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Tumor and Stem Cell Biology

Activationof the Lin28/let-7Axis byLossof ESE3/EHF Promotes a Tumorigenic and Stem-likePhenotype in Prostate CancerDomenico Albino1, Gianluca Civenni1, Cecilia Dallavalle1, Martina Roos2, Hartmut Jahns2,Laura Curti1, Simona Rossi1, Sandra Pinton1, Gioacchino D'Ambrosio3, Fausto Sessa4,Jonathan Hall2, Carlo V. Catapano1,5,6, and Giuseppina M. Carbone1,5

Abstract

Although cancer stem-like cells (CSC) are thought to be themost tumorigenic, metastatic, and therapy-resistant cell subpop-ulation within human tumors, current therapies target bulktumor cells while tending to spare CSC. In seeking to under-stand mechanisms needed to acquire and maintain a CSCphenotype in prostate cancer, we investigated connectionsbetween the ETS transcription factor ESE3/EHF, the Lin28/let-7 microRNA axis, and the CSC subpopulation in this malig-nancy. In normal cells, we found that ESE3/EHF bound andrepressed promoters for the Lin28A and Lin28B genes whileactivating transcription and maturation of the let-7 microRNAs.In cancer cells, reduced expression of ESE3/EHF upregulated

Lin28A and Lin28B and downregulated the let-7 microRNAs.Notably, we found that deregulation of the Lin28/let-7 axiswith reduced production of let-7 microRNAs was critical for celltransformation and expansion of prostate CSC. Moreover,targeting Lin28A/Lin28B in cell lines and tumor xenograftsmimicked the effects of ESE3/EHF and restrained tumor-initiatingand self-renewal properties of prostate CSC both in vitro andin vivo. These results establish that tight control by ESE3/EHFover the Lin28/let-7 axis is a critical barrier to malignant trans-formation, and they also suggest new strategies to antagonizeCSC in human prostate cancer for therapeutic purposes. Cancer Res;76(12); 3629–43. �2016 AACR.

IntroductionProstate cancer is the most common malignancy and the

second most frequent cause of cancer-related mortality in menin developed countries (1, 2). Several studies have providedevidence of the presence of self-renewing tumor-initiatingstem-like cancer cells in human cancers, including prostate cancer(3–5). Cancer stem-like cells (CSC) can derive from transforma-tion of tissue/adult stem cells or from more differentiated pro-genitor cells that acquire stem-like properties (6, 7). CSCs withinthe primary tumors are likely a major source of tumor heteroge-neity, disease progression, and treatment failure. Our knowledge

of the factors governing the behavior of CSCs in prostate cancer islimited (5, 8). Understanding the pathways controlling the expan-sion and maintenance of prostate CSCs could be an importantstep toward development of more effective CSC-directed strate-gies for treatment of prostate cancer.

ETS transcription factors are important elements in differenti-ation and developmental programs inmany tissues. Expression ofETS factors is tightly regulated according to tissue-specific andtime-dependent programs (9, 10). Deregulated expression of ETSfactors has oncogenic consequences altering tissue developmentalprograms and is one of the most frequent findings in humantumors. About 50% of prostate cancers exhibit gene rearrange-ments and ectopic expression of ETS genes, like ERG and ETV1(11–14). ESE3/EHF is an ETS factor normally expressed in epi-thelial cells, including prostate epithelial cells (10). We reportedpreviously that ESE3/EHF is downregulated frequently in prostatetumors (15, 16). More recently, we showed that ESE3/EHF has akey role in controlling the differentiation program of prostateepithelial cells (17). Loss of ESE3/EHF altered cell differentiationand conferred to prostate epithelial cells a CSC-like phenotypealong with tumor-initiating and metastatic capability. In clinicalsamples loss of ESE3/EHF expression marked a subset of prostatetumors with enrichment of CSC transcriptional features andcharacteristics of clinically aggressive prostate tumors (17). Mech-anistically, ESE3/EHF acts as transcriptional activator and repres-sor of a large network of target genes (17). We showed thatin normal prostate epithelial cells ESE3/EHF induces genes ofthe epithelial cell differentiation and represses genes connectedwith self-renewal and the CSC phenotype, like Nanog, POU5F1(Oct4), BMI-1, EZH2 (17). Expression of these genes was

1Tumor Biology and Experimental Therapeutics Program, Institute ofOncology Research (IOR), Bellinzona, Switzerland. 2Institute of Phar-maceutical Sciences, Department of Chemistry and Applied Bios-ciences, ETH Zurich, Zurich, Switzerland. 3IRCCS Multimedica, Milan,Italy. 4Department of Pathology, University of Insubria, Varese, Italy.5Oncology Institute of Southern Switzerland (IOSI), Bellinzona, Swit-zerland. 6Department of Oncology, Faculty of Biology and Medicine,University of Lausanne, Lausanne, Switzerland.

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

C.V. Catapano and G.M. Carbone share co-senior authorship for this article.

Corresponding Authors: Giuseppina M. Carbone, Institute of OncologyResearch, Via Vela, 6, Bellinzona CH-6500, Switzerland. Phone: 41-091-820-0366; Fax: 41-091-820-0367; E-mail: [email protected]; and Carlo V.Catapano, Institute of Oncology Research, [email protected]

doi: 10.1158/0008-5472.CAN-15-2665

�2016 American Association for Cancer Research.

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conversely upregulated in transformed prostate epithelial cells,prostate cancer cell lines and human tumors with loss of ESE3/EHF expression, indicating their contribution to the acquisition ofCSC properties in prostate cancer.

Lin28 is ahighly conservedRNA-bindingprotein andoneof thekey embryonic stem cell factors (18, 19). Lin28A (LIN28) and itsparalog Lin28B (LIN28B) repress the processing of pri- and pre-miRNAs of the let-7 family into mature microRNAs (miRNAs),thus preventing differentiation of embryo stem cells and main-taining self-renewal and pluripotency (20–25). Lin28A andLin28B are frequently overexpressed in human cancers (26–28).Lin28 promotes neoplastic transformation by repressing let-7miRNAs, which act as tumor suppressors inhibiting expression ofkey oncogenes, like RAS, MYC, and HMGA2 (20, 21, 29). Inaddition, the Lin28/let-7 axis has a critical role by regulatingtumor-initiating and self-renewal properties of CSCs in humancancers (30–33).

In the attempt to identify relevant mediators of the prostateCSC phenotype and actionable targets for drug discovery andtherapeutic intervention, we examined the relationship betweenESE3/EHF and the Lin28/let-7 axis in prostate cancer. We showhere that ESE3/EHF represses transcription of Lin28A and Lin28Band concomitantly sustains transcription and processing of let-7pri-miRNAs to mature let-7 miRNAs. The dual transcriptionaland posttranscriptional control exerted by ESE3/EHF on theLin28/let-7 axis ensures tight control of this key developmentalprogram and proper balance between cell differentiation and self-renewal in the normal prostate epithelium. Targeting the Lin28/let-7 axis in prostate cancer cell lines and tumor xenografts withESE3/EHF deregulation antagonizes tumor-initiating and self-renewal properties of prostate CSCs and leads to reduced tumorgrowth, suggesting that it could be a valid strategy for treatment ofclinically aggressive prostate cancers.

Materials and MethodsCell lines, transfection, and selection of cell clones

Immortalized human prostate epithelial cells (PrECs; ref. 15)and RPWE-1 with stable knockdown of ESE3/EHF by shRNAswere established as previously described (16). LNCaP, DU145,and PC3 were obtained from the ATCC, which performs cell linecharacterization based on DNA profiling (short tandem repeatanalysis), and maintained in RPMI-1640 (Gibco) supplementedwith 10% FBS. DU145 cells expressing ESE3/EHF were generatedafter transfection with ESE3/EHF expression vector and selectionwithG418 (15). Cells were usedwithin 6months of culturing andregularly checked for Mycoplasma contamination using theMycoAlert Mycoplasma Detection Kit (Lonza).

