v-Src Oncogene Induces Trop2 Proteolytic Activation via ... · signal transducer 2 (TACSTD2/Trop2/M1S1) is enriched in the prostate, promoting prostate stem cell self-renewal upon

Tumor and Stem Cell Biology

v-Src Oncogene Induces Trop2 ProteolyticActivation via Cyclin D1Xiaoming Ju1,2, Xuanmao Jiao1,2, Adam Ertel1,2, MathewC. Casimiro1,2, Gabriele Di Sante1,2,Shengqiong Deng1,2, Zhiping Li1,2, Agnese Di Rocco1,2,Tingting Zhan2,3, AdamHawkins2,4,Tanya Stoyanova5, Sebastiano And�o6, Alessandro Fatatis2,7, Michael P. Lisanti2,8,Leonard G. Gomella2,9, Lucia R. Languino1,2, and Richard G. Pestell1,2,4

Abstract

Proteomic analysis of castration-resistant prostate cancer dem-onstrated the enrichment of Src tyrosine kinase activity in approx-imately 90% of patients. Src is known to induce cyclin D1, and acyclin D1–regulated gene expression module predicts poor out-come in human prostate cancer. The tumor-associated calciumsignal transducer 2 (TACSTD2/Trop2/M1S1) is enriched in theprostate, promoting prostate stem cell self-renewal upon proteo-lytic activation via a g-secretase cleavage complex (PS1, PS2) andTACE (ADAM17), which releases the Trop2 intracellular domain(Trop2 ICD). Herein, v-Src transformation of primary murineprostate epithelial cells increased the proportion of prostatecancer stem cells as characterized by gene expression, epitope

characteristics, and prostatosphere formation. Cyclin D1 wasinduced by v-Src, and Src kinase induction of Trop2 ICD nuclearaccumulation required cyclin D1. Cyclin D1 induced abundanceof the Trop2 proteolytic cleavage activation components (PS2,TACE) and restrained expression of the inhibitory componentof the Trop2 proteolytic complex (Numb). Patients with pros-tate cancer with increased nuclear Trop2 ICD and cyclinD1, and reduced Numb, had reduced recurrence-free survivalprobability (HR ¼ 4.35). Cyclin D1, therefore, serves as atransducer of v-Src–mediated induction of Trop2 ICD by enhanc-ing abundance of the Trop2 proteolytic activation complex.Cancer Res; 76(22); 1–12. �2016 AACR.

IntroductionThe cyclin D1 gene encodes the regulatory subunit of the

holoenzyme that phosphorylates and inactivates the retinoblas-toma (pRb) protein, promoting G1–S phase cell-cycle entry.Cyclin D1 enhances prostate cellular proliferation in vivo and

endogenous cyclin D1 maintains prostate cancer cellular prolif-eration in vitro (1). In human prostate cancer, cyclin D1 abun-dance is increased in many patients and a cyclin D1–regulatedgene expression signature predicts poor outcome (1). The abun-dance of cyclin D1 is induced by activating mutations of Src viatranscriptional mechanisms (2). The abundance of cyclin D1 israte limiting in growth of a variety of tumors in vivo, includingErbB2-induced breast cancer (3, 4) and a 50% reduction in cyclinD1 abundance in cyclin D1 heterozygote mice is sufficient toreduce the onset and progression of gastrointestinal tumorigen-esis induced by the Apc/Min mutation (5).

The Src family of kinases (SFK) includes nonreceptor tyrosinekinases with nine homologous members that encode an SH4domain governing cytoplasmicmembrane association. Src kinaseconveys a functional role in both initiation and progression ofmurine prostate cancer (6, 7). Coexpression of wild-type Src andthe androgen receptor (AR) enhances the formation of murineprostate adenocarcinoma (7). Genomic analysis of human pros-tate carcinoma demonstrated that mutations of activating tyro-sine kinases are rare. However, phosphotyrosine peptide analysiswith quantitative mass spectrometry demonstrated increased Srckinase activity inmost patients with prostate cancer, and >80% ofpatients are candidates for Src-inhibitor treatment (8). In contrast,very few patients were positive for activated states of receptors forMET, ErbB2, or EGFR, despite their detection in prostate cancercell lines (8). Given the frequency of Src kinase activation inhuman prostate cancer, an enhanced understanding of Src-medi-ated transformation of prostate epithelium is fundamental toimproving prostate cancer patient treatments.

A variety of independent analyses have provided supportingevidence for a role of stem cells in the onset and progression of

1Department of Cancer Biology, Thomas Jefferson University, Phila-delphia, Pennsylvania. 2Sidney Kimmel Cancer Center,Thomas Jeffer-son University, Philadelphia, Pennsylvania. 3Division of Biostatistics,Department of Pharmacology and Experimental Therapeutics,Thom-as Jefferson University, Philadelphia, Pennsylvania. 4Department ofMedical Oncology, Thomas Jefferson University, Philadelphia, Penn-sylvania. 5Department of Microbiology, Immunology, and MolecularGenetics University of California, Los Angeles, California. 6Faculty ofPharmacy, Nutrition, and Health Science, University of Calabria, Arca-vacata,RendeCS, Italy. 7DepartmentofPharmacologyandPhysiologyand Laboratory Medicine, Drexel University, Philadelphia, Pennsylva-nia. 8StemCell Biology and RegenerativeMedicine,Thomas JeffersonUniversity, Philadelphia, Pennsylvania. 9Department of Urology,Thomas Jefferson University, Philadelphia, Pennsylvania.

