The FASEB Journal • Research Communication

Thymosin �4 targeting impairs tumorigenic activity ofcolon cancer stem cells

Lucia Ricci-Vitiani,*,1 Cristiana Mollinari,†,‡,1 Simona di Martino,* Mauro Biffoni,*Emanuela Pilozzi,§ Alfredo Pagliuca,* Maria Chiara de Stefano,† Rita Circo,�

Daniela Merlo,†,¶ Ruggero De Maria,*,�,2 and Enrico Garaci#,2

*Department of Hematology, Oncology, and Molecular Medicine and †Department of Cell Biologyand Neuroscience, Istituto Superiore di Sanita, Rome, Italy; ‡Department of Neurobiology andMolecular Medicine, Consiglio Nazionale delle Ricerche (CNR), Rome, Italy; §Department ofLaboratory Medicine and Pathology, Sant’Andrea Hospital, University La Sapienza, Rome, Italy;�Mediterranean Institute of Oncology, Catania, Italy; ¶Istituto di Ricovero e Cura a CarattereScientifico (IRCCS) San Raffaele Pisana, Rome, Italy; and #Department of Experimental Medicineand Biochemical Science, University of Rome Tor Vergata, Rome, Italy

ABSTRACT Thymosin �4 (T�4) is an actin-bindingpeptide overexpressed in several tumors, includingcolon carcinomas. The aim of this study was to investi-gate the role of T�4 in promoting the tumorigenicproperties of colorectal cancer stem cells (CR-CSCs),which are responsible for tumor initiation and growth.We first found that CR-CSCs from different patientshave higher T�4 levels than normal epithelial cells.Then, we used a lentiviral strategy to down-regulateT�4 expression in CR-CSCs and analyzed the effects ofsuch modulation on proliferation, survival, and tumor-igenic activity of CR-CSCs. Empty vector-transducedCR-CSCs were used as a control. Targeting of the T�4produced CR-CSCs with a lower capacity to grow andmigrate in culture and, interestingly, reduced tumorsize and aggressiveness of CR-CSC-based xenografts inmice. Moreover, such loss in tumorigenic activity wasaccompanied by a significant increase of phosphataseand tensin homologue (PTEN) and a concomitantreduction of the integrin-linked kinase (ILK) expres-sion, which resulted in a decreased activation of proteinkinase B (Akt). Accordingly, exogenous expression ofan active form of Akt rescued all the protumoralfeatures lost after T�4 targeting in CR-CSCs. In con-clusion, T�4 may have important implications for ther-apeutic intervention for treatment of human coloncarcinoma.—Ricci-Vitiani, L., Mollinari, C., di Martino,S., Biffoni, M., Pilozzi, E., Pagliuca, A., Chiara deStefano, M., Circo, R., Merlo, D., De Maria, R., Garaci,E. Thymosin �4 targeting impairs tumorigenic activityof colon cancer stem cells. FASEB J. 24, 4291–4301(2010). www.fasebj.org

Key Words: actin cytoskeleton � cell cycle � tumor growth � tar-get therapy

Cell transformation is accompanied by a loss ofactin filaments that contribute to alter cell morphologyand reduce cell-cell contact and adhesion (1). Eventhough the precise mechanisms of how actin is involved

in cancer transformation are not yet well understood,the involvement in the tumorigenic process of a seriesof actin-binding proteins that govern the organizationof the actin structures is becoming evident. One of themajor group of actin-binding proteins comprises the�-thymosins, a class of small peptides, with thymosin �4(T�4) being the most abundant member.

Initially believed to be a thymic hormone (2), T�4was subsequently identified as an ubiquitously ex-pressed intracellular G-actin-sequestering molecule.T�4 plays a pivotal role in modulating actin dynamic.Depending on the cell type, its overexpression caninduce either polymerization of stress fiber or decreasethe number of actin fibers (3–6). T�4 has been shownto be involved in a number of cell functions, such asadhesion and spreading of fibroblasts (5, 7), differen-tiation of endothelial and neural cells (8, 9), direc-tional migration of endothelial cells and keratinocytes(10–12), angiogenesis (13, 14), wound healing (11, 15,16), hair follicle growth (12), and apoptosis (17, 18).The current opinion is that T�4 influences cell prolif-eration, migration, and differentiation by maintaininga dynamic equilibrium between G-actin and F-actin,critical for the rapid reorganization of the cytoskeleton.However, recent observations indicate that T�4 canexpress its activity toward different cell types also byinfluencing signaling cascades or directly acting intothe nucleus as transcription factor (19, 20). Moreover,T�4 can promote cell survival through interaction witha complex involving the integrin-linked kinase (ILK)with subsequent activation of AKT (21, 22).