RNA extraction, quantitative real-time RT-PCR, miRNAprecursor, mature miRNA, antagomiR, and siRNAs

RNA was extracted by the Direct-zol RNA MiniPrep Kit (ZymoResearch). Quantitative real-time RT-PCR (qRT-PCR) was carriedout using 20 ng of RNA as template for the SYBR Green Fast OneStep Kit (Qiagen). qRT-PCR primers are reported in Supplemen-tary Table S1. For let-7b miRNA overexpression, cells were tran-siently transfected with 50 nmol/L of the specific miRNA precur-sor (pre–miR let-7b, Ambion) or negative control (Control #1,Ambion). For let-7b inhibition cells were transiently transfectedwith 40 nmol/L of the specific LNA antagomiR (Mercury LNAPower Inhibitor; Exiqon) or a scrambled control (Negative Con-

trol A; Exiqon). FormiRNA expression analysis, 400 ng of purifiedRNA was retrotranscribed using the TaqMan MicroRNA ReverseTranscription Kit (Applied Biosystem) with specific primers andthe cDNA was subjected to TaqMan Probe-based Real Time PCRusing TaqMan MicroRNA Assays and TaqMan Universal PCRMaster Mix (Applied Biosystem). The expression was normalizedto RNU6 (Control miRNA assay; Applied Biosystem) and 18SEukaryotic (18S rRNA Endogenous Control; Applied Biosystem).For transient gene knockdown cells were transfected with siRNAsdirected to Lin28A and Lin28B (siRNA Silencer select; Ambion) orcontrol (siGL3) siRNA (17) using jetPRIME (Polyplus).

ImmunoblottingCell lysates were prepared using RIPA buffer with protease

inhibitor cocktail (Roche) and phosphatase inhibitor cocktail(PhosStop; Roche). Total cell extracts were separated by SDS-PAGE and transferred to nitrocellulose membranes (PROTRAN).Antibodies directed to the following proteins were used forimmunoblot analysis: total Lin28 (11724-1-AP, Proteintech),Lin28A (16177-1-AP Proteintech), Lin28B (16178-1-AP Protein-tech), ESE3/EHF (sc-367574, Santa Cruz Biotechnology), glycer-aldehyde-3-phosphatedehydrogenase (GAPDH; Millipore) andtubulin (CP06; Calbiochem).

Soft agar and in vitro prostatosphere forming andself-renewal assay

Soft-agar assays were performed as previously described (34).The prostatosphere assay was previously described (17). Thesphere-forming efficiency (SFE) was determined as the percentageof prostatosphere relative to the number of cells plated at the startof the experiment. Each experiment was carried out in triplicateand repeated at least three times.

Expression vectors, reporter constructs, and luciferase assaysThe pEF5/FRT/Lin28A-V5 and pCMV6-XL4 LIN28B plasmid

were kindly provided by Wilbert and colleagues (35) and byLoughlin and colleagues (36), respectively, and were transfectedin cells using jetPRIME (Polyplus). To analyze Lin28 and let-7bpromoter activity, we used the following luciferase reporters:pGL3-basic hLin28A-NRSE, provided byGunsalus and colleagues(37), pGL3-basic-Lin28b P1-IRES, provided by Chang and col-leagues (38), and pGL3-let7b-1.5K, provided by D. Wang andcolleagues (39). Luciferase reporter assays were performed aspreviously described (17). Results were normalized to Renillaluciferase and expressed as Relative Luciferase Activity (RLA). Eachexperiment was performed in triplicate and repeated at least threetimes.

Synthesis of siRNA for in vivo studiesGuide and passenger sequences of phosphodiester oligoribo-

nucleotides (ORN) for siLin28B-2 were AAAUCCUUCCAU-GAAUAGUTT (mass calc.: 6599.1; mass obs.: 6598.2) andACUAUUCAUGGAAGGAUUUTT (mass calc.: 6656.1; mass obs.:6655.3; ref. 40). Chemicals for ORN synthesis were from Aldrichand TCI (Sigma-Aldrich Chemie GmbH, D-89555 Steinheim).Phosphoramidites were from Thermo Fisher Scientific. The acti-vator 5-benzylthiotetrazole (BTT) was from Biosolve. ORNs weresynthesized on an MM12 synthesizer from Bio Automation Inc.on 500 Å UnyLinker CPG from ChemGenes. Coupling time forphosphoramidites was 2 � 90 seconds. The ORNs were purified

Albino et al.

Cancer Res; 76(12) June 15, 2016 Cancer Research3630




on an Agilent 1200 series preparative HPLC fitted with a Water-sXBridge OST C-18 column, 10 � 50 mm, 2.5 mm at 60�C. TheRNA phosphoramidites were prepared as a 0.08 mol/L solutionsin dry acetonitrile (ACN); BTT was prepared as a 0.24 mol/Lsolution in dry ACN. Oxidizer was prepared as a 0.02 mol/L I2solution in THF/Pyridine/H2O (70:20:10, w/v/v/v). Cappingreagent Awas: THF/lutidine/acetic anhydride (8:1:1) and cappingreagent B was: 16% N-methylimidazole/THF. Deblock solutionwas a 3% dichloroacetic acid in dichloroethane. The cleavagefrom the solid support and the deprotection of the nucleotideswas affected by incubation of the CPG-support for 2 hours, at65�C in gaseous methylamine at 1.8 bar. Deprotection of20-TBDMS (tert-butyldimethylsilyl-) was carried out at 1.5 hours,at 70�C in a mixture of dry 1-N-methyl-2-pyrrolidone/triethyla-mine/trimethylamine.3HF. Running buffer for HPLC purifica-tion of ORNs: buffer A (0.1 mol/L triethylammonium acetate),buffer B (methanol): gradient for the DMT-on purification:20% to 60% buffer B over 5 minutes; gradient for the DMT-off purification: 5% to 35% buffer B over 5 minutes. Fractionscontaining the product were collected and dried in a miVacduo SpeedVac from Genevac. ORNs were analyzed by LC-MS(Agilent 1200/6130 system) on a Waters Acquity OST C-18column, 2.1 � 50 mm, 1.7 mmol/L, 65�C. Buffer A: 0.4 mol/LHFIP, 15 mmol/L triethylamine; buffer B: MeOH. Gradient: 7%to 35% B in 14 minutes; flow-rate: 0.3 mL/min.

Animals and tumor xenograftsMice were purchased from the Harlan Laboratories. Mice were

maintained under pathogen-free conditions with food and waterprovided ad libitum and their general health status was monitoreddaily. All protocols involving animals were conducted in confor-mity with the institutional guidelines for animal experimentationand in compliancewith national and international policies. Studyprotocol was approved by the Swiss Veterinary Authority. Forsubcutaneous tumor xenografts and in vivo self-renewal experi-ments prostatosphere-derived and bulk adherent growingESE3KD-PrECs cells were inoculated (2 � 105 cells/site) withMatrigel in the flank of NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG)mice (n ¼ 4/group). For in vivo self-renewal experiments primarytumor xenografts derived from ESE3KD-PrECs prostatosphereswere dissociated into single cell suspensions and dissociatedtumor cells were implanted (2 � 105 cells/site) subcutaneouslyas above for two generations (n¼ 4/group). In vivo serial dilutionsexperiments were performed with adherent and prostatosphere-derived ESE3KD-PrECs cells by inoculating 2 � 105, 1 � 105 and0.5� 105 cells/site subcutaneously in NSGmice (n¼ 4/group) asabove. To assay in vivo tumorigenic capacity following in vitroknockdown, ESE3KD-PrECs, DU145 or PC3 cells (2 � 106 cells/site) transfected with control or Lin28-targeting siRNAs wereinjected in the flank of NSG mice (n ¼ 4/group). Tumor engraft-ment and growth were monitored twice a week. For systemictreatment with siRNA, DU145 cells with stable expression ofluciferase reporter gene were injected (5 � 106 cells/site) withMatrigel in the flank of athymic nude mice (Balb/c nu/nu, 4–6-weeks-old; n ¼ 4/group). Mice with subcutaneous tumor xeno-grafts were then injected intraperitoneally with siLin28B-2 orcontrol siRNA formulated with in vivo jetPEI (Polyplus Transfec-tion) at the dose of 2 mg/kg/body weight/d three times a week.Tumor growth was monitored as above. To assess in vivo self-renewal capability of tumor cells from DU145 xenografts, cellsdissociated from control and siLin28B-2–treated tumors were

re-injected (5 � 105 cells/site) with Matrigel in the flank ofathymic nude mice (n ¼ 2/group).