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

X. Ju and X. Jiao contributed equally to this article.

Current address for T. Stoyanova: Department ofRadiology, StanfordUniversity,Palo Alto, California; current address for M.P. Lisanti, The Breakthrough BreastCancer Research Unit, University of Manchester, Manchester, England, UnitedKingdom.

Corresponding Author: Richard G. Pestell, Thomas Jefferson University, 233South 10th Street, Suite 908, Philadelphia, PA 19107. Phone: 215-503-5649; Fax:215-503-9334; E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-15-3327

�2016 American Association for Cancer Research.

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tumorigenesis (9, 10). The tumor-associated calcium signal trans-ducer 2 (Trop2) identifies a subpopulation of prostate cells withstem cell characteristics in bothmurine and human prostate (11).Trop2 is also highly expressed in the proximal region of theprostate. Trop2 has oncogenic activity (12) demonstrated bychimeric cyclin D1–Trop2 fusions in many cancer types, andsilencing the fusion protein inhibits tumor growth (13). Therelated Trop2 familymember, EpCAM, is considered a therapeutictarget through regulation of cell–cell adhesion (14–16). Trop2associates with the a5 integrin subunit, and thereby displacesfocal adhesion kinase from focal contacts to promote an invasivephenotype. Consistent with this finding, Trop2 is upregulated inhuman prostate cancer with extracapsular extension (stages pT3/pT4) as compared with organ-confined (stage pT2) prostatecancer (16). Intramembrane proteolysis of Trop2 occurs by theTNFa converting enzyme (TACE), followed by g-secretase. Twocleavage products are generated: the extracellular (ECD) and theintracellular domains (ICD). The Trop2 ICD accumulates in thenucleus, colocalizing with b-catenin to promote prostatic intrae-pithelial neoplasia (PIN) and self-renewal (17). The molecularmechanisms governing the expression and activation of Trop2 arepoorly understood. The current studies were undertaken to inves-tigate themechanisms governing Trop2 activity in prostate cancerand given the importance of Src kinase, to determine the potentialrole for Src in Trop2 activity.

Materials and MethodsCell culture, DNA transfection, and luciferase assays

LNCaP cell line was obtained from ATCC, the v-Src-PEC andthe NeuT-PEC cell lines were previously described (18). Orig-inal cells were expanded and stored in liquid nitrogen at anearly passage. During the experiments, the morphology of allcell lines was checked routinely under phase contrast micro-scope. For the LNCaP cell line, proliferation and AR abundancein response to DHT stimulation were tested by MTT assay andWestern blot analysis. For v-Src-PEC and NeuT-PEC cell lines,the proliferation in response to Src kinase inhibitor or NeuTinhibitor was tested. v-Src or NeuT expression in these cellswas checked by Western blot analysis for verification. All of thenewly revived cells were treated with BM cyclins (Roche) andmycoplasma contamination was determined with Hoechst33258 staining under high magnification fluorescent micro-scope routinely. DNA transfection and luciferase assays wereperformed as previously described (1, 18). The CBF-Luc and�3,400 cyclin D1-Luc reporter plasmids were previouslydescribed (19, 20). The Src kinase inhibitor PP1 (4-amino-5-(4-methylphenyl)-7-(t-butel)pyrazolo-d-3-4-pyrimidine; Cal-biochem) and dasatinib (BMS-354825, Selleckchem), the CDKinhibitor abemaciclib (MedChem Express), palbociclib (Sigma-Aldrich), ribociclib (Selleckchem), and the EGFR inhibitorcanertinib (Selleckchem) were used at the indicated doses.

Mice, Western blotting, and immunohistochemical stainingExperimental procedures with mice were approved by the

ethics committee of Thomas Jefferson University. Mouse ventralprostates were fixed in 4% paraformaldehyde, and then used forsectioning and hematoxylin and eosin (H&E) staining. Antibo-dies used for Western blot analysis and immunohistochemicalstaining in this studywere as follows: anti-cyclinD1, anti-vinculin,anti-presenilin1 (PS1), anti-presenilin2 (PS2), anti-TACE (Santa

Cruz Biotechnology), and anti-Numb (Cell Signaling Technolo-gy), anti-Notch 1 (Millipore 07-1232), anti-p-Src (Upstate 07-020, Tyr 416), anti-Src (Oncogene OP07). Anti-Trop2- ICD anti-body was from Professor Owen Witte (University of California,Los Angeles, Los Angeles, CA).

FACS analysis of stem cellsFACS analysis for prostate cancer stem cells was based on prior

publications (1, 19, 21–23). Before labeling, the cells wereblocked with normal mouse IgG in 1:100 dilution for 30minutesand then incubated with fluorescein isothiocyanate (FITC) orphycoerythrin (PE)-labeled rat anti-mouse Sca-1 (clone E13-161.7; Pharmingen; 1/100–1/200), PE-labeled rat anti-mouseCD133 (1:10; clone MB9-3G8; Miltenyi Biotec), PE/Cy5-labeledrat anti-human/mouse CD44 (1:200; clone IM7; BioLegend), PE/Cy5-labeled rat anti-human/mouse CD49f (1/10), and/or FITC-labeled goat anti-mouse Trop2 (FAB1122F; R&D) for 1 hour. Allexperiments were conducted at 4�C. Cell sorting was performedon a FACSCalibur Cell Sorter (BD Biosciences). The data wereanalyzed with FlowJo software (TreeStar, Inc).

