imatinib spares ckit-expressing prostate neuroendocrine ...matteo bellone4, regina tardanico5,...

Cancer Biology and Signal Transduction

Imatinib Spares cKit-Expressing ProstateNeuroendocrine Tumors, whereas Kills SeminalVesicle Epithelial–Stromal Tumors by TargetingPDGFR-bElena Jachetti1, Alice Rigoni1, Lucia Bongiovanni2, Ivano Arioli1, Laura Botti1,Mariella Parenza1, Valeria Cancila2, Claudia Chiodoni1, Fabrizio Festinese3,Matteo Bellone4, Regina Tardanico5, Claudio Tripodo2, and Mario P. Colombo1

Abstract

Prostate cancer is a leading cause of cancer-related death inmales worldwide. Indeed, advanced and metastatic diseasecharacterized by androgen resistance and often associated withneuroendocrine (NE) differentiation remains incurable. Usingthe spontaneous prostate cancer TRAMP model, we have shownthat mast cells (MCs) support in vivo the growth of prostateadenocarcinoma, whereas their genetic or pharmacologic tar-geting favors prostate NE cancer arousal. Aiming at simulta-neously targeting prostate NE tumor cells and MCs, bothexpressing the cKit tyrosine kinase receptor, we have tested thetherapeutic effect of imatinib in TRAMP mice. Imatinib-treatedTRAMP mice experience a partial benefit against prostate ade-nocarcinoma, because of inhibition of supportive MCs. How-ever, they show an unexpected outgrowth of prostate NE

tumors, likely because of defective signaling pathway down-stream of cKit receptor. Also unexpected but very effective wasthe inhibition of epithelial–stromal tumors of the seminalvesicles achieved by imatinib treatment. These tumors normallyarise in the seminal vesicles of TRAMP mice, independently ofthe degree of prostatic glandular lesions, and resemble phyl-lodes tumors found in human prostate and seminal vesicles,and in breast. In both mice and in patients, these tumors arenegative for cKit but express PDGFR-b, another tyrosine kinasereceptor specifically inhibited by imatinib. Our results imply apossible detrimental effect of imatinib in prostate cancerpatients but suggest a promising therapeutic application ofimatinib in the treatment of recurrent or metastatic phyllodestumors. Mol Cancer Ther; 16(2); 365–75. �2016 AACR.

IntroductionProstate cancer is one of the leading causes of cancer-related

death worldwide (1), and effective therapies for advanced, hor-mone-refractory, and metastatic disease are still lacking. Prostatecancers are heterogeneous, multifocal, and characterized by awide spectrum of biological behaviors ranging from indolent tohighly malignant stages. Adenocarcinoma comprises the vastmajority of prostate malignant lesions, but various other sub-

types, including neuroendocrine (NE) cancers may occur asfocal areas into adenocarcinomas or as pure forms (2). Prostatecancers with NE phenotype account for a small percentage ofcases at diagnosis, but NE differentiation often appears afterandrogen ablation (3) and correlates with aggressiveness, poorprognosis, and resistance to current therapies (4).

A well-characterized spontaneous model of prostate canceris the TRAMP mouse, which express SV40 small and largeT antigens under the control of the androgen-driven rat probasinregulatory element (5). Therefore, TRAMP mice on C57BL/6Jbackground progressively develop prostatic intraepithelial neo-plasia (8–16 weeks of age), adenocarcinoma (after 16 weeks ofage) and metastasis (6). As in humans, prostate tumors with NEphenotype can arise in about 15% to 20% of C57BL/6J TRAMPmice (7) and the frequency of these cancers increase aftercastration (8). Moreover, TRAMP mice also develop epitheli-al–stromal tumors in the seminal vesicles (9, 10), which ariseindependently from prostatic lesions (9) and resemble humanphyllodes tumors found in breast (11, 12), prostate (13), andseminal vesicles (14). In humans, these rare neoplasms arecommonly managed with surgical removal; however, localrecurrence is common and metastatic spread associated witha poor prognosis has been reported in some patients (12, 14).

The study of tumor microenvironment and of the interplaybetween tumor, stromal, and immune cells is of fundamen-tal relevance for understanding cancer biology and for the

1Molecular Immunology Unit, Department of Experimental Oncology andMolec-ular Medicine, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy.2Tumor Immunology Unit, Department of Health Sciences, University ofPalermo, Palermo, Italy. 3Pharmacy Unit, Fondazione IRCCS Istituto Nazionaledei Tumori, Milan, Italy. 4Division of Immunology, Transplantation and InfectiousDiseases, Cellular Immunology Unit, San Raffaele Scientific Institute, Milan, Italy.5Department of Molecular and Translational Medicine, Section of Pathology,University of Brescia, Brescia, Italy.

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

CorrespondingAuthor:Mario P. Colombo, Fondazione IRCCS Istituto Nazionaledei Tumori, via Amadeo 42, Via G. Venezian 1, Milano 20133, Italy. Phone: 3902-2390-2252; Fax: 3902-2390-2630; E-mail:[email protected]

doi: 10.1158/1535-7163.MCT-16-0466

�2016 American Association for Cancer Research.