1 These authors contributed equally to this work.2 Correspondence: R.D., Department of Hematology,

Oncology, and Molecular Medicine, Istituto Superiore diSanita, Viale Regina Elena 299, 00161 Rome, Italy. E-mail:demaria@iss.it; E.G., Department of Experimental Medicineand Biochemical Science-University of Rome “Tor Vergata,” viaMontpellier, 00133, Rome, Italy. E-mail: garaci@tiscali.it

doi: 10.1096/fj.10-159970

42910892-6638/10/0024-4291 © FASEB

Initial studies have shown that T�4 is overexpressedin several tumors, such as kidney and nonsmall celllung cancers (23–25). Later on, up-regulation of T�4 inhuman colon carcinomas and a variety of malignantcell lines and tumors have been reported (26, 27).Elevated T�4 expression has been associated with in-creased tumorigenicity and metastatic potential (7).The increased expression of T�4 correlates with theinvasiveness of the cells, the degree of morphologicaltransformation, and the disintegration of actin fila-ments. Moreover, increased T�4 expression has beencorrelated with enhanced cell growth in some cell types(18, 28) but not in others (7, 14).

Colorectal carcinoma (CRC) is one of the leadingcauses of cancer death. CRC development results froma progressive transformation of colorectal epithelialcells following the accumulation of mutations in anumber of oncogenes and tumor suppressor genes(29). Aberrant expression of T�4 has been recentlyfound to be associated with CRC progression inducinga reduction of E-cadherin expression, accumulation of�-catenin in the nucleus, and activation of the Tcf/LEFpathway (18, 28). In this respect, the aberrant expres-sion of T�4 could be responsible for alterations in thegrowth and differentiation of enterocytes, as well asmotility and invasion of tumor cells.

Cancer is increasingly being viewed as a stem celldisease. According to the cancer stem cell (CSC) hy-pothesis, cancer is initiated and maintained by a smallpopulation of tumor-initiating cells endowed with theability to self-renew and differentiate in nontumori-genic cells. The CSC model has been supported bystudies showing that the growth of several tumorsdepends on a small subset of stem-like cancer cellsdisplaying many features in common with their non-transformed counterparts. We and others recently dem-onstrated that a small number of undifferentiated cellswithin CRC are able to proliferate indefinitely andreproduce the tumor in immunocompromised mice,while generating a progeny of more differentiated cellsdevoid of tumorigenic potential (30–33). The identifi-cation of such colorectal CSCs (CR-CSCs) providesstrong support for the hierarchical organization ofhuman colon cancer, implying the necessity to definethe mechanisms responsible for unrestrained prolifer-ation and high malignancy of CR-CSCs.

Recently, the possible up-regulation of T�4 in tumor-initiating cells has been hypothesized on the basis ofthe analysis of the CSC-containing side population inthe breast cancer cell lines MCF7 and MDA-MB231(34). To elucidate the role of T�4 in CRC, here, wehave examined the expression of this gene in CR-CSCsand determined in vitro and in vivo the effects of T�4modulation on cell cycle, migration, and tumor growth.We found that T�4 plays a key role in CR-CSC prolif-eration and migration via activation of the Akt signal-ing pathway, thus promoting tumor growth and aggres-siveness.

MATERIALS AND METHODS

Cell culture and lentiviral infection

CR-CSCs were obtained from human tumor samples, asdescribed previously (31). T�4 cDNA in pCDNA3.1 vectorwas kindly provided by Dr. Hynda Kleinman (U.S. NationalInstitutes of Health, Bethesda, MD, USA) and subcloned intoa modified pCDNA3 (KpnI-XhoI sites; provided by FabienneHans, Albert Bonniot Institute, Grenoble, France) containingan HA-tag. Thus, the HA-T�4 cDNA was subcloned in theantisense orientation, under the CMV promoter of a lentiviralvector, which carried the EGFP reporter gene under the PGKpromoter. Constitutively active Akt (Myr-Akt, HA-tagged) wascloned under the CMV promoter of a lentiviral vector carry-ing the puromycin resistance gene under the hPGK pro-moter. Recombinant lentiviruses were produced as describedpreviously (35). Transduced cells were sorted for their fluo-rescence (FACS Aria; Becton Dickinson, Franklin Lakes, NJ,USA) or selected by exposure to puromycin (1 �g/ml).

Xenograft mouse models

Transduced CR-CSCs (5 � 105), resuspended in Matrigel,were subcutaneously injected in the flanks of nude or severecombined immunodeficiency (SCID) mice (Charles RiverLaboratories, Calco, Italy). Tumor size was assessed by cali-pers. After 10- to 12-wk-old mice were sacrificed by cervicaldislocation, tumors were removed, fixed in 10% neutralbuffered formalin solution (Sigma, St. Louis, MO, USA), andparaffin embedded for histological analysis. Animal experi-ments were performed in accordance with relevant institu-tional and national regulations.