Prostatosphere formation assayv-Src-PEC cells were plated at a density of 10,000 cells/mL on

ultra-low attachment Corning cell culture plates and grown inDMEM/F12withB27, 20 ng/mLEGF, 20 ng/mLFGF, and4 mg/mLheparin. Prostatospheres were collected by gentle centrifugation(800 rpm) after 7 to 10 days and counted under the microscopeusing a 96-well plate (1, 24).

siRNA transfection and shRNA infectionThe transfection of siRNA to cyclin D1 and control siRNA into

the v-Src cell line, and the infection of cyclin D1 shRNA intoLNCaP cells, were performed as previously described (1). pTRIPZtet-inducible shRNA Vector, which use TurboRFP as shRNAexpression reporter, was from Qiagen Biotechnology.

ImmunofluorescenceCells were grown in 4-well chamber slides and were fixed

with 4% paraformaldehyde in PBS for 20 minutes at roomtemperature. The slides were rinsed with PBS and permeatedwith 0.05% NP-40 in PBS. The primary antibodies were rabbitpolyclonal anti-Trop2-ICD (1/200) and mouse anti-b-catenin(mouse, Santa Cruz Biotechnology; SC-7963, 1/100). The sec-ondary antibodies used were rhodamine red X–conjugated goatanti-rabbit IgG (Jackson ImmunoResearch Laboratories; 1/100)and Alexa Fluor 633–conjugated F (ab0) 2 fragment of goat anti-mouse immunoglobulin G (IgG; Molecular Probes; 1/250).Fluorescence imaging was acquired with a 40� objective of aZeiss LSM510/META laser confocal microscope. ImageJ wasused to quantify fluorescence intensity of the whole cell andnucleus. Thirty cells were measured for each sample and theresults were shown as relative intensity per mm2.

Microarray analysis and comparison with cancer stem celldataset

Transcript expression profiling was previously performed onthe v-Src-PEC cell lines alongwithparental prostate epithelial cells(PEC) using Affymetrix MoGene 1.0ST microarrays and themicroarray data were deposited to GEO (GSE 37428; ref. 18).Genes differentially expressed in the v-Src-PECs relative to paren-tal PECs were compared with transcript profiles of CD133þ

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prostate cancer stem cells from a previously published study (25).The analysis is described in detail in the Supplementary Methodsbased on ref. 26.

Enrichment analysis of gene ontology biological process termsThe Database for Annotation, Visualization and Integrated

Discovery (DAVID; ref. 27) functional annotation tool was usedto analyze the genes identified in common between CD133þ andv-Src-PEC differentially expressed gene lists for enriched GeneOntology biological process (GO-BP) terms (28). GO-BP termswere reported at a 10%FDR cutoff and ranked on the basis of genecount for visualization in a bar chart.

Trop2, cyclin D1, and Numb correlation with recurrence ofprostate cancer

The tissue microarray (TMA) was constructed at Thomas Jef-ferson University Hospital. All patients had undergone radicalprostatectomies. Ethical approval was obtained from the ThomasJeffersonUniversity Institutional Ethical Review Board. A detaileddescription is provided in the Supplementary Methods section.

IHC staining was conducted using the cyclin D1 antibody(Thermo Fisher Scientific, RB-010-P, 1:2000), rabbit Trop-2 ICDantibody (gift from Dr. Owen Witte, dilution 1:400), Numbantibody (Abcam, ab14140, 1:250), and presenilin 2 antibody(Santa Cruz Biotechnology, sc-1456, 1:150) by a DAKO Auto-stainer Plus equipment with an enzyme labeled biotin–strepta-vidin system. The slides were scanned on the BLISS system (BacusLaboratory) and quantified on the basis of the staining intensityand the proportion of cells stained. All comparisons of stainingintensities were made at �200 magnification.

Kaplan–Meier analysis was used to evaluate the difference inrecurrence-free survival associated with high expression versus lowexpression of Trop2 ICD, cyclin D1, and Numb proteins, respec-tively, in the 126 samples that had both a clinical record and IHC

staining. TheCox regressionfitting proportional hazardsmodels tocensored survival data was used to evaluate the association of allthreemarkers to the risk of recurrence. Stratificationwas performedrecursively. On the basis of the risk score, patients were assigned tohigh-, medium-, low-risk groups, and the difference in recurrence-free survival was evaluated among the three groups.

Statistical analysisComparisons between groups were analyzed by the two-sided t

test. A difference of P < 0.05 was considered to be statisticallysignificant. All analyses were done with SPSS 11.5 software. Dataare expressed as mean � SEM.