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development of novel therapeutic strategies. In this scenario,mast cells (MCs) have been recognized capable of proangio-genic (15) and protumorigenic activities (16, 17). We havepreviously demonstrated that MC-produced MMP9 is necessaryfor the growth of incipient prostate adenocarcinoma, while itbecomes dispensable for tumors undergoing epithelial-to-mes-enchymal transition that produce MMP9 autonomously (18).However, treatment of TRAMP mice with cromolyn, an agentcommonly used to prevent MCs degranulation (19, 20),reduced the development of adenocarcinoma with the draw-back of increasing the frequency of prostate NE tumors (18),which suggested a protective role of MCs against NE differ-entiation. These results implied that any attempt to targetMC-induced support of adenocarcinoma development shouldbe coupled with drug(s) killing the NE variants. Alternatively, asingle drug able to hit a common target on both MCs andNE tumor cells could be taken into account. This latter possi-bility is explored in this article under the observation that NEtumor cells express the cKit receptor, which is also needed forMCs survival and functional activation (21). Therefore, wehypothesized that imatinib mesylate, a drug-targeting cKit,should be able of eliminating both adenocarcinoma, via MCsinhibition, and the NE variants, via direct effect on NE tumorcell. Imatinib is a well-known tyrosine kinase inhibitor, cur-rently approved for the treatment of chronic myelogenousleukemia (22) and gastrointestinal stromal tumors (23), whichtargets the intracellular ABL kinase and the fusion oncogeneBCR-ABL of chronic myeloid leukemia (24) and the surfacereceptors cKit and PDFGRs (25). In this study, we have ana-lyzed tumor outcome in TRAMPmice treated with imatinib anddemonstrated its activity against adenocarcinoma but not NEtumors, which we found to have a defect in cKit signaling. Wealso have uncovered a new application for imatinib in treatingepithelial–stromal tumors of seminal vesicles.

Materials and MethodsMice, cell lines, and treatments

TRAMP mice on C57BL/6J background (C57BL/6-tg(TRAMP)8247Ng/J) were kindly provided by Dr. Vincenzo Bronte (IRCCSIstituto Oncologico Veneto, Padova, Italy), under agreement withDr. NormanMichael Greenberg (formerly working at FredHutch-inson Cancer Research Centre, Seattle, WA, at the time whenMaterial Transfer Agreement was signed). Heterozygous experi-mental TRAMPmicewere obtained by breedingwild-typeC57BL/6 male mice and heterozygous female TRAMPmice and screenedaccording to ref. 5. MC-deficient C57BL/6-KitW-sh/W-sh (KitWsh;ref. 26) mice were purchased from Jackson Laboratories andintercrossed over 12 generations with TRAMP mice to obtainMC-deficient KitWsh-TRAMP mice. Mice were maintained underpathogen-free conditions at the animal facility of FondazioneIRCCS Istituto Nazionale dei Tumori, and experiments wereauthorized by the Institute Ethical Committee and performed inaccordance to institutional guidelines and national law (D.lgs 26/2014). Cromolyn (10 mg/kg dissolved in saline; Sigma Aldrich)or imatinib (50mg/kg dissolved in saline; Novartis; provided as adonation by the family of a deceased patient) were administeredintraperitoneally in TRAMP mice for 5 days/week. Treatmentsstarted at 8 or 16 weeks, as indicated in text and figures, andcontinued for the duration of the experiment.Micewere sacrificedat 25 weeks and their urogenital apparatus collected for IHC.

OPT-7714 cells were obtained in our laboratory from aNE tumor occurred in an osteopontin-deficient TRAMPmouse (27). OPT-7714, PC3 human prostate cancer cells(CRL-1435, LCG-Promochem), and B16 F10 murine melano-ma cells were grown in DMEM (Gibco-Thermo Fisher) with10% of FBS (Gibco-Thermo Fisher), 2 mmol/L L-glutamine,150 U/mL streptomycin, and 200 U/mL penicillin (Cambrex),10 mmol/L HEPES, and 10 mmol/L sodium pyruvate (Gibco-Thermo Fisher). TNE-PCSC and TSV-PCSC were obtainedfrom a NE tumor or from a seminal vesicle tumor of TRAMPmice, respectively, and cultured in serum-freeDMEMF12 (Gibco-Thermo Fisher) containing EGF and FGF2 (PeproTech) as de-scribed in ref. 28.