Immunohistochemistry and immunocytochemistryIHC on histologic tissues samples was performed using an

antibody detecting total Lin28 protein (Proteintech, 1:25 dilu-tion, code n. 11724-1-AP Rb Poly), and an anti-Ki67 antibody(Lab Vision Corporation, Clone SP6, cat. RT-9106-R7). The spec-ificity of the antibodies was previously confirmed byWestern blotanalysis. Cell nuclei were counterstained with hematoxylin solu-tion. Positive samples for each antibody and negative samples, inwhich the primary antibody was omitted, were used as controls.Slides were evaluated by three investigators. Average of valuesscored by the three investigators was calculated. Following ameeting, each sample was defined positive and negative and/orcytoplasmic and nuclear in full concordance between the threereaders. For immunocytochemistry (ICC), harvested cells werewashed in PBS by centrifugation and then the concentrationadjusted to 5 � 106 cells/mL in PBS. Cells were attached to slidesusing Cytospin Cytocentrifuge (Thermo Scientific) at 800 rpm for4 minutes. Cells were fixed and permeabilized with Acetone:Methanol, 1:1. After blocking with 5% BSA, cells were incubatedwith anti-Lin28 antibody (Proteintech, 1:75, code n. 11724-1-APRb Poly). Cell nuclei were counterstained with hematoxylinsolution and finally, the sections were dehydrated and mountedin a suitable organic mounting medium.

Flow cytometryAll steps for flow cytometry were performed in PBS supple-

mentedwith 0.5%BSA, and 2mmol/L EDTA. Fluorescein di-beta-D-galactopyranoside (FDG) was purchased from Invitrogen andused for analysis in accordance with the manufacturer's instruc-tions. Cell sorting was performed with a FACSAria III sorter (BDBiosciences).

Chromatin immunoprecipitationComputational search for ETS binding sites on selected gene

promoters was performed usingMotifviz (biowulf.bu.edu/Motif-Viz). Chromatin immunoprecipitation (ChIP) was performedwith anti-ESE3 (Clone 5A5.5; Lab Vision); anti-acetylated H3(Upstate, Millipore); anti-H3K9 2met (Upstate, Millipore);anti-H3K27 3met (Upstate, Millipore), and IgG control antibody.Samples were analyzed as previously described (16) by qRT-PCR.Primer sets are reported in Supplementary Table S1.

Patient samplesTissue samples were collected at Department of Pathology

(IRCCS Multimedica, Italy) with the approval of the EthicsCommittee of Regione Lombardia, Italy, and patient writteninformed consent. Primary and metastatic tumor samples wereobtained from patients with organ-confined disease treated withradical prostatectomy and frompatientswith advancedmetastaticdisease undergoing transurethral resection, respectively. Tissuemicroarrays containing samples from normal and adenocarcino-ma were prepared from paraffin-embedded tissues.

ESE3/EHF, Lin28A, and Lin28B expression correlation analysisA human prostate cancer dataset (41) was retrieved from GEO

(GSE21034). The dataset was based on the microarray platform"Affymetrix Human Exon 1.0 ST Array." Only raw intensity data

ESE3/EHF and Lin28/let7 Control Prostate CSC

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for prostate cancer samples were considered. Data were processedin R using the Bioconductor package "oligo" for Affymetrix arrays:sets were separately RMA normalized (with background correc-tion) and quantile normalized at the probe level. Log2, normal-ized expression values for ESE3/EHF and Lin28A/B genes wereextracted and their correlation was tested with the test for asso-ciation between paired samples with Pearson's product momentcorrelation coefficient.

ResultsESE3/EHF loss is linked to upregulation of Lin28A andLin28B in prostate tumors

We previously showed that stable knockdown of ESE3/EHF inimmortalized normal prostate epithelial PrECs and RWPE-1cells (Supplementary Fig. S1A) leads to the acquisition of CSCtraits and the upregulation of CSC-related genes (17). Parentalprostate epithelial cells and their ESE3KD counterpart representgood models to investigate the pathways activated in prostateCSCs and driving CSC-enriched aggressive prostate tumors.In this context, we examined whether, similar to other CSCgenes, expression of Lin28A and Lin28B was upregulated inESE3KD cells. We observed a significant increase of Lin28A andLin28B mRNAs in both ESE3KD-PrECs and ESE3KD-RWPE-1cells (Fig. 1A). The total level of Lin28 protein, measured withan antibody that recognized both Lin28A and Lin28B, was alsoincreased in ESE3KD-PrECs and ESE3KD-RWPE-1 cells (Fig. 1B).Consistently, ESE3KD-PrECs and ESE3KD-RWPE-1 cells exhib-ited lower levels of let-7 family miRNAs compared with theparental cells (Supplementary Fig. S1B). We reported previouslythat ESE3KD cells acquire CSC properties and form a largernumber of prostatospheres (PS) than parental PrECs andRWPE-1 cells (17). Interestingly, we found that CSC-enriched PSderived from ESE3KD cells had substantially higher levels (3–6-fold) of Lin28A and Lin28B mRNAs compared with bulkESE3KD-PrECs and ESE3KD-RWPE-1 cells growing as adherentmonolayers (Fig. 1C). Consistently, total Lin28 protein washigher in PS of ESE3KD-PrECs compared with adherent growingESE3KD-PrECs (Fig. 1D). This was confirmed by ICC that showedvery intense Lin28 staining in PS derived from ESE3KD-PrECs(Fig. 1D).

Consistent with previous results (17), CSC-enriched PSderived from ESE3KD-PrECs (2� 105 cells/site) formed tumors(G1 xeno) when implanted subcutaneously in NSG micewhereas adherent growing ESE3KD-PrECs in the same condi-tions did not (Fig. 1E). Serial dilution experiments confirmedthe higher efficiency of tumor initiation by CSC-enriched PScells compared with adherent growing ESE3KD-PrECs (Fig. 1F).Tumors were detected in 75% (3/4) and 25% (1/4) of miceinjected with 2 � 105 and 1 � 105 PS-derived cells. In contrast,no tumors developed in mice injected with the same numbersof adherent growing cells. The minimal number of bulkESE3KD-PrECs capable of forming tumors in 100% of micewas 2 � 106 cells as determined in additional experiments(see Fig. 3G). Thus, PS-derived ESE3KD-PrECs retained in vivohigh tumor-initiating and stem-like properties. Furthermore,cells isolated from the PS-derived xenografts (G2 and G3 xeno)were highly tumorigenic when re-engrafted in NSG mice forconsecutive generations, an indication that they retainedtumor-initiating and self-renewal capability as expected forstem-like cancer cells (Fig. 1E).

Interestingly, we observed high expression of Lin28A andLin28B mRNAs in the PS-derived xenografts at the first andconsecutive passages in vivo in line with the in vitro data with thePS-enriched cell cultures (Fig. 1G). These findings were intriguingand suggested a link between loss of ESE3/EHF, upregulation ofLin28A/B, and expansion and maintenance of the CSC compart-ment in prostate cancer cell lines both in vitro and in vivo.Furthermore, these data pointed to Lin28A andLin28Baspossiblecritical factors driving the CSC phenotype and self-renewingproperties in aggressive prostate cancers.