ResultsSrc kinasemaintains PEC growth and nuclear tumor-associatedcalcium signal transducer 2 [TACSTD2/TROP2/MISI] Trop2ICD translocation

To determine the functional significance of Src kinase activity inthe maintenance of cellular proliferation, v-Src PEC–stable pros-tate cancer cell lines were analyzed. The addition of the Srcinhibitor PP1 reduced cellular proliferation by 20% to 50% ina dose-dependent manner (Fig. 1A). The Src inhibitor dasatinibsimilarly reduced cellular proliferation by 10% (P < 0.05) to 40%(P < 0.001) in a dose-dependent manner (Fig. 1B). Phospho-Srcwas downregulated upon treatment with dasatinib and PP1(Fig. 1C). Cyclin D1 promotes DNA synthesis of the murineprostate and human prostate cancer cell lines (1). The relativeabundance of cyclin D1 was reduced 40% by 25 to 100 nmol/Ldasatinib (Fig. 1C) associated with a reduction in cellular prolif-eration. PP1 reduced cyclin D1 abundance by 50% (Fig. 1C).In recent studies, Trop2, which is known to be expressed in asubpopulationofprostate basal cellswith stemcell characteristics,was shown to correlate with poor prognosis in prostate cancer

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Src kinase activity promotes v-Src prostatecancer cell line proliferation. Cellularproliferation assays were conducted by cellcounting in the presence of either control or Srcinhibitor PP1 (A), or the Src inhibitor dasatinib(B), with doses as indicated. C, Western blotanalysis of v-Src prostate cancer cell linestreated with the Src inhibitors dasatinib or PP1.The antibodies were directed to the proteins asindicated.

Src Activates Trop2 via Cyclin D1

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(11, 16, 29). Trop2 is activated by regulated intra-membraneproteolysis (RIP), a mechanism involved in processing and acti-vation of other transmembrane proteins, including N-cadherinand E-cadherin (30). Nuclear ICD of Trop2 is found in humanprostate cancer, but not in the adjacent benign tissue (17). Todetermine whether Src kinase activity regulates the relative abun-dance of Trop2 ICD,Western blot analysis was conducted with anICD-specific antibody. The relative abundance of Trop2 ICD wasreduced 50% in the presence of the Src kinase inhibitors, dasa-tinib, and PP1 (Fig. 1C). To examine the kinetics with which Srckinase inhibition by dasatinib reduced the abundance of theTrop2 ICD, a time course analysis was conducted. Cyclin D1levels were reduced >50% by 24 hours, and >90% by 48 hours.The reduction in Trop2 ICD abundance was reduced 50% at 48hours (Supplementary Fig. S1).

v-Src induces gene expression of prostate cancer stem cellepitopes

To examine the molecular signaling pathways activated uponv-Src transformation of prostate epithelial cells, gene expressionprofiling was conducted to compare v-Src–transformed PECsand parental PECs. Experiments were conducted on threeseparate PEC-Src lines (Fig. 2A). In previous studies, CD133þ

cells were considered enriched for prostate cancer stem cells(31, 32). A correlative analysis was conducted examining thegenes that were differentially expressed between the CD133þ

versus CD133� prostate cancer cells (25). We therefore com-pared the genes enriched by v-Src in PECs with the genesexpressed in CD133þ prostate cells. The relative abundanceof genes in the CD133þ prostate cancer stem cell signature isshown in Fig. 2A (left panel). The genes differentially expressedwithin v-Src prostate cancer cell lines, relative to nontransformedparental prostate epithelial cells, are shown as the differentiallyexpressed genes (right panel; ref. 18). A substantial overlap wasseen between the genes enriched in CD133þ prostate cellswhen compared with genes regulated by v-Src. Genes inducedwithin CD133þ cells were also induced by v-Src, and genesrepressed by CD133þ were repressed by v-Src (Fig. 2A). The Pvalue for thedegreeof similarity of theCD133þ stemcell signaturewith v-Src–regulated genes was significant (P ¼ 2.148 � 10�10

; Fig. 2A).Seventy-seven genes regulated by v-Src (77/466, �17%) were

regulated by CD133þ in a common manner (P ¼ 5.52 � 10�14

; Fig. 2B). These studies suggest an enrichment of Src-regulatedgenes among the CD133 enriched genes. GO terms were used toidentify gene pathways regulated upon v-Src transformation ofPECs. Additional pathways enricheduponv-Src transformationofPECs, included "cell cycle, DNA damage repair, DNA metabolicprocess and chromosomal organization" (Fig. 2C). To determinewhether CD133þ cells were enriched for Src kinase activity, theoncogene-transformed PECs were FACS sorted for CD133 andthen characterized for Src activity using the antibody directed toactivated Src (Srcp Tyr 416; Fig. 2D). CD133þ cells were enriched3-fold for Srcp Tyr 416 compared with CD133� cells (Fig. 2E).

v-Src transformation induces epitope markers of prostatecancer stem cells

Prostatosphere formation assays, a surrogate measure of pros-tate cancer stem cells, were performed on the v-Src-PEC cell line.Approximately 16 of 1,000 cells formed prostatospheres in v-Src-PECs, whereas in parental PECs, only 1 of 1,000 formed prostato-

spheres (Fig. 3A). The v-Src-PEC line–derived prostatosphereswere consistently larger than the nontransformed, and only thev-Src-PEC lines gave rise to secondary prostatospheres (Fig. 3A).Treatment with the Src kinase inhibitors dasatinib or PP1 reducedthe number (Fig. 3B), but not the size of the prostatospheres.

In view of the finding that v-Src transformation inducedexpression of genes associated with CD133þ, a prostate stemcell marker, we examined further the association of v-Srctransformation with the expression of prostatic cancer stemcell epitopes. FACS was conducted for the relative proportion ofother epitope markers of prostate cancer stem cells. v-Srctransformation of prostate epithelium was associated with anapproximately 6-fold increase of Sca1high cells (Fig. 3C). Sim-ilarly, the proportion of CD44þ/CD133þ cells was increased 6-fold (Fig. 3D). The proportion of CD49fþ Sca1þ Trop2þ cellswas increased 2.2-fold in v-Src–transformed PECs comparedwith parental PECs (Fig. 3F).