Histology and IHCHuman primary tumor samples were obtained from the

pathology archives of the Human Pathology Section, Depart-ment of Health Sciences (University of Palermo, Palermo, Italy)or from the Department of Molecular and Translational Med-icine, Section of Pathology, University-Spedali Civili of Brescia.Human tumor specimens and murine urogenital apparatus,including prostate and seminal vesicles, were fixed in formalinand embedded in paraffin. Five-micron sections were stainedwith Mayer-hematoxylin and eosin (H&E; BioOptica) and eval-uated by an expert pathologist in a blind fashion. Prostateslesions of TRAMP mice were scored as hyperplasia or intrae-pithelial neoplasia (HYP-PIN), adenocarcinoma (ADENO), andneuroendocrine tumor (NE). The actual malignant nature of theepithelial proliferation in TRAMP adenocarcinoma samples istestified by the stromal invasion by highly atypical cells positivefor cytokeratin 8 (CK8) and organized in the formation ofdistorted glands. For evaluation of MC infiltration, sectionswere stained with toluidine blue (BioOptica). Alternatively,after rehydratation and antigen retrieval, IHC was performedusing the streptavidin–biotin–peroxidase complex method, and3,30-Diaminobenzidine tetrahydrochloride as chromogenic sub-strate. Primary antibodies were: rabbit anti-human and mousecKit polyclonal antibody (catalog no. AP20497PU-N, Acrisantibodies); rabbit anti-human and mouse PDGFR-b antibody(Y92; catalog no. Ab32570, Abcam); rabbit anti mouse Ki-67(catalog no. AB15580, Abcam), and rabbit anti-mouse CK8(catalog no. AB59400, Abcam). Slides were analyzed under aLeica DM2000 optical microscope equipped with a LeicaDFC320 digital camera (Leica Microsystems).

Bone marrow–derived MCs and degranulationBone marrow–derived MCs were obtained by in vitro differ-

entiation of bone marrow cells taken from mouse femursand tibias, in RPMI (Gibco-Thermo Fisher) with 10% FBS,2 mmol/L L-glutamine, 150 U/mL streptomycin, and 200 U/mLpenicillin (Cambrex), 10 mmol/L HEPES, 10 mmol/L sodiumpyruvate, and 5 mmol/L b-mercaptoethanol (Gibco-ThermoFisher), in the presence of IL3 and SCF (20 ng/mL each;Peprotech), as described in ref. 29. After 5 weeks of culture,BMMCs were monitored for FceRI and c-Kit expression by flowcytometry, and used if purity was more than 90%. For degran-ulation experiments, MCs were starved 2 hours in mediumwithout IL3 and SCF, then sensitized 2 hours with 1 mg/mL ofDNP-specific IgE and stimulated 30 minutes with 100 ng/mLof DNP-HSA (100 ng/mL; Sigma-Aldrich). Alternatively, starv-ed MCs were stimulated for 30 minutes with 100 ng/mL of

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SCF. When indicated, imatinib or Cromolyn were addedduring stimulation (10 mmol/L each). Degranulation was mea-sured as assessment of surface CD107a expression by flowcytometry (30).

In vitro cell proliferationFor proliferation experiments, cells were plated in 96-well

plates (5,000 cells/well) in presence of different concentrationsof imatinib, as indicated (1, 5, 10 mmol/L). After 96 hours, cellproliferation was assessed using the XTT Cell-Viability Kit(Applichem) according to the manufacturer's protocol.

Flow cytometryFor surface detection of cKit, PDGFR-b, or CD107a, single-cell

suspensions were incubated 10 minutes with FcR blocker (BDBiosciences), labeled for 15 minutes at 4�C with fluorochrome-conjugated mAbs or isotype controls (all from BD Biosciences oreBiosciences). For intracellular detection of AKT, STAT1, or theirphosphorylated forms, cells were stimulated for 20 minutes with100 ng/mL of SCF or PDGF-bb, respectively. When indicated,imatinib (10 mmol/L) was added during stimulation. Cells werethen fixed with BD-FixI Buffer, permeabilized with BD-PermIIIBuffer, and stained with desired antibodies or isotype controlsaccording to the BD-Phosflow Kit protocol (BD Biosciences). Allsamples were acquired with BD LSRII Fortessa instruments andanalyzed with the FlowJo software.

Statistical analysisStatistical analysis was performed with the GraphPad Prism

software (GraphPad Software); we used Fisher test for com-parison of tumor frequencies and the Student t, or one-wayANOVA followed by Tukey tests for other experiments.Values were considered statistically significant for �, P < 0.05;��, P < 0.01; ��, P < 0.001; ��, P < 0.0001.

ResultsImatinib treatment restrains growthof adenocarcinomabutnotof NE prostate tumors

Aiming at targeting the cKit receptor in prostate cancers ofTRAMP mice, we tested its expression in adenocarcinoma andNE prostate cancers spontaneously occurring in 25-week-olduntreated TRAMP mice. Tumor histology was evaluated onH&E-stained sections and scored as adenocarcinoma when weobserved stromal invasion by highly atypical cells, positive forcytokeratin 8 (CK8) immunostaining, which have distorted theglands organization (Supplementary Fig. S1A). The differentproliferative attitude of TRAMP adenocarcinomas and NEtumors is evidenced by immunostaining for Ki-67, whichreveals that NE tumors display a higher fraction of activelyproliferating malignant elements in comparison with adeno-carcinomas (Supplementary Fig. S1B).

Accordingly to our previously published data (18), MCs infil-trating the stroma of adenocarcinoma showed strong positivityfor cKit staining, whereas epithelial and other stromal cells werenegative (Fig. 1A, left). Tumor cells in seldom occurring sponta-neous NE tumors of TRAMP mice displayed homogeneous andmild positivity for cKit (Fig. 1A, right). Results obtained in themouse model were confirmed also in prostate cancer patients,with MCs infiltrating the adenocarcinoma strongly positive for

cKit (Fig. 1B, left), and tumor cells of the focal NE areas showingdiffuse but mild positivity for cKit (Fig. 1B, right).