To determine whether the link between ESE3/EHF and Lin28observed in our cell line models was seen in clinical samples, weanalyzed gene-expression data from a large (n ¼ 131) humanprostate cancer dataset (Fig. 2A; ref. 41). We found that Lin28Aand Lin28B were positively correlated with each other in primaryprostate tumors (Pearson correlation ¼ 0.33; P ¼ 9.76E�05).Furthermore, there was a significant inverse correlation of ESE3/EHF with Lin28B (Pearson correlation¼�0.516; P¼ 2.89E�10)and Lin28A (Pearson correlation ¼ �0.216; P ¼ 0.013). Weevaluated Lin28 protein expression also by IHCusing an antibodythat recognized both Lin28A and Lin28B in primary prostatetumors (n ¼ 28) and matched normal tissues (n ¼ 17) and inan independent cohort of metastatic (n ¼ 24) tumor samples.Lin28 staining was low or absent in normal prostate. Primarytumors were positive for Lin28 with weak or moderate Lin28staining and a slight prevalence of cytoplasmic (35%) overnuclear (27%) staining (Fig. 2B and C). We did not find anycorrelation between Lin28 expression and Gleason score in pri-mary tumors. However, metastatic tumors exhibited a higherpercentage of tumors with positivity for Lin28 and a slightprevalence of cases with nuclear (61%) over cytoplasmic(54%) staining (Fig. 2D and E). Although the in vitro data showhigher Lin28 expression in CSC-enriched prostatospheres, it ishighly likely that CSCs represent only a small fraction of theLin28-positive cancer cells detected by IHC both in primary andmetastatic tumors. We evaluated Lin28 protein expression also intumor xenografts derived from human prostate cancer cell lineswith different levels of ESE3/EHF expression. We observed highernumbers of positive cells and prevalence of nuclear staining inxenografts of DU145 and PC3 cells, whereas low frequency andprevalently cytoplasmic staining was seen in xenografts of LNCaPcells (Supplementary Fig. S2). Therefore, the inverse correlationwith ESE3/EHF and different expression patterns of Lin28 weremaintained also in the tumor xenografts.

Lin28A and Lin28B promote malignant transformation andCSC-like phenotype in prostate epithelial cells

To determine the role played by Lin28 in prostate CSC, Lin28Aand Lin28B were downregulated in ESE3KD-PrECs and ESE3KD-RWPE-1 cells using specific siRNAs. The extent of the knockdownwas assessed by qRT-PCR and Western blot and the impact onLin28 function demonstrated by the increased level of maturelet-7b miRNA (Supplementary Fig. S3A–S3C). Knockdown ofLin28A and Lin28B reduced the ability of the cells to formcolonies in soft-agar (Fig. 3A). Moreover, the ability to form PSin non-adherent growth conditions was significantly impaired fortwo consecutive generations, indicating a prolonged effect on theCSC-like PS forming cells upon a single siRNA transfection (Fig.3B). We showed previously that induction of cell senescence, aform of growth arrest that antagonizes CSC expansion, coincidedwith loss of self-renewal capability in prostate CSCs upon c-Myc

Albino et al.





knockdown (42). We found that knockdown of Lin28A andLin28B increased the percentage of senescent cells detected byFDG staining in ESE3KD-PrECs and ESE3KD-RWPE-1 cells (Fig.3C). Thus, induction of cell senescence could contribute to theloss of clonogenic and PS forming potential of transformedESE3KD cells. Importantly, knockdown of either Lin28A orLin28B produced similar effects, indicating that both had relevant

and independent roles in the induction of these cancer cellproperties.

To further support the hypothesis that Lin28A and Lin28B arekey factors in the induction of the prostate CSC phenotype, weassessed the effects of their overexpression in immortalized nor-mal prostate epithelial cells. Forced overexpression in RWPE-1cells, as shown by qRT-PCR andWestern blotting (Fig. 3D), led to

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Figure 1.ESE3/EHF loss leads to upregulation of Lin28A and Lin28B in cancer stem cells in vitro and in vivo. A and B, Lin28A and Lin28B mRNA and protein levelevaluated by qRT-PCR (A) and Western blot (B) in control and ESE3KD-PrECs and RWPE-1 cells. b-Actin and GAPDH were used as reference for loadingcontrol. Data are presented as fold change in mRNA relative to control PrECs and RWPE-1 cells. Lin28/GAPDH ratio determined by band intensity is reported forWestern blot. C, Lin28A and Lin28B mRNA evaluated by qRT-PCR in adherent and prostatosphere cells derived from ESE3KD-PrECs (left) and ESE3KD-RWPE-1(right) cells. Data are presented as fold change relative to the corresponding adherent cells. D, Lin28 protein level in adherent and prostatosphere cellsderived from ESE3KD-PrECs assessed by Western blot and ICC on intact prostatospheres. E, in vivo growth of adherent cells and prostatosphere derivedfrom ESE3KD-PrECs. Cells (2 � 105 cells/site) were injected subcutaneously in NSG mice (n ¼ 4/group). Tumors formed by ESE3KD-PrECs prostatospherecells (G1 xeno) were dissociated and re-implanted (2 � 105 cells/site) in NSG mice (n ¼ 4/group) for two consecutive generations (G2 and G3 xeno).Experimental plan (top), tumor volume determined by caliper (bottom). F, tumor initiation by prostatospheres and adherent ESE3KD-PrECs injected in NSG miceat decreasing cell numbers. The number of palpable tumors and average time to tumor formation are shown. G, Lin28A and Lin28B mRNA evaluated byqRT-PCR in xenografts of adherent ESE3KD-PrECs and ESE3KD-PrECs–derived prostatospheres at first and consecutive passages in vivo. P values weredetermined using the t test; � , P < 0.05; �� , P < 0.01. Data are representative of three independent experiments, with at least three replicates per experiment.






a substantial increase in colony formation in soft agar (Fig. 3E)and PS (Fig. 3F), indicating that Lin28A and Lin28B promoted theacquisition of tumorigenic and CSC-like properties. Similareffects were observed in PrECs (Supplementary Fig. S3D–S3F)and LNCaP cells (Supplementary Fig. S3G–S3I), in which over-expression of Lin28 increased substantially the formation ofcolonies in soft-agar and PS.

Impairment of CSC functions could result in persistent reduc-tion of in vivo tumor-initiating capability of cancer cells. Weevaluated the effects of Lin28 knockdown on the ability of bulkESE3KD-PrECs to form tumors when implanted subcutaneouslyin NSG mice. ESE3KD-PrECs cells (2 � 106 cells/site) transfectedwith control (siGL3) siRNA formed tumors that grew exponen-tially after the initial lag phase (Fig. 3G). In contrast, cells

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Figure 2.Lin28 expression is inversely correlatedwith ESE3/EHF and increases inprostate cancer progression. A, heatmaps correlating expression of Lin28A,Lin28B, and ESE3/EHF in human primaryprostate tumors in the Taylor dataset(n ¼ 131). B and C, representative imagesof Lin28 staining (B) in normal andprimary prostate tumors andimmunohistochemical scores (C) inprimary tumors. D and E, representativeimages of Lin28 immunohistochemicalstaining (D) and relative scores (E) inmetastatic prostate tumors.

Albino et al.