Src kinase activity induces nuclear Trop2 ICD abundance incultured prostate cancer cells and in vivo

Although the total Trop2 ICD abundance was reduced by Srckinase inhibitor, the nuclear pool of Trop2-ICD is considered tobe the biologically active moiety, therefore we next sought todetermine whether Src kinase governs the abundance of nuclearTrop2 ICD. Comparison was made between parental and v-Src–transformed PECs by immunostaining for Trop2-ICD (red),b-catenin (green), and nucleus (DAPI, blue; Fig. 4A). The relativeabundance of nuclear Trop2 ICDwas enhanced 4-fold upon v-Srctransformation (Fig. 4B). The addition of the Src inhibitor, either100 nmol/L dasatinib or 10 mmol/L PP1, reduced nuclear Trop2ICD abundance by approximately 40% (Fig. 4C and D). Incontrast, b-catenin immunostaining was not significantly reducedby dasatinib.

The v-Src-PEC cell lines were derived from FVBmurine prostateepithelium and can therefore be reintroduced into immunocom-petent FVB mice (18). In view of the importance of the immunesystem in the onset and progression of prostate cancer, weexamined the abundance of the Trop2 ICD in v-Src tumorsimplanted into immunocompetent mice in vivo. The presence ofnuclear Trop2 ICDwas demonstrated in the v-Src prostate tumorsin vivo (Fig. 4E). The relative staining of the Trop2 ICD in the v-Srcprostate tumors was increased 2-fold compared with the non-transformed murine prostate gland (Fig. 4F). The induction ofTrop2 abundance assessed byWestern blot analysis was increasedapproximately 10-fold in a series of v-Src tumors derived afterimplantation in FVB mice (Fig. 4G). RNA extracted from v-Srctumor and normal ventral prostate tissues was assessed bymicro-array analysis. The relative mRNA abundance of Trop2 wasinduced 3.5-fold. We examined the abundance of several othergenes associated with the prostate cancer stem cell signature (33).Notch1 was induced 3.5-fold. CD44 mRNA was induced morethan 5-fold (Fig. 4H); CD44 is a cell adhesion glycoprotein thatparticipates in presentation of cytokines and associates with stemcell functions (34). The abundance of Wnt7A was induced 50%(Fig. 4H), consistent with recent studies suggesting heterotypicsignaling from the interstitium maintains prostate cancer pro-gression (35).

Cyclin D1 is required for Trop2 ICD nuclear accumulationPrevious studies in human breast cancer cells demonstrated

that cyclin D1 enhances Notch1 activity through inducing

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g-secretase activity (19). The g-secretase cleavage complex com-ponents PS1 and PS2 contribute to the g-secretase activity govern-ing Trop2 cleavage (17). We therefore examined the potentialimportance of cyclin D1 in the induction of the Trop2 ICD. Todetermine the mechanism by which Src induces cyclin D1 abun-dance, we first examined the relative abundance of cyclin D1 in v-

Src versus parental PECs. Cyclin D1mRNAwas induced 2-fold byv-Src transformation (18). The �3,400 bp cyclin D1 promoterfragment linked to a luciferase reporter gene was induced 1.7-foldby v-Src (Fig. 5A) and reduced 70% by dasatinib in v-Src PECs(Fig. 5B). Immunohistochemical staining for Trop2 ICD wasconducted in cyclin D1þ/þ and cyclin D1�/� mouse prostate. We

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Microarray analysis of v-Src PEC cellscompared with the gene expressionprofile enriched in CD133þ prostatecancer cells. A versus B, heatmapsshow the intersection of differentiallyexpressed genes from pairedCD133þ versus CD133� sample data(left; ref. 25) and genes that aredifferentially expressed in thev-Src oncogene cell line (right).A Venn diagram represents thedegree of overlap among differentiallyexpressed genes in CD133þ cells andv-Src, 767 genes specific to v-Src celllines, and 466 genes specific to theCD133þ cells, with a 77-gene overlap.C, GO biological process was analyzedfor the overlapping 77 genes; theinduced functional pathways areshown with the fold enrichmentand the number of genes for eachGO term. D, FACS analysis onNeuT-transformed PEC cells showedthat there were less CD133þ cells withlower Src activity than CD133� cells.






observed a 4-fold decrease of nuclear Trop2 ICD in cyclin D1knockout prostates when compared with normal wild-type pros-tate (Fig. 5C). To determine whether cyclin D1–mediated induc-tion of Trop2 ICD nuclear abundance is regulated in transformedprostate cancer cells, a cyclin D1 shRNA linked to a tet-inducible

red fluorescent protein (RFP) was introduced into the LNCaPprostate cancer epithelial cell line (Fig. 5D). The addition ofdoxycycline induced the expression system and thereby RFP(Fig. 5D). Western blot analysis demonstrated cyclin D1 shRNAreduced the relative abundance of cyclin D1 by >50%, associated

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v-Src transformation induces epitopemarkers of prostate cancer stemcells. A, first and second generationof prostatosphere formationassay was performed on thev-Src–transformed PEC cells. B, theeffects of Src-inhibitor dasatinib andPP1 on prostatosphere formation ofv-Src–transformed PEC cells. C–E, therelative abundance of prostate cancerstem cell markers were assessed byFACS analysis, including Sca1high cellsincreased 8-fold in v-Src-PEC cells (C),CD44þ CD133þ cells increased 6-fold(D), and CD49þ Sca1þ Trop2þ cellsincreased 2.4-fold compared withparental PEC (E).