To test whether cKit inhibition could impair both adenocar-cinoma-promoting MCs activity and NE tumor cell growth, wetreated TRAMP mice with imatinib from 8 to 25 weeks of age,and collected the urogenital apparatus, at necropsy, for histo-pathology. The treatment significantly reduced the incidence ofadenocarcinomas (47.1% vs. 76.9% of untreated TRAMPmice; Fig. 1C) but had no effect against NE tumors, whichinstead significantly increased in frequency (23.5% vs. 15.4%of untreated TRAMP mice; Fig. 1C). The expression of cKitwas maintained in NE tumors treated with imatinib (Fig.1D), which ruled out that loss of receptor expression couldunderlie imatinib resistance.

These results were compared with those we obtained withcromolyn, a compound inhibiting MCs degranulation. As wepreviously reported (18), and similar to Imatinib, the treatmentwith cromolyn reduced adenocarcinoma incidence in favor of NEtumor occurrence in TRAMP mice (Supplementary Fig. S2A).

The cKit signaling pathway is inactive in NE tumor cellsAs IHC detected cKit expression confined to MCs infiltrating

adenocarcinomas (Fig. 1A and B), and as imatinib and cromo-lyn treatments had comparable outcomes in TRAMP mice(ref. 18; Fig. 1C and Supplementary Fig. S2A), we tested whetherthe two drugs similarly inhibited MCs activity. It is known thatthe administration of cromolyn or imatinib does not affect thenumber of MCs in vivo (20, 21). Accordingly, neither thetreatment with imatinib nor that with cromolyn decreased MCsinfiltration of prostate adenocarcinomas in TRAMP mice (Fig.2A; Supplementary Fig. S2B). When tested in vitro, these drugssignificantly inhibited MCs degranulation, evaluated as CD107amembrane expression, in response to IgE/Ag stimulation (Fig.2B). A mild stimulus, like that provided by stem cell factor(SCF) triggering of cKit, induces less degranulation, which wasstill inhibited by imatinib but not by cromolyn (Fig. 2B). Theseresults indicate that imatinib inhibits MCs degranulation. Nev-ertheless, imatinib did not reduce in vitro proliferation of bonemarrow–derived MCs (Fig. 2C).

We next sought to understand why NE tumors were insensitiveto imatinib treatment, despite their expression of cKit. We testedtwo different cell lines, namely TNE-PCSC, endowed also withcancer stem cells properties (28), and OPT 7714 (27), bothderived from NE tumors of TRAMP mice, and able to generatetumors upon transplantation into syngeneic mice (Supplemen-tary Fig. S3 and refs. 27, 28). Confirming the in vivo data, imatinibwas ineffective on in vitro proliferation of TNE-PCSC and OPT-7714,whereas active against B16-F10melanoma cells, whichwereused as positive control (Fig. 2C; Supplementary Fig. S4). Inaccording to in vivo data, TNE-PCSC and OPT 7714 cells expresscKit, though at lower levels if compared with bone marrow–derived MCs (Fig. 2D). Results obtained with murine cells lineswere confirmed using human PC3 prostate cancer cells, whichhave NE features (31), express cKit on cell surface (ref. 31;Supplementary Fig. S5A), but were insensitive to imatinib treat-ment in vitro (Supplementary Fig. S5B).

We then investigated whether cKit expressed on NE tumorcells was able to induce signaling upon stimulation with SCF(Fig. 2E). AKT phosphorylation, which was effectively inducedby SCF in MCs used as positive control (32), did not occurin TNE-PCSC and OPT 7714 cells upon SCF-mediated cKit

Imatinib Kills Seminal Vesicle Stromal Tumor

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engagement (Fig. 2E). In MCs, imatinib was effective in block-ing SCF activation of cKit and the downstream AKT phosphor-ylation (Fig. 2E). These results indicate that cKit signalingpathway is defective in NE tumor cells.

As imatinib can target tyrosine kinase receptors other thancKit, specifically the PDGFR-b, we tested its expression inprostates of 25-week-old TRAMP mice. Immunostainingmarked pericytes of tumor vessels but not tumor and stromalcells of adenocarcinoma or NE tumors (Fig. 3A). A correlationwas found with human specimens showing PDGFR-b expres-sion confined to pericytes and scattered stromal cells, andnegativity for staining of tumor cells of either adenocarcinomaor NE histotype (Fig. 3B).

Also, the TNE-PCSC and OPT 7714 cells as well as bonemarrow–derived MCs showed very low expression of PDGFR-bby flow cytometry (Fig. 3C), and consistently did not respond toPDGF-bb stimulation measured as phosphorylation of STAT1(Fig. 3D; ref. 33).