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Figure 3.Lin28A and Lin28B promote transformation and stemness in the ESE3KD prostate epithelial cells. A, colony formation in soft agar by ESE3KD-PrECs (top) andESE3KD-RWPE-1 cells (bottom) after knockdown of Lin28A or Lin28B by siRNA. B, in vitro SFE of ESE3KD-PrECs and RWPE-1 cells evaluated at first (G1) and second(G2) generation following Lin28A and Lin28B knockdown. C, flow-cytometry analysis of cell senescence based on FDG staining following Lin28A and Lin28Bknockdown. D, Lin28A and Lin28B expression evaluated by qRT-PCR (left) and Western blot (right) following transfection with Lin28A and LIN28B expressionvectors in RWPE-1 cells. E, colony formation in soft agar by RWPE-1 cells after transfection with Lin28A and Lin28B expression vectors or control vector (pcDNA).F, SFE of RWPE-1 cells transiently expressing Lin28A and Lin28B. G, in vivo tumor-initiating ability of ESE3KD-PrECs cells transfected in vitro with control(siGL3), Lin28A (siLin28A), and Lin28B (siLin28B) targeting siRNAs and implanted subcutaneously (2� 106 cells/site) in NSGmice (n¼ 4/group). Experimental plan(top), tumor volume determined by caliper (middle); histopathology and IHC staining for Lin28 in the ensuing tumor masses (bottom). H, expression of Lin28A,Lin28B, let-7b, and the indicated CSC genes determined by qRT-PCR in tumor tissues from ESE3KD-PrEC xenografts shown in G. I, ex vivo SFE of cells derived fromtumor tissues from the ESE3KD-PrEC xenografts shown in G at first (G1) and (G2) second generation. P values were determined using the t test; � , P < 0.05;�� , P < 0.01. Data are representative of three independent experiments.






transfected in vitro with Lin28A or Lin28B targeting siRNAsformed smaller masses that barely expanded over time. The levelof Lin28A and Lin28B mRNA was reduced in the xenografts fromcells treated with siLin28A and siLin28B compared with controlxenografts (Fig. 3H). Interestingly, the level of total Lin28 proteindetermined by IHC was reduced in the siLin28-treated xenografts(Fig. 3G). Concomitantly, the expression of various CSCmarkers,like Nanog, Sox2, POU5F1, KLF4, and BMI-1 (17, 42), wassignificantly reduced in xenografts treated with siLin28A andsiLin28B, consistent with a reduction of the CSC subpopulation(Fig. 3H). These findings suggested the activation of feedbackloops that extend the effects of the transient knockdown of Lin28,leading to persistent repression of Lin28 and other CSC genesduring the in vivo growth. Accordingly, ex vivo PS-forming assaysperformedwith cells isolated from the tumor xenografts showed asignificant and persistent reduction of PS-forming and self-renew-al cells in xenografts treated with siLin28A and siLin28B com-pared with control xenografts (Fig. 3I). Thus, targeting Lin28A/Bhad a profound effect on the CSCs and their self-renewal capa-bility in vivo. Furthermore, these results indicated that Lin28A/Bplayed key roles in the cell transformation upon loss of ESE3/EHFand sustained the CSC-like phenotype and expansion of the CSCcompartment in prostate tumors. Targeting Lin28A/B reversed theCSC properties in prostate cancer cell models in vitro and in vivo,suggesting that it could re-activate a latent differentiation/senes-cence program and that it might be a valid approach for selectiveCSC ablation in prostate tumors.

ESE3/EHF exerts dual transcriptional and posttranscriptionalcontrol on the Lin28/let-7 axis

ESE3/EHF and Lin28A/B expressions were inversely correlatedin cell lines and prostate tumors. Furthermore, Lin28A/B had animportant role in determining tumorigenic properties of trans-formed prostate epithelial cells. To better define the relationship

with ESE3/EHF, we searched for ETS binding site (EBS) in thepromoters of the Lin28A and Lin28B genes. Computational ana-lysis of transcription factor–binding sequences revealed the pres-ence of multiple EBS in both promoters. To test whether ESE3/EHF bound to the promoters and controlled Lin28A/B transcrip-tion, we selected the EBSs with the highest scores and nearest thetranscription start sites (TSS) of the respective genes and perform-ed ChIP assays (Fig. 4A). ESE3/EHF bound to the Lin28A andLin28Bpromoters inRWPE-1 cells (Fig. 4B).However, no bindingwas detected in ESE3KD-RWPE-1 cells, in which both Lin28A/Bproteins were highly expressed. Consistent with a repressive func-tion on the Lin28A/B promoters, we found enrichment of repres-sive (H3K9me and H3K27me) histone marks in RWPE-1 cells(Fig. 4C). We performed luciferase reporter assays in RWPE-1 andESE3KD-RWPE-1 cells using Lin28A and Lin28B promoter con-structs (Fig. 4D). Activity of both promoter reporters was signif-icantly reduced in RWPE-1 comparedwith ESE3KD-RWPE-1 cells,consistent with transcriptional repression by ESE3/EHF.

Lin28A/B block the production of mature let-7 miRNAs(20–25). Accordingly, we observed reduced levels of the matureforms of miRNAs of the let-7 family in ESE3KD-PrECs andESE3KD-RWPE-1 cells comparedwith the corresponding parentalcells (Supplementary Fig. S1B). This was consistent with reducedprocessing of let-7miRNAs in the presence of high Lin28A/B levelin cells with lowESE3/EHF expression. In linewith this notion,wemonitored in greater details the level of mature let-7b in differentcells and conditions. Mature let-7b was significantly reduced inESE3KD cells and in RWPE-1 and LNCaP cells upon forcedoverexpression of Lin28A (Fig. 5A and B). We hypothesized thatlet-7 miRNAs could be important mediators of ESE3/EHF pro-differentiation functions in prostate epithelial cells. Accordingly,antagonizing let-7b with a locked nucleic acid (LNA) anti-miRNAincreased PS-forming ability of PrECs and RWPE-1 cells inducingan effect similar to Lin28A/B overexpression or ESE3/EHF

B

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Figure 4.ESE3/EHF represses Lin28A and Lin28B transcription. A, predicted ETS-binding sites (EBS) in the human Lin28A (top) and Lin28B (bottom) promoter. Positionrelative to the TSS of the gene, sequence, and corresponding score are indicated for each site. B, binding of ESE3/EHF to the Lin28A and Lin28B promoterdetermined by ChIP in RWPE-1 and ESE3KD-RWPE-1 cells. C, chromatin marks at the Lin28A and Lin28B promoters evaluated by ChIP in RWPE-1 and ESE3KD-RWPE-1 cells. D, transcriptional activity of Lin28A (left) and Lin28B (right) promoter reporters in RWPE-1 and ESE3KD-RWPE-1 cells evaluated by dualluciferase assay. P values were determined using the t test; � , P < 0.05; �� , P < 0.01. Data are representative of three independent experiments.

Albino et al.





knockdown (Fig. 5C). Conversely, forced overexpression ofpre-let-7b reduced PS formation in DU145 cells (SupplementaryFig. S4A and S4B), phenocopying the effects of ESE3/EHFre-expression (17) or Lin28A/B ablation in these cells.

Interestingly, we observed that ESE3KD cells had also a reducedlevel of pri-let-7b (Fig. 5D). This suggested that ESE3/EHF couldcontrol the production of let-7 miRNAs also at the stage oftranscription. Consistently, a high scoring EBS was found nearthe TSS in the let-7b promoter (Fig. 5E). ChIP assays demonstrat-ed the presence of ESE3/EHF bound to the let-7b promoter inRWPE-1 cells and its absence in ESE3KD-RWPE-1 cells (Fig. 5F).The differential occupancy of the let-7b promoter by ESE3/EHFcorrelated with enrichment of activating (AcH3) histonemarks inRWPE-1 cells compared with ESE3KD RWPE-1 cells (Fig. 5G). Toconfirm transcriptional activation of the let-7b promoter, we

performed a luciferase reporter assay. Activity of the reporter wassignificantly higher in RWPE-1 compared to ESE3KD-RWPE-1cells consistent with increased transcriptional activity in cellsexpressing ESE3/EHF (Fig. 5H). Interestingly, we found EBS inthe promoters of other let-7 miRNAs (Supplementary Fig. S5),suggesting that other members of this miRNA family could betranscriptionally activated by ESE3/EHF.