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with a reduction in Trop2 ICD by approximately 50% (Fig. 5E).Vinculin, used as a control for normalization of protein abun-dance, was unchanged (Fig. 5E). Immunofluorescence stainingfor nuclear Trop2 in LNCaP cells treated with cyclin D1 shRNAdemonstrated the reduction innuclear Trop2 ICDupon inductionof cyclin D1 shRNA (Fig. 5F and G). CBF-1 (C-promoter bindingfactor-1), the mammalian homolog of Suppressor of Hairless(Drosophila) and CSL protein in C. elegans, also known as RBP-JK,is activated by the Trop2 ICD. As our studies showed v-Srcenhanced Trop2 cleavage, we determined whether CBF activitywas maintained by v-Src kinase activity in prostate cancer cells.CBF activity was assessed using a synthetic CBF luc reporter in v-Src-PEC cell line. The Src kinase inhibitors dasatinib and PP1

reduced CBF activity in a dose-dependent manner (Fig. 5H).Cyclin D1 siRNA reduced CBF (CBF8luc) activity approximately47% in v-Src-PEC cell line (Fig. 5I).

Cyclin D1 induces expression of the Trop2 cleavage complexIntramembrane proteolysis of Trop2 occurs via a g-secretase

cleavage complex (which includes PS1 and PS2) and TACE(ADAM17). To determine the mechanism by which cyclinD1 enhances Trop2 ICD accumulation, we considered thatcyclin D1 may increase the abundance of the Trop2 cleavagecomplex. Immunohistochemical staining of cyclin D1þ/þ andcyclin D1�/� mouse prostate glands was conducted to examinethe abundance of the components regulating Trop2 ICD

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v-Src transformation enhancesnuclear Trop2 ICD expression in vitroand in vivo. Immunofluorescencestaining for Trop2 ICD of v-Src–transformed cells versus parental PECcells (A), and the intensity of nuclearTrop2 ICDwas quantified (B). C andD,the treatment of v-Src-PEC lines withthe Src inhibitors PP1 and dasatinibshows the reduction in nuclear Trop2staining upon treatment with Srckinase inhibitor (C), the relativeintensity of Trop2 ICD (D). E, thepresence of nuclear Trop2 ICD wasshown in the normal ventral prostateand v-Src prostate tumors. F, therelative staining of the Trop2 ICD in thev-Src prostate tumors was increased2-fold comparedwith nontransformedmurine prostate gland. G, theinduction of Trop2 abundanceassessed byWestern blot analysis in aseries of v-Src-PEC–induced tumors.H, the relative mRNA abundance ofTrop2 and several other genesassociated with the cancer stem cell inv-Src tumors and normal murineprostate.






abundance. TACE and PS2 are the key inducers of Trop2cleavage (17). Therefore, we examined TACE and PS2 in cyclinD1þ/þ and cyclin D1�/� prostate (Fig. 6A). The deletion of thecyclin D1 gene reduced TACE and PS2 abundance in theprostate by 50% (Fig. 6B and C). Previous studies had dem-onstrated that cyclin D1 represses Numb to thereby induceNotch1 activity in breast cancer cells (19). As Notch can alsoactivate CBF, we considered the possibility that endogenouscyclin D1 may induce CBF through repression of Numb inprostate epithelium. Consistent with prior findings in the mam-mary gland, we found cyclin D1 gene deletion enhanced Numbabundance in the prostate in vivo (Fig. 6D). To determinewhether the mRNA levels of the Trop2 cleavage complexwere induced by cyclinD1,mRNA levels of the Trop2 proteolyticcleavage components were assessed in prostate tissues of cyclin

D1þ/þ and cyclin D1�/� mice by RT-PCR (Fig. 6E). Cyclin D1reduced Numb and increased PS2 and TACE (Fig. 6E).To determine whether cyclin D1 maintains expression ofthe Trop2 cleavage complex in transformed prostate cells, West-ern blot analysis was conducted on cyclin D1–transduced v-SrcPECs. Commensurate with the approximately 50% reductionin cyclin D1 abundance, Trop2 ICD abundance was reducedapproximately 50%, and PS2 was reduced approximately 50%(Fig. 6F).

Cyclin D1 is known to convey kinase-dependent and -inde-pendent functions. To determine whether the induction of Trop2by cyclin D1 was kinase dependent, we examined the effect ofinhibiting cyclin D1/cdk activity using the cdk inhibitors abema-ciclib, palbociclib, and ribociclib. The cdk inhibitors reducedpRbp at 24 hours, and reduced Trop2 ICD abundance at 48 and

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Cyclin D1 is required for v-Src–inducedTrop2 ICD. �3,400 bp cyclinD1-Luc activity was inducedapproximately 2-fold by v-Src in 293Tcells (A) and reduced 80% bydasatinib in v-Src-PECs (B). C, thenuclear abundance of the Trop2 ICDis reduced in the cyclin D1�/�

prostate epithelium compared withcyclin D1þ/þ. D and E, cyclin D1shRNA induced by doxycycline inLNCaP cells (D), with Western blotanalysis demonstrating thereduction in cyclin D1 abundanceassociated with a decrease in theTrop2 ICD (E). Vinculin was used as aprotein loading control. F and G, incyclin D1 shRNA-treated LNCaP cells,the Trop2 ICD was reduced, shownby immunofluorescence staining.H, the Src kinase inhibitors dasatiniband PP1 reduced CBF activityin a dose-dependent manner inv-Src-PECs. I, CBF-Luc activityactivity is shown in cyclinD1 siRNA-treated v-Src-PECs.