Results collected so far, together with our published datashowing that infiltrating MCs support the growth of prostateadenocarcinoma by releasing MMP9 (18), suggest that thereduced incidence of prostate adenocarcinoma in TRAMP micetreated with imatinib is likely due to the inhibition of MCsdegranulation. Moreover, adenocarcinoma and stromal cells

were negative for both cKit and PDGFR-b. This likely excludeda direct effect of imatinib on these cell types in treated mice.Our data also indicate that imatinib is not effective againstNE prostate cancer, which bear unresponsive cKit receptor andlack PDGFR-b.

Imatinib treatment inhibits the growthof PDGFR-b–expressingepithelial–stromal tumors of seminal vesicles

Along with prostate adenocarcinomas, TRAMP mice alsodevelop spontaneous epithelial–stromal tumors of seminalvesicles (9, 10). These tumors develop as independent clonesunder the same transgenic SV40 large T antigen oncogene(9, 10) and resemble phyllodes epithelial–stromal tumorsobserved in seminal vesicles (14), prostate (13), and breast(11) in humans. Therefore, we investigated whether in theabove-described cohort of TRAMP mice imatinib exerted anyeffect on seminal vesicles tumors. Histologically, untreated25-week-old TRAMP mice showed enlarged seminal vesicleswith neoplastic lesions composed by an epithelial componentand by a subepithelial proliferating stromal component pro-truding inside the lumen (Fig. 4A), developing in 70% of micein our cohort (Fig. 4C). Strikingly, in mice treated with ima-tinib, the seminal vesicles were normal, with lumen surfacelined by columnar epithelium that ramified in folds (Fig. 4B).

Figure 1.

Imatinib treatment hampersadenocarcinoma but not NE prostatetumor outgrowth in TRAMPmice.A andB, Representative cKit staining ofprostates of 25-week-old TRAMP mice(A) or human patients (B) bearingadenocarcinoma (ADENO; left) or NEtumors (right). Magnification �40.Arrows indicate cKit-positive MCswithin adenocarcinoma.C,TRAMPmicewere either left untreated (n ¼ 13) ortreated from 8 to 25 weeks of age withimatinib (n ¼ 17). Urogenital apparatawere collected at necropsy at 25 weeksand embedded in paraffin forhistopathologyanalysis onH&E-stainedsections. Graph depicts the relativepercentage of lesions scored by apathologist as hyperplasia-prostaticintraepithelial neoplasia (HYP-PIN,white area), adenocarcinoma (ADENO,light blue area), or neuroendocrine (NE,violet area). Numbers within barrepresent relative percentages. Resultsare a pool of two independentexperiments. D, Representative cKitstaining of NE tumors grown in TRAMPmice treated with imatinib.Magnification �40. Fisher test:�� , P < 0.0001.

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Only 1 of 17 (5.9%) TRAMP mice treated with imatinib from 8weeks of age developed a phyllodes seminal vesicle tumor(Fig. 4C). Such efficacy was strengthened by the demonstrationthat imatinib was curative when given to TRAMP mice of16 weeks of age, already in presence of incipient lesions inthe seminal vesicles (Supplementary Fig. S6A). Interestingly,Cromolyn-treated TRAMP mice did not show any change inthe frequency of seminal vesicles tumors (Fig. 4C), suggestingthat MC activity was dispensable for the growth of phyllodestumors and that imatinib directly affected tumor cells. Indeed,imatinib inhibited the proliferation of TSV-PCSC (Fig. 4D), acancer stem cell–like cell line obtained from a seminal vesicletumor of a TRAMP mouse (28).

We tested the expression of both cKit and PDGFR-b receptorsin seminal vesicle tumors, as possible targets of imatinib. IHCshowed cKit expression on epithelial cells and in scatteredinfiltrating MCs, but not in the proliferating phyllodes com-partment (Fig. 5A). On the contrary, PDGFR-b was markedlyexpressed in stromal sub-epithelial–proliferating cells but notin the epithelial compartment (Fig. 5B). Normal adjacentseminal vesicle tissue was negative for PDGFR-b (Supplemen-tary Fig. S6B).

Human counterpart represented by phyllodes tumor of theprostate showed mixed mesenchymal and epithelial prolifera-tion, characterized by the presence of spindle-shaped or ovalmesenchymal cells with irregular and slightly atypical nucleiintermingling with epithelial cells with an irregular contour andpapillary epithelial projections. Remarkably, the mesenchymalcomponent resulted negative for cKit, which was expressed onlyby scattered MCs (Fig. 5C), whereas it was intensely positive forPDGFR-b (Fig. 5D).

Interestingly enough, as also reported by others (34),human breast phyllodes tumors showed marked positivity toPDGFR-b in the stromal proliferating compartment (Supple-mentary Fig. S7).