To provide further support for this hypothesis, we assessed theeffects of modulating ESE3/EHF expression on let-7b and pri–let-7b in DU145 cells. Expression of both pri–let-7b and mature let-7b was higher in ESE3/EHF-expressing DU145 cells comparedwith control DU145 cells, in agreement with dual transcriptionaland posttranscriptional control of let-7b by ESE3/EHF (Fig. 5I).Thus, ESE3/EHF could sustain expression of let-7 miRNAs bypromoting directly pri–let-7 miRNA transcription and indirectly

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Figure 5.ESE3/EHF controls pri-let7b and mature let7b miRNA. A, level of mature let-7b miRNA determined by qRT-PCR in the indicated cell lines. B, expression of maturelet-7b in RWPE-1 (left) and LNCaP (right) cells transiently transfected with Lin28A (pLin28A) or control plasmid (pcDNA). C, SFE of PrECs and RWPE-1 cellstransfected with control (Scrambled) or let-7b anti-miRNA (LNA-let-7b). D, Level of pri–miR-let7b (pri–let-7b) determined by qRT-PCR in the indicated celllines. E, predicted ETS-binding site (EBS) in the pri–miR-let-7bpromoter. Sequence, score, and position relative to the geneTSS are indicated. F, bindingof ESE3/EHFto the pri–miR-let-7b promoter in RWPE-1 and ESE3KD-RWPE-1 cells determined by ChIP. G, chromatin marks at the pri–miR-let-7b promoter evaluated byChIP. H, transcriptional activity of the pri–miR-let-7b promoter reporter evaluated by luciferase assay in control RWPE-1 (Ctrl) and ESE3KD-RWPE-1 cells. I,expression of pri–miR-let-7b and mature let-7b miRNA in control (DU145pcDNA) and ESE3/EHF expressing (DU145pESE3) DU145 cells. P values weredetermined using the t test; � , P < 0.05; �� , P < 0.01. Data are representative of three independent experiments.






the processing of let-7 precursors to the mature forms throughrepression of Lin28A/B expression. Interestingly, co-expression ofESE3/EHF in RWPE-1 cells partially rescued the effects of forcedoverexpression of Lin28A in soft-agar and PS-forming assays(Supplementary Fig. S6A and S6B). This could be attributed tothe ability of ESE3/EHF to induce pri-let7 miRNA transcriptionand, independently of posttranscriptional inhibition of pre-miRNA processing, to partially restore the level of mature let-7b (Supplementary Fig. S6C).

Targeting of Lin28 in prostate cancer cells blocks CSCself-renewal and tumor growth in vivo

We showed previously that re-expression of ESE3/EHF inmetastatic DU145 cells resulted in suppression of the malignantphenotype, inhibiting anchorage-independent growth, PS forma-tion, and tumor growth in vivo (17). We reasoned that interferingwith the Lin28/let7 axis could have similar effects and reverse themalignant phenotype of prostate cancer cells. DU145 and PC3cells are models of aggressive, androgen-independent castration-resistant prostate cancer. These cells are highly tumorigenic inmice and have a high fraction of tumor-initiating and CSC-likecells (17, 42). DU145 and PC3 cells do not express ESE3/EHF andexhibit high levels of both Lin28A and Lin28B compared withnormal PrECs (Fig. 6A). As seen with ESE3KD cell models, thelevels of Lin28A and Lin28B further increased in CSC-enriched PScomparedwith bulk adherentDU145 andPC3 cells (Fig. 6B). Thiswas particularly evident inDU145 cells with >200-fold increase ofLin28B expression in PS compared with adherent cells. Highexpression of Lin28 in PS derived from DU145 and PC3 cellswas also demonstrated by ICC staining (Fig. 6B). We inhibitedLin28A and Lin28B with siRNAs in DU145 and PC3 cells (Sup-plementary Fig. S7A). Knockdown of Lin28A and Lin28B wasassociated with reduced formation of colonies in soft-agar (Fig.6C) and PS (Fig. 6D) compared with control cells. Furthermore,Lin28A and Lin28B downregulation led to increased levels of let-7b, confirming inhibition of Lin28 function and restoration of let-7 miRNA synthesis (Supplementary Fig. S7B). Interestingly, wefound a significant increase of senescent cells upon Lin28A andLin28B knockdown (Supplementary Fig. S7C).

To assess the effect of Lin28 depletion on in vivo tumorigenicityof DU145 and PC3 cells, cells were first transfected in vitro withcontrol siRNA and the siRNA directed against Lin28B (siLin28B),and then implanted subcutaneously in nude mice. Growth oftumors formed by Lin28B-targeted cells was delayed comparedwith control cells, leading to a significant difference in tumor sizeand weight (Fig. 6E). As seen in the ESE3KD-PrEC xenografts, thelevels of total Lin28 protein and the proliferation marker Ki67were significantly reduced in DU145 and PC3 xenografts fromLin28B-targeted cells examined at the endof the in vivo experiment(Fig. 6F). Furthermore, expression of Lin28B and other CSC geneswas decreased whereas levels of let-7b increased in Lin28B-tar-geted tumor xenografts (Fig. 6G). These results indicated thattargeting Lin28B affected the CSC-like component and antago-nized the development of aggressive prostate tumors.

To further evaluate the potential of Lin28-targeting therapy, weassessed the ability of a siRNAdirected to Lin28B to inhibit growthof established tumor xenografts upon systemic delivery. Thispreviously characterized siRNA (siLin28B-2) was selected for itshigh knockdown efficiency (40). Transfection of siLin28B-2reduced Lin28B mRNA and concomitantly increased let-7bexpression in DU145 cells (Supplementary Fig. S8A and S8B). It

also inhibited PS-forming ability of DU145 cells in vitro (Supple-mentary Fig. S8C). For in vivo systemic delivery siLin28B-2 and thecontrol siRNA were complexed with in vivo jetPEI and adminis-tered by intraperitoneal injections at the dose of 2 mg/kg/d. Micewith established DU145 tumor xenografts were treated withsiLin28B-2 or control siRNA three times per week for 3 weeks.Treatment with siLin28B-2 reduced tumor growth compared withtreatment with the control siRNA (Fig. 7A). In addition, tumorsize assessed by in vivo imaging and tumor weight at the end of thetreatment were significantly reduced compared with control mice(Fig. 7A). Tumors from siLin28B-2–treated mice showeddecreased Lin28 protein and Ki67 staining, indicative of repres-sion of Lin28, and reduced fraction of proliferating tumor cells(Fig. 7B). Furthermore, the expression of several CSC markergenes was significantly reduced in siLin28B-2–treated xenograftsalongwith reduced Lin28BmRNAandupregulationof let-7b (Fig.7C). Consistent with an effect on the CSCs component, cellsextracted from siLin28B-2–treated xenografts exhibited reducedex vivo PS-forming ability compared with cells from xenograftstreated with the control siRNA (Fig. 7D). Furthermore, whentumor cells derived from siLin28B-2–treated xenografts werere-implanted in mice they retained lower in vivo tumor-initiatingand ex vivo self-renewal capability, in line with the persistentchange in the cell phenotype and loss of CSC properties inducedby restoring the normal function of the Lin28/let-7 axis (Fig. 7E).Thus, targeting Lin28B resulted in significant contraction of theCSC compartment impairing tumor initiation, tumor growth, andself-renewal capabilities of prostate cancer cells. Furthermore,depletion of Lin28B was sufficient to affect tumor growth in vivo,in line with the notion that Lin28A and Lin28B have non-redun-dant functions and cooperate to sustain the tumorigenic and CSCphenotype.Collectively, these data indicate that targeting Lin28A/B function is a valid strategy to antagonize tumor growth andtarget the CSC compartment in aggressive prostate cancers.