Ju et al.





72 hours. At 48 hours, the reduction in Trop2 ICD was 10% withabemaciclib (500 nmol/L), whereas palbociclib (>60%) andribociclib (>60%) was more substantial.

To examine further the specificity of the effect mediated byoncogenic Src, we examined the possibility that growth factorsignaling via the EGFRmay induce Trop2 ICD.We therefore tested

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Cyclin D1 induces expression of the Trop2 cleavage complex. A, IHC staining of cyclin D1þ/þ and cyclin D1�/� mouse prostate glands. B–D, the deletion of cyclin D1induced Numb abundance (B), reduced TACE (C) and PS2 (D) by approximately 50%. E, RT-PCR demonstrating cyclin D1 reduced Numb and increasedPS2 and TACE in vivo. F, Western blot analysis for v-Src-PECs transduced with cyclin D1 siRNA. The reduction in cyclin D1 abundance correlated with a reductionin Trop2 ICD. G, the effects of the CDK inhibitor abemaciclib, palbociclib, and ribociclib on Trop2 ICD cleavage. CDK inhibitors decreased Trop2 ICD abundanceafter the second day of CDK inhibitor treatment.






the EGFR antagonist canertinib. Canertinib is a 3-chloro 4-fluoro4-anilinoquinazoline compound. It is a low molecular weightirreversible pan-EGFR family TKI and has been shown to inhibitcell proliferation via restraint of ERK/MAPK in a number ofdifferent cell types. Canertinib treatment of the v-Src-PEC inhib-ited cell proliferation (Supplementary Fig. S2A). Canertinib treat-ment of the v-Src-PEC did not affect levels of Trop2 ICD, and didnot affect cyclin D1 levels. These findings suggest that dissociablepathways conduct selective signaling to Trop2-ICD abundance.Together, these studies demonstrate that v-Src induces cyclin D1,and that cyclinD1 induces expressionof components of the Trop2cleavage complex, directly through increasing PS2 and TACE, andindirectly through reducing Numb, thereby enhancing the abun-dance of the Trop2 ICD, and CBF activity in prostate cancer cells(Supplementary Fig. S3).

Cyclin D1, Trop2 ICD, and Numb predict outcome of prostatecancer patients

To determine whether the abundance of cyclin D1, Trop2 ICD,PS2, andNumb correlate with outcome in human prostate cancerpatients, an annotated TMA of patients with prostate cancer wasanalyzed. IHC staining was conducted of a 126 prostate cancerpatient TMA. The expression of cyclin D1, Trop2 ICD, PS2, andNumb was quantified (Supplementary Fig. S4). Kaplan–Meieranalysis was used to evaluate the difference in recurrence-freesurvival associated with high expression versus low expression ofcyclin D1, Trop2 ICD, PS2, and Numb. Survival probability ofcyclinD1high expression groupwas approximately 60%,whereasthe low expression group is more than 90%. The difference wassignificant (P¼ 0.049; Fig. 7A). The survival probability of Trop2ICD high expression group was approximately 25%, which was

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Recurrence-free survival analysis.Kaplan–Meier analysis was used toevaluate the difference in recurrence-freesurvival associated with high expressionversus low expression of Trop2, cyclin D1(A), Trop2 ICD (B), PS2 (C), andNumb (D).E, combined the three target genes;patients were assigned to high, medium,and low recurrence risk groups. Therecurrence-free survival curves ofdifferent groups and HR between groupsare shown.

Ju et al.





significantly increased to 73% in the low expression group (P ¼0.008; Fig. 7B). The change in survival probability between PS2groups was not significant (Fig. 7C). In the low Numb expressiongroup, the survival probability was reduced to 0% compared with72%for thehigh expressiongroup (P<0.001; Fig. 7D). These threetarget genes were combined and recurrence-free survival analysiswas conducted. Using the Cox model based on the risk scoredetermined by recursive partitioning with the predictors, cyclinD1, Numb, and Trop2, patients were assigned to high, medium,low recurrence risk groups, and the recurrence-free survival curvesof different groups and HRs between the groups were analyzed.Compared with the low-risk group, the HR for survival was 3-foldincreased in the medium-risk group and 4.3-fold in the high-riskgroup (P < 0.001; Fig. 7E).

DiscussionSrc kinase is increased in approximately 80% of human pros-

tate cancers (8). The current studies provide several lines ofevidence that v-Src transformation of PECs enriches for prostatecancer stemcells. First, v-Src transformation induced expressionofgenes identified in prostate cancer stem cells. Genes associatedwith the prostate cancer stem cell signature (33) includedNotch1,which was induced 3.5-fold. CD44 mRNA, which encodes a celladhesion glycoprotein that participates in presentation of cyto-kines and associates with stem cell functions (34), was inducedmore than5-fold (Fig. 4H). The abundance ofWnt7Awas induced50%, consistent with recent studies suggesting heterotypic sig-naling from the interstitium maintains prostate cancer progres-sion (35). CD133þ cells are enriched for cancer stem cells fromprostate cancer (36). Together with a2b1 integrin, CD133 is usedto enrich for stem cells that have increased proliferation potentialand undergo full prostatic differentiation in vivo (37–39). Approx-imately 17% of genes enriched in the CD133 population werealso induced by oncogenic Src transformation. Second, v-Srcinduced the abundance of the epitopes characteristic of prostatecancer stem cells. Third, v-Src induced prostatosphere formationand Src kinase inhibitors reduced the number of prostatospheres.Fourth, the abundance of the Trop2 ICD, which is known topromote stem cell expansion (17, 19), was enhanced by Src kinaseactivity.