Figure 2.NE tumor cells are insensitive to imatinib treatment and have a defective cKitsignaling pathway. A, MC count carried on toluidine blue–stained sections ofprostates of 25-week-old TRAMP mice either untreated or treated withcromolyn or imatinib as reported in Fig. 1 and Supplementary Fig. S2. Resultsare a pool of two independent experiments. B, Bone marrow–derived MCswere left untreated, stimulated with SCF (100 ng/mL), or sensitized withDNP-specific IgE and stimulated with DNP antigen (IgE/Ag). Cromolyn(10 mmol/L) or imatinib (10 mmol/L) were added during stimulation. MCsdegranulation was tested as membrane CD107a expression by flowcytometry. Results are representative of three independent experiments. C,TNE-PCSC, OPT 7714 cells, or bone marrow–derived MCs were cultured for96 hours in the presence of different concentrations of imatinib, as indicated.Proliferation was assessed with the Cell proliferation Kit XTT (Applichem).Results are representative of three independent experiments. B16-F10 tumorcells were used as positive control (CTR). Statistical comparison wasperformed to confront for each cell line the proliferation at different imatinibconcentrations with respect to control (cells without imatinib). Student t test:�� , P < 0.001; �� , P < 0.0001. D, Flow cytometry analysis of cKit expressionin TNE-PCSC, OPT 7714 cells, or bone marrow–derived MCs. RFI: relativefluorescence intensity calculated as ratio of mean fluorescence intensitybetween stained sample (red histogram) and isotype control sample (blackhistogram). E, TNE-PCSC, OPT 7714 cells, or BMMCs were left untreated,stimulated with SCF (100 ng/mL), or with SCF (100 ng/mL) and imatinib(10 mmol/L), and then analyzed by flow cytometry for expression of AKT orphosphorylated AKT (pAKT). Graph reports ratio of RFI between pAKT andtotal AKT. Results are representative of three independent experiments.ANOVA followed by Tukey test: � , P < 0.05; �� , P < 0.01.






In line with in vivo results, TSV-PCSC cells displayed verylow levels of cKit (RFI: 4.5) but high levels of PDGFR-b (RFI:61.7; Fig. 5E). Accordingly, SCF stimulation did not trigger AKTphosphorylation (Fig. 5F), but PDGF-bb induced STAT1 phos-phorylation, which was inhibited by concomitant presence ofimatinib (Fig. 5G).

Altogether, these results indicate that both in murine modeland in patients, urogenital epithelial–stromal tumors expresshigh levels of functional PDGFR-b and candidate imatinib astreatment of choice for these diseases.

DiscussionDespite recent approval of several new drugs and biotherapy,

including docetaxel, abiraterone, enzalutamide, carbazitaxel,223Radium dichloride, and Sipuleucel-T (35), the survival ofcastration-resistant and metastatic prostate cancer patientsremains limited (35). The search for optimal treatment is com-plicated by the multifocal nature of this disease, and by the likelyoccurrence of NE differentiation (4), which is associated withresistance to therapy and dismal prognosis (4).

The different components of the tumormicroenvironment thathave the capacity of promoting tumor growth are viewed aspotential alternative targets. In this context, we have previouslyidentified MCs as a supporting component of the prostate micro-environment endowed with the ability to produce MMP9, whichin turn is necessary for the progression of incipient adenocarci-noma (18). Unfortunately, in the spontaneous TRAMP mousemodel of prostate cancer, the use of cromolyn to inhibit MCfunction while reducing the frequency of adenocarcinoma alsopromoted the rise of NE variants (18). This result underscores apossible protective role of MCs against NE prostate cancer var-iants, likely through the release of soluble factors, and warnsabout the need of coupling MCs targeting with drugs directedagainst NE tumors to foresee any possible translation in prostatecancer patients.

The common expression of cKit on MCs, which support thegrowthof adenocarcinoma, andonNE tumors, as shownhere andin and other recent publications (36–38), prompted the rationaleof testing the cKit inhibitor imatinib in TRAMP mice. Similar tocromolyn, imatinib reduced the incidence of adenocarcinoma,likely by neutralizing MCs activity. Contrary to our expectation,

Figure 3.

MCs and NE tumor cells do not expressPDGFR-b. A and B, RepresentativePDGFR-b staining of prostates of25-weeks-old TRAMP mice (A) orhuman patients (B) bearingadenocarcinoma (left) or NE tumors(right). Magnification �20x. C, Flowcytometry analysis of PDGFR-bexpression in TNE-PCSC, OPT 7714cells, or BMMCs. RFI, relativefluorescence intensity calculated asratio of mean fluorescence intensitybetween stained sample (redhistogram) and isotype control sample(black histogram). D, TNE-PCSC, OPT7714 cells, or BMMCs were leftuntreated or stimulated with PDGF-bb(100 ng/mL) and then analyzed byflow cytometry for expression ofSTAT1 and phosphorylated STAT1(pSTAT1). Graph reports ratio of RFIbetween pSTAT1 and total STAT1.Results are representative of twoindependent experiments.

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the NE variants generated as a consequence of MCs inhibitionwere insensitive to imatinib treatment, despite expressing cKit.Indeed, the intracellular pathway downstream cKit receptor inNE

tumor cells was found inactive, suggesting this pathway dispens-able forNE tumor cells proliferation and survival. Thisfindingwasunexpected, as usually activating mutations render cKit an

Figure 4.