DiscussionThis study provides novel insights on the roles and the regu-

lation of the Lin28/let-7 axis in prostate cancer. We report thatLin28A and Lin28B are highly expressed in prostate cancer andthat they sustain the expansion andmaintenance of prostate CSCsin cell cultures and tumor xenografts.We found that the epithelial-specific ETS factor ESE3/EHF directly controls transcription ofLin28A, Lin28B and let-7 pri-miRNAs, such as pri–let-7b, innormal prostate epithelial cells. Furthermore, targeting Lin28Aand Lin28B had a significant impact on the CSC compartmentimpairing tumor initiation, tumor growth, and self-renewalcapability.

CSCs are a critical source of intratumor heterogeneity withinthe primary tumor mass and likely the main factors responsiblefor tumor progression, metastasis, and treatment failures (3).Understanding the molecular mechanisms underlying the induc-tion and expansion of CSCswill help to define new approaches toselectively target these cells and thereby improve cancer treat-ments (43). Lin28A and Lin28B, which are highly expressedduring normal embryogenesis, are frequently upregulated inhuman cancers and likely play important roles in CSC expansionin many tumors (26, 28, 32). These RNA-binding proteins neg-atively regulate the processing and maturation of let-7 miRNAs(20, 28, 29, 44, 45). Lin28 proteins bind to let-7 precursors andprevent their processing by DICER and DROSHA. Lin28A has

Albino et al.





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Figure 6.Targeting of Lin28 in prostate cancer cell lines blocks CSC expansion and tumor growth. A, Lin28A and Lin28B mRNA evaluated by qRT-PCR in the PrECs, LNCaP,DU145, and PC3 cells. B, Lin28A and Lin28B mRNA (left) and Lin28 protein (right) evaluated by qRT-PCR and ICC, respectively, in adherent cultures andprostatospheres from DU145 and PC3 cells (scale bar, 50 mm). C, colony formation in soft agar by DU145 (top) and PC3 (bottom) cells after Lin28A and Lin28Bknockdown. D, SFE of DU145 (top) and PC3 cells (bottom) at first (G1) and second (G2) generation after Lin28A and Lin28B knockdown. E, growth of tumorxenografts of control (siGL3) and siLin28B-transfected DU145 (top) and PC3 (bottom) cells implanted (2 � 106 cells/site) in NSG mice (n ¼ 4/group). Left, tumorvolume (percentage relative to day 19). Right, average tumor weights at the end of the experiment. F, immunohistochemical staining for Lin28 and Ki67 oftumor xenografts formed by control (siGL3) and siLin28B transfected DU145 (top) and PC3 (bottom) cells. G, Lin28B, let-7b and the indicated CSC genes evaluatedby qRT-PCR in tumor tissues from the xenografts shown in E. P values were determined using the t test; � , P < 0.05; ��, P < 0.01. Data are representative ofthree independent experiments.






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Figure 7.Lin28B ablation by systemic treatment with Lin28B-2 siRNA stably reverses tumor initiation, tumor growth, and self-renewal properties of DU145 cells. A, growth ofDU145 (5 � 106 cells) tumor xenografts in athymic nude mice receiving intraperitoneal injections of control siRNA (Ctrl) or siRNA targeting Lin28B (siLin28B-2;n ¼ 4/group).Treatment scheme (top). Bottom, tumor volume determined by caliper (left), tumor weight (middle), and tumor size by in vivo bioluminescenceimaging (right). B, Lin28 and Ki67 IHC staining in tumor tissues derived the xenografts shown in A. C, Lin28B, let-7b, and the indicated CSC genes evaluatedby qRT-PCR at the end of the experiment in tumor tissues from the xenografts shown in A. D, ex vivo SFE of cells isolated from tumor tissues derived from DU145xenografts shown in A. E, growth of DU145 cells isolated from tumor tissues from the primary xenografts shown in A and re-injected (5 � 105 cells/site)subcutaneously in mice (n¼ 2/group). Left, tumor volume; middle, tumor weight and size by in vivo bioluminescence; right, ex vivo SFE of tumor cells isolated fromsecondary tumor xenografts at the end of the experiment. F, transcriptional and posttranscriptional control of the Lin28/let-7b axis by ESE3/EHF and impacton differentiation and tumorigenesis in prostate epithelial cells. P values were determined using the t test; � , P < 0.05; �� , P < 0.01. Data are representative ofthree independent experiments.


Albino et al.




been shown to recruit Zcchc11/TUT4 to let-7 precursors andblockprocessing byDICER in the cytoplasm,whereas Lin28Bbinds pri–let-7miRNAs andblockprocessing in thenucleus (22, 28, 44–46).More recent data, however, indicate that Lin28A accumulate alsoin the nucleus upon methylation to block pri–let-7 biogenesis(47). Intranuclear localization of both Lin28A and Lin28Bappears to enhance their oncogenic effects, perhaps providing amore complete block of let-7 miRNA processing. This is in linewith our finding of increased intranuclear localization of Lin28proteins inmore aggressive prostate cancer xenograftsmodels andhuman metastatic tumors. The Lin28 and let-7 interaction is alsoan example of the complex interplay between miRNAs and theirregulators. The existence of a negative feedback loopbetween let-7miRNA and Lin28 proteins is well established, whereby increasedlevels of let-7 miRNAs prevent accumulation of Lin28 proteinsand promote further miRNA maturation.

Lin28 proteins function as oncogenes in a variety of humancancers (26). However, the factors regulating the expression ofLin28A and Lin28B in normal and cancer cells are largelyunknown (19). Few transcription factors have been identifiedthat regulate the expression of Lin28A or Lin28B (19). Wereported previously that the transcription factor ESE3/EHF main-tains the differentiation state and restrains transformation andself-renewal in normal prostate epithelial cells (17). Here, weshow that ESE3/EHF exerts these functions in part by controllingthe level and activity of distinct components of the Lin28/let-7axis. ESE3/EHF represses transcription of both Lin28A andLin28Bin normal prostate epithelial cells by binding to their promotersand inducing a repressive chromatin state. Thus, ESE3/EHF is thefirst transcription factor known to repress both Lin28A andLin28B genes. Coordinate repression of Lin28A and Lin28B byESE3/EHF allows maturation of let-7 miRNAs to proceed innormal cells. In addition to prevent the posttranscriptional blockby Lin28, we demonstrate that ESE3/EHF also controls transcrip-tion of let-7 miRNAs. ESE3/EHF binds to the promoter of pri–let-7b and activates its transcription, thus increasing production ofmature let-7b miRNA. The promoters of other let-7 miRNAscontain similar EBS, suggesting that ESE3/EHF might positivelyregulate the transcriptionofmanymembers of thismiRNA family.Notably, let-7 miRNAs are known to downregulate posttranscrip-tionally the level of Lin28 protein, thus reinforcing the repressionof Lin28 transcriptionbyESE3/EHF. Therefore, in cells depleted ofESE3/EHF both transcriptional and posttranscriptional controlsof the Lin28/let-7 axis are altered shifting the balance towardupregulation of Lin28A/Lin28B and diminished production ofmature let-7 miRNAs. We show that this is associated with theacquisition of the transformed and stem-like state in normalprostate epithelial cells. Consistent with this, we found thatsilencing Lin28A and Lin28B or addition of let-7 precursors weresufficient to reverse the stem-like and tumor-initiating phenotypeinduced by loss of ESE3/EHF. Notably, similar effects were alsoseen in the prostate cancer cell lines DU145 and PC3, which havelow levels of ESE3/EHF and mimic the behavior of aggressive,castration-resistance prostate cancer.