In the current studies, cyclinD1was required for v-Src–inducedTrop2 ICD accumulation. The TNFa-converting enzyme (TACE),and g-secretase, both participate in Trop2 ICD accumulation.TACE mediates the initial proteolysis and ectodomain shedding,followed by intramembrane proteolysis carried out by the g-secre-tase complex. Herein, endogenous cyclin D1 was shown toenhance expression of the Trop2 cleavage complex by increasingthe abundance of TACE (ADAM17) andPS2. TACE is amember ofthe ADAM family of proteases. Our prior studies demonstratedthat endogenous cyclin D1maintains ADAM protease expressionin the mammary gland (40). In the current studies, cyclin D1induced the abundance of TACE in the normal prostate gland. Inprostate epithelial cells in culture and in transformed prostatecancer cells, cyclinD1 also induced expression of PS2, the catalyticsubunit of the g-secretase complex that conducts the sequentialintramembrane proteolysis of Trop2 (40). Thus, consistent withthe finding in the mammary gland in vivo, in which cyclin D1induced the ADAM proteases, the current studies demonstratecyclin D1 induces expression of the ADAM17 protease (TACE) inthe prostate in vivo.

In the current studies endogenous cyclin D1 augmented CBFactivity in a Src kinase–dependent manner. Trop2 and Notchaugment activity of the transcription factor CBF-1 (suppressorof hairless, LAG1, also known as RBP-RJ; ref. 41) and LEF-1.Notch activity is restrained by endogenous Numb. In thecurrent studies, endogenous cyclin D1 repressed Numb inprostate cancer cells. These findings are consistent with priorstudies in breast cancer epithelial cells in which cyclin D1augmented ErbB2-induced Notch1 activity, through repressionof Numb (19). These prior studies demonstrated the inductionof Notch signaling by cyclin D1 (19). Using cyclin D1 knockoutmice, it was demonstrated that g-secretase cleavage of Notch1was enhanced by endogenous cyclin D1 (19). In a reciprocalfeedback, Notch is known to induce cyclin D1 expression andcyclin D1 is required for Notch-induced transformation (42),consistent with findings that Notch1 and cyclin D1 expressioncorrelate during embryogenesis (42).

The current studies demonstrated that a reduction in Numb, anincrease in Trop2 ICD and an increase in cyclin D1 abundance,each predict poor outcome in patients with prostate cancer.Whencombined, these three genes assigned to high, medium, or lowrecurrence risk groups, gave ahigh-risk groupwith aHR4.35 timesgreater than the low-risk group. Although cyclin D1 protein isoverexpressed in a subset of prostate cancers, correlating withpoor outcome, it is the cyclin D1-mediated gene expressionsignature that provides additional prognostic value to theGleasonscore (1, 43). The current studies are consistent with a model inwhich the molecular targets of cyclin D1–mediated signaling inthe prostate are important predictors of poor outcome in prostatecancer.

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

Authors' ContributionsConception and design: X. Ju, X. Jiao, G. Di Sante, R.G. PestellDevelopment of methodology: X. Ju, X. Jiao, G. Di Sante, Z. Li, A. HawkinsAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): X. Ju, X. Jiao, G. Di Sante, S. Deng, A. HawkinsAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): X. Ju, X. Jiao, A. Ertel, G. Di Sante, T. Zhan,L.G. Gomella, L.R. Languino, R.G. PestellWriting, review, and/or revision of themanuscript: X. Ju, X. Jiao, T. Stoyanova,S. And�o, A. Fatatis, M.P. Lisanti, L.G. Gomella, R.G. PestellAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): X. Ju, X. Jiao, M.C. Casimiro, A. Di Rocco,A. Hawkins, T. StoyanovaStudy supervision: X. Ju, X. Jiao, M.C. Casimiro

Grant SupportThis work was supported in part by the NIH grants R01CA070896,

R01CA075503, R01CA132115, R01CA107382, R01CA086072 (R.G. Pes-tell), and R01CA109874 (L.R. Languino), the Sidney Kimmel Cancer CenterNIH Cancer Center Core grant P30CA056036 (R.G. Pestell), generous grantsfrom the Dr. Ralph and Marian C. Falk Medical Research Trust (R.G. Pestell),and a grant from Pennsylvania Department of Health (R.G. Pestell). M.P.Lisanti and his laboratory were supported via the resources of ThomasJefferson University.

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 December 15, 2015; revised July 18, 2016; accepted August 18,2016; published OnlineFirst September 15, 2016.






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Ju et al.




Published OnlineFirst September 15, 2016.Cancer Res Xiaoming Ju, Xuanmao Jiao, Adam Ertel, et al. Cyclin D1v-Src Oncogene Induces Trop2 Proteolytic Activation via

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v-Src Oncogene Induces Trop2 Proteolytic Activation via ... · signal transducer 2 (TACSTD2/Trop2/M1S1) is enriched in the prostate, promoting prostate stem cell self-renewal upon