or60�. Quantification of the TMA images including the number of samples evaluated and percentage of total area double positive and was carried out withSoftware NIS Elements BR3.2 64-bit (Nikon). PCa, prostate cancer.

Mishra et al.





Acquisition of data (provided animals, acquired and managed patients, providedfacilities, etc.): A. Mishra, S. McGee, J. Kim, J.E. Berry, A. HavensAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): A. Mishra, J. Wang, Y. Jung, J. Joseph, K.J. Pienta,R.S. TaichmanWriting, review, and/or revision of the manuscript: A. Mishra, Y. Shiozawa, S.McGee, J. Kim, J.E. Berry, K.J. Pienta, R.S. TaichmanAdministrative, technical, or material support (i.e., reporting or organizing data,constructing databases): A. Mishra, Y. Jung, J. JosephStudy supervision: Y. Shiozawa, K.J. Pienta, R.S. Taichman

AcknowledgmentsThe authors thank Chris Strayhorn for assistance with the histology and the

University ofMichigan FlowCytometry Core, Vector Core, and the Imaging Core fortheir expertise.

Grant SupportThis work is directly supported by the National Cancer Institute (CA163124,

CA093900, to K.J. Pienta and R.S. Taichman), the Fund for Cancer Research (to R.S.Taichman), the Department of Defense (to Y. Shiozawa, K.J. Pienta, and R.S.Taichman), and the Prostate Cancer Foundation (to Y. Shiozawa, K.J. Pienta, andR.S. Taichman). K.J. Pienta receives support as an American Cancer Society ClinicalResearch Professor, NIH SPORE in prostate cancer grant P50 CA69568, and theCancer Center support grant P30 CA46592.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be herebymarked advertisement in accordance with18 U.S.C. Section 1734 solely to indicate this fact.

Received December 5, 2011; revised April 9, 2012; accepted April 9, 2012;published OnlineFirst April 19, 2012.

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Mishra et al.





2012;10:703-712. Published OnlineFirst April 19, 2012.Mol Cancer Res Anjali Mishra, Jingcheng Wang, Yusuke Shiozawa, et al. CancerHypoxia Stabilizes GAS6/Axl Signaling in Metastatic Prostate

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