Imatinib treatment strongly inhibits the growth of epithelial–stromal tumor of seminal vesicles in TRAMP mice. A and B, Representative H&E staining of seminalvesicles of 25-week-old TRAMP mice untreated (A) or treated daily with imatinib (B) as in Fig. 1. Magnification �4 and �40 as indicated. C, TRAMP micewere either left untreated (n ¼ 13) or treated from 8 to 25 weeks of age with cromolyn (n ¼ 12) or imatinib ("Imatinib preventive"; n ¼ 17), as reported in Fig. 1and Supplementary Fig. S2. An additional group of TRAMPmice was treated with imatinib starting from 16 weeks of age ("Imatinib therapeutic"; n¼ 13). Urogenitalapparata were collected at necropsy at 25 weeks and embedded in paraffin for histopathology analysis on H&E-stained sections. Graph depicts the relativepercentage of epithelial–stromal tumor of seminal vesicles (SV tumors) as scored by a pathologist. Results are a pool of two independent experiments. Fisher test:�� ,P<0.0001.D, TSV-PCSCswere cultured for 96hours in the presence of different concentrations of imatinib, as indicated. Proliferationwas assessedwith theCellproliferation Kit XTT (Applichem). Results are representative of three independent experiments. Statistical comparison was performed for the proliferation atdifferent imatinib concentrations with respect to control (cells without imatinib); Student t test: � , P < 0.05; �� , P < 0.001.






Figure 5.

Imatinib sensitivity of phyllodes tumors of seminal vesicles is due to PDGFR-b expression. A and B, Representative cKit (A) or PDGFR-b (B) stainingof phyllodes epithelial–stromal tumors arising in seminal vesicles of 25-week-old TRAMP mice. Magnification �20; insets �40. Arrows indicate cKitpositive MCs. C and D, Representative cKit (C) or PDGFR-b (D) staining of human phyllodes tumor infiltrating the prostate. E, Flow cytometry analysisof cKit (top) or PDGFR-b (bottom) expression in TSV-PCSC. RFI: relative fluorescence intensity calculated as ratio of mean fluorescence intensitybetween stained sample (red histogram) and isotype control sample (black histogram). (Continued on the following page.)

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oncogene, like in gastrointestinal stromal tumors (GIST; ref. 39).Nevertheless, our results are in accordance with the data pub-lished by Tetsu and colleagues (40), who found the cKit pathwayinhibited because of missense mutations in a subset of adenoiccystic carcinomas (ACC)of the salivary glands (40). The persistentproliferation of ACC carrying the cKit-inactivating mutationssuggested that cKit was probably necessary at early stages oftransformation whereas dispensable in late-stage tumors, andthat cKit inhibition is probably useless for treating ACC patients(40). Similarly, we suppose that cKit is dispensable for the growthof both adenocarcinoma and NE prostate cancer cell, being cKitnegative or defective for cKit signaling, respectively.

The origin of prostate NE tumor is still poorly understood;trans-differentiation from adenocarcinoma or distinct clonalevolution from basal/progenitor cells are the most creditedhypothesis (4, 41, 42). The issue is further complicated by theexistence of NE morphologic variants, namely: "de novo"small-cell NE carcinoma and prostate adenocarcinoma withNE differentiation. Moreover, "de novo" small cells NE tumorscan rarely occur as sole tumor in absence of adenocarcinoma(less than 1% of cases) or more frequently after therapy ofpre-existing adenocarcinoma (roughly in 25% of patientswith advanced prostate cancer; ref. 4). Both can express mar-kers of basal/progenitor cells (i.e., cKit and p63; ref. 4), andfall into a basal stem-like gene signature (42). Our datasuggest that cKit is a marker of these "de novo" NE tumorslikely reflecting their basal origin rather than a functionaldriver for their progression.

In our murine model, imatinib defeated adenocarcinoma,via MCs neutralization, but was ineffective against NE tumors.Inefficacy of imatinib in prostate cancer patients has beendocumented also in several clinical trials (43). Phase I trialswere mainly performed in metastatic castration resistant pros-tate cancer, and only few of them warranted further phase IItrials (43). To our knowledge, results of seven different phase IItrials testing imatinib in prostate cancer patients have beenpublished so far; however, six of them were discouraging, withno evidences of therapeutic benefits (43–46). Evidences of PSAresponse rate and increased progression-free survival have beenreported in one recent study, in which imatinib was given incombination with pioglitazione, etoricoxib, treosulfsan, anddexamethasone (47), a result that however does not explain theweight of imatinib in this five-drug combination. Taken togeth-er, these studies lead to the conclusion that imatinib is mostlyineffective in prostate cancer patients (43). We think that ourexperimental data may in part explain these discouragingresults in the clinical setting. As NE differentiation in recurrentand metastatic disease is often not analyzed its frequency couldgo underestimated (4), and the potential benefic effects ofimatinib on adenocarcinoma via MCs inactivation could havebeen masked by unrestrained NE tumor cells outgrowth, asobserved in our preclinical model. Indeed, in these trials, thefinal outcome of disease progression does not specify theprevalent histotype that remains mostly unknown.