Lin28A and Lin28B function on distinct steps of the processingpathway of pre-miRNAs tomature let-7miRNAs (28).However, itis unclear whether the two proteins exert complementary orredundant functions with respect to let-7 processing and tumor-igenesis (26, 28, 29). In some tumor types prevalent upregulationof either Lin28A or Lin28B has been reported (26, 28, 29). Inprostate cancer we found a positive correlation between expres-

sionof Lin28A andLin28B across human tumors,while bothwereinversely correlated to ESE3/EHF. Thus, consistent with the cellline data, ESE3/EHF apparently set the level of expression of bothLin28A and Lin28B in human prostate cancers and loss of ESE3/EHF was associated with upregulation of both. The frequent co-expression of Lin28A and Lin28B indicates that, although theirfunctions might be partially redundant, both are capable andapparently needed for tumorigenic transformation in prostatecancer. Notably, expression of both Lin28A and Lin28Bincreased in prostate epithelial cells upon knockdown ofESE3/EHF. In addition, both Lin28A and Lin28B were furtherelevated in the CSC subpopulation derived from ESE3KDepithelial cells and prostate cancer cell lines compared withthe bulk tumor cell population. Thus, loss of ESE3/EHF leads toupregulation of Lin28A and Lin28B, which is more pronouncedin CSC-enriched subpopulation and has a direct impact on thegrowth and self-renewal of CSCs. In this context, therefore,Lin28A and Lin28B appear to have complementary, non-redun-dant functions and to cooperate to induce cell transformation,tumorigenic, and stem-like phenotype. More in depth studieswould allow to dissect the functions of Lin28A and Lin28B indifferent cell context and clarifying these issues. This would bewarranted also in light of the novel roles of Lin28 proteins thatmight be independent of let-7 miRNA regulation (35, 47, 48).On the other hand, our data show that targeting either Lin28Aor Lin28B is sufficient to reverse these malignant phenotypes.The activation of negative feed-back loop between Lin28 andlet-7 miRNAs might explain the effects of the knockdown ofindividual Lin28 on the entire pathway as well as the long-termeffects of the transient downregulation of Lin28 proteins. Asdescribed in other models (25, 27), increased levels of let-7miRNAs would lead to posttranscriptional repression of Lin28protein, thus extending indefinitely the effects of the initialtransient depletion by siRNAs. This seems the case in the in vivoexperiments that showed significant depletion of Lin28 proteinin the tumor xenografts after knockdown of either Lin28A orLin28B.

Our data point to the Lin28/let-7 axis as a relevant target fordeveloping therapeutics directed to the CSC compartment.Current therapies target and kill preferentially proliferating,partially differentiated tumor cells that constitute the bulk ofthe tumor, while sparing the rare CSC population (43). Con-siderable evidence demonstrates that surviving CSCs may causedisease recurrence and drug resistance in prostate cancer (5).Targeting Lin28A and Lin28B might directly antagonize theexpansion of the CSC compartment, thus preventing survival ofCSCs, generation of therapy-resistant clones, and disease recur-rence. Consistently with this notion, Lin28A and Lin28B abla-tion in ESE3/EHF-KD cells and cancer cell lines resulted indecreased tumorigenic and stem-like properties in vitro. Tofurther test this hypothesis, we knocked down Lin28B in vitroin DU145 cells and then engrafted the cells in mice. Alterna-tively, we performed systemic treatment of tumor bearing micewith a Lin28B-targeting siRNA by repeated intraperitonealinjections. Both approaches resulted in significant reductionof tumor initiation and growth in mice. Moreover, tumor cellsisolated from Lin28B-targeted xenografts had impaired abilityto generate PS ex vivo and, notably, failed to form tumors whenre-engrafted in mice. These results were consistent with apersistent change of the tumor cell phenotype induced bytransient (in vitro) or prolonged (in vivo) targeting of Lin28.






Furthermore, they were detectable over multiple generationsand passages of the cells in vitro and in vivo.

These data have important implications for therapeutic appli-cations. Collectively, our results indicate that Lin28A and Lin28Bsupport tumor-initiating properties and their inhibition success-fully reverts the tumorigenic and stem-like state and tumordevelopment in vivo. The selectivity of Lin28-targeting therapeu-tics may rely on the increased expression of Lin28 proteins in theprostate CSCs comparedwith normal cells andbulk tumor cells aswell as their role in maintaining the stem-like state of CSCs.Furthermore, the induction of prolonged effects on the cancercell phenotype even after transient knockdown of Lin28 proteinssuggests that continuous exposure to the inhibitors and persistenttarget inhibition might not be required to achieve beneficialtherapeutic effects. Notably, transient short-term inhibition ofLin28 was sufficient to induce persistent effects on the CSCfunction. This persistent impairment of CSCsmight be associatedwith the inductionof cell senescence and irreversible growth arrestthat might occur upon Lin28 knockdown. Thus, in combinationswith drugs directed to the bulk tumor cells, Lin28-targetingtherapeuticsmight deplete the CSC compartment and allowmorepersistent control of the tumors, prolong disease-free survival, andreduce disease recurrence. Furthermore, in addition to chemicallyimproved RNAi effectors more suitable for in vivo applications(40), the highly structured interaction between Lin28 proteinsand let-7 precursors provides an interesting area for drugdiscoveryand development of innovative approaches for targeting thispathway (23, 36, 45, 49).

In conclusion, our study identifies a relevant upstream mech-anism controlling the Lin28/let-7 axis in normal prostate epithe-lial cells and prostate cancer. The dual control exerted by ESE3/EHF on transcription of Lin28A/Lin28B and let-7 precursors isfundamental to sustain production of let-7miRNAs andmaintainthe differentiation state in normal epithelial cells. On the otherhand, deregulation of this mechanism in tumors by loss of ESE3/EHF promotes tumorigenesis and affects prominently the CSCcompartment, thus pointing to specific strategies to target the

CSCs in prostate tumors and prevent disease progression andrecurrence.

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

Authors' ContributionsConception and design: D. Albino, G. Civenni, J. Hall, C.V. Catapano,G.M. CarboneDevelopment of methodology: D. Albino, G. Civenni, C. Dallavalle, M. Roos,L. Curti, C.V. Catapano, G.M. CarboneAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): D. Albino, H. Jahns, S. Pinton, G. D'Ambrosio,F. Sessa, G.M. CarboneAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): D. Albino, H. Jahns, S. Rossi, C.V. Catapano,G.M. CarboneWriting, review, and/or revisionof themanuscript:D.Albino,M. Roos, J. Hall,C.V. Catapano, G.M. CarboneAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): M. Roos, H. Jahns, S. Pinton, G. D'Ambrosio,F. Sessa, G.M. CarboneStudy supervision: J. Hall, C.V. Catapano, G.M. Carbone

AcknowledgmentsThe authors thank Enrica Mira Cat�o for her assistance with in vivo experi-

ments. The authors declare no competing financial interests.

Grant SupportThis work was supported by grants from the Swiss Cancer League (KLS-2648-

08-2010, KLS-3243-08-2010, and KLS 3243-08-2013), Swiss National ScienceFoundation (SNF 310030-146560-2013), ETH Zurich (ETH-01 11-2 andETH-14 09-3), Swiss Bridge Award, Ticino Foundation for Cancer Research,Virginia Boeger Foundation, and Fidinam Foundation.

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

Received September 30, 2015; revised March 16, 2016; accepted March 30,2016; published OnlineFirst May 2, 2016.

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2016;76:3629-3643. Published OnlineFirst May 2, 2016.Cancer Res Domenico Albino, Gianluca Civenni, Cecilia Dallavalle, et al. Tumorigenic and Stem-like Phenotype in Prostate CancerActivation of the Lin28/let-7 Axis by Loss of ESE3/EHF Promotes a

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ActivationoftheLin28/let-7AxisbyLossofESE3/ EHF Promotes ......from the solid support and the deprotection of the nucleotides was affected by incubation of the CPG-support for 2 hours,