The use of imatinib in those trials was justified by the presenceof PDGFR-b in bone metastasis of prostate cancer (44, 45).However, data on PDGFR-b expression in primary prostate cancerremain contradictory, as some authors reportedmild positivity inthemajority of specimens tested (45, 46),whereas others reportedmoderate or strong PDGFR-b expression only in 5% of clinicallylocalized and 16% of metastatic prostate cancer cases (48). Theselast evidences are in line with our data showing no expression ofPDGFR-b in adenocarcinoma or NE prostate tumor cells in bothmice and human specimens, and offer a further possible expla-nation for the poor results obtained in clinical trials with imatinibin prostate cancer patients.

Our results have evidenced that imatinib can potentially hitdifferent cell types within the tumor microenvironment, depend-ing on their expression of targeted receptors. The final effect mayresult in tumor suppression or promotion according to thefunction of the targeted cell as exemplified by MCs that fosteradenocarcinoma while, likely, could prevent NE variants. More-over, our finding that NE tumor cells bear unresponsive cKitunderscores that the mere receptor expression might not alwaysimply drug responsiveness.

The TRAMP mouse is also a good model for studying theprimary epithelial stromal tumors of seminal vesicles (9, 10), arare disease in humans (14). In TRAMP mice this tumor developindependently from prostatic lesions (9) and resemble humancounterparts observed in seminal vesicles (14), prostate (13) andbreast (11). Human primary epithelial–stromal tumors of theseminal vesicles count for a total of 24 documented cases in theliterature, and they have been described with different names,including "cystadenoma," "cystic epithelial–stromal tumors,""cystomoma," "Mullerian adenosarcoma-like tumors," "phyl-lodes tumor," and "cystosarcoma phyllodes" (14). Follow-upafter surgery is available for only 20 patients, among them 1 hadrecurrence and 2 had lung metastasis (14). Patients with lungmetastasis either received systemic chemotherapy and was alivewith no recurrence for the known 6 months follow up (49), ordied 11 month after surgery (50).

Here, we confirm that, similar to human breast phyllodestumors (ref. 34 and this article), both murine and human epi-thelial–stromal tumors of seminal vesicles express PDGFR-b. Weshow for the first time that imatinib can effectively restrain thesetumors, at least in the mouse model, suggesting the use ofimatinib to treat the rare patients thatmight experience recurrenceor metastasis. Interestingly our data suggested that MCs, despitebeing infiltrating, are likely innocent bystanders in epithelial–stromal tumors of seminal vesicles, and that imatinib directlyinhibits the growth of PDGFRb-positive tumor cells.

As for the seminal vesicles, no effective therapy other thansurgery is available for humanbreast phyllodes tumors,which canbe classified as benign, borderline or malignant, all at risk ofrecurrence. Those classified borderline and malignant have alsoincreased risk of metastasis, with no suitable therapies (12).Expression of PDGFR-b also in these tumors (this article andref. 34) suggests of considering imatinib for treating phyllodes

(Continued.) F, TSV-PCSC were left untreated or stimulated with SCF (100 ng/mL) and then analyzed by flow cytometry for expression of AKT orphosphorylated AKT (pAKT). Graph reports ratio of RFI between pAKT and total AKT. Results are representative of three independent experiments. G,TSV-PCSC were left untreated, stimulated with PDGF-bb (100 ng/mL), or with PDGF-bb (100 ng/mL) and imatinib (10 mmol/L) and then analyzed byflow cytometry for expression of STAT1 and phosphorylated STAT1 (pSTAT1). Graph reports ratio of RFI between pSTAT1 and total STAT1. Results arerepresentative of two independent experiments; Student test: � , P < 0.05; �� , P < 0.01.






breast tumormetastases. Indeed, we think that the use of imatinibor other tyrosine kinase inhibitors in phyllodes tumors deservesclinical investigation.

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

Authors' ContributionsConception and design: E. Jachetti, M.P. ColomboDevelopment of methodology: E. JachettiAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): E. Jachetti, A. Rigoni, L. Bongiovanni, I. Arioli,L. Botti, M. Parenza, V. Cancila, C. Chiodoni, M. Bellone, R. Tardanico,C. TripodoAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): E. Jachetti, A. Rigoni, L. Bongiovanni, V. Cancila,C. Tripodo

Writing, review, and/or revision of the manuscript: E. Jachetti, C. Tripodo,M.P. ColomboAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): L. Bongiovanni, F. FestineseStudy supervision: E. Jachetti, M.P. Colombo

Grant SupportThis work was supported by grants from Fondazione Italo Monzino

and from Associazione Italiana per la Ricerca sul Cancro (I.G. n 14194; toM.P. Colombo). E. Jachetti is supported by a fellowship from FondazioneUmberto Veronesi.

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

Received July 18, 2016; revised October 31, 2016; accepted November 19,2016; published OnlineFirst December 15, 2016.

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2017;16:365-375. Published OnlineFirst December 15, 2016.Mol Cancer Ther Elena Jachetti, Alice Rigoni, Lucia Bongiovanni, et al.

βTargeting PDGFR-Stromal Tumors by−whereas Kills Seminal Vesicle Epithelial

Imatinib Spares cKit-Expressing Prostate Neuroendocrine Tumors,

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