Immunohistochemistry and immunofluorescence

Immunohistochemical analysis was carried out on forma-lin-fixed paraffin-embedded or frozen tissue. Paraffin sec-tions were dewaxed in xylene and rehydrated with distilledwater. The slides were subsequently incubated with thefollowing antibodies: anti-MUC2 (ABR, Alameda, CA,USA) and anti-Ki67 (DakoCytomation, Glostrup, Den-mark). The reaction was performed using Elite VectorStain ABC systems (Vector Laboratories, Burlingame, CA,USA) and DAB substrate chromogen (DakoCytomation)followed by hematoxylin counterstaining. Immunofluores-cence was performed on cells fixed in 2% paraformalde-hyde-PBS. The following primary antibodies were used:polyclonal anti-T�4 (Acris, Herford, Germany) and anti-�-tubulin (Sigma). Secondary antibodies, including Cy3-conjugated anti-rabbit and Cy2-conjugated anti-mouse IgG,(Jackson Immunoresearch, West Grove, PA, USA) wereused at 2.5 �g/ml. Images were collected with a LaserScanning Microscope (FV-1000; Olympus, Tokyo, Japan).

Real-time PCR

Total RNA was retrotranscribed into cDNA using the Super-script II system (Superscript; Invitrogen, Carlsbad, CA, USA)and pd(N)6 random nucleotide. Relative quantitative real-time PCR was performed in a real-time Thermocycler (MX3000; Stratagene, La Jolla, CA, USA) using the Brilliant SYBRGreen QPCR Master Mix, according to manufacturer’s in-structions. All PCR reactions were coupled to melting-curveanalysis to confirm the amplification specificity. Nontemplatecontrols were included for each primer pair to check for any

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significant levels of contaminants. Gene-specific primers arelisted in the Supplemental Table 1.

Western blot analysis

Cellular pellets were lysed in RIPA buffer: 150 mM NaCl, 10mM Tris-HCl, 1 mM EDTA, and 1% Triton-X100 and pro-tease inhibitors (Sigma), 1 mM PMSF pH 7.4. Samples wereresolved in SDS-PAGE gels (13% for T�4 detection). Thepurified T�4 peptide (10 mM) (kindly provided by Prof.Allan Goldstein, George Washington University, Washington,DC, USA) was run as reference for protein migration. ForT�4 detection, the acrylamide gel was incubated in 10%glutaraldehyde (Sigma) before transfer to nitrocellulose. Af-ter blocking, the membrane was incubated overnight at 4°Cwith a polyclonal antibody to T�4 (T�4 1–43; Acris). Western blotsfor the evaluation of other proteins were carried out without thestep of glutaraldehyde, by using the following antibodies: mouseanti-�-tubulin (Sigma), mouse anti-�-actin (Sigma), rabbit anti-�-catenin (Cell Signaling Technology, Beverly, MA, USA), rabbitanti-ILK 1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA),mouse anti-p21 (Santa Cruz Biotechnology), mouse anti-p27(Santa Cruz), mouse anti-total Akt (Cell Signaling Biotechnology),rabbit anti-Ser-473 Akt (Cell Signaling Biotechnology) and mouseanti-PTEN (BD Pharmingen, San Diego, CA, USAS). The quanti-tation of protein expression was determined after normalization to�-tubulin by measuring the optical density of respective band blotsusing the Quantity One software (Bio-Rad, Hercules, CA, USA).

In vitro growth curve

Spheres were mechanically dissociated after a short incuba-tion in diluted trypsin. Cells were then plated in 96-well platesin triplicate, and incubated at 37°C in a 5% CO2 incubator.Cells proliferation was monitored by counting the cell andconfirmed by using the CellTiter-Blue Viability Assay (Pro-mega, Madison, WI, USA).

Cell cycle assay

Cell proliferation was measured by bromo-2�-deoxyuridine(BrdU) incorporation. Briefly, transduced CR-CSCs weremechanically dissociated and incubated with BrdU (10 mM;BD Pharmingen). After 24 h, cells were harvested and fixedwith cold methanol (90% in PBS) for 10 min at �20°C. Afterwashing with PBS, cells were incubated for 30 min at RT, with2 N HCl, 0.5% Triton X-100 to obtain DNA denaturation.Neutralization was performed with 0.1 M sodium tetraborate.Cells were then incubated with FITC-conjugated anti-BrdUantibody (BD Pharmingen), according to the manufacturerprotocol, and DNA was stained by propidium iodide (PI, 50�g/ml, Sigma). Samples were analyzed with a FACS Cantoand evaluated by either FACS Diva (Becton Dickinson) orFlowJo software (Tree Star, Ashland, OR, USA). Mitosis arrestwas induced by incubating CR-CSCs with nocodazole (100ng/ml; Sigma) for 24 h before PI staining.

Soft agar colony formation assay

Assays of colony formation in soft agar were done usingstandard protocols. Briefly, transduced CR-CSCs (5–10�104

cells/well) were suspended in 0.35% Noble agar and wereplated onto a layer of 0.7% Noble agar in 24-well tissueculture plates (Corning, Corning, NY, USA). The agar-contain-ing cells was allowed to solidify overnight at 37°C in 5% CO2humidified atmosphere. Additional medium was overlaid on theagar, and the cells allowed to grow undisturbed for 2 wk. Plates

were stained with 0.5 ml of 0.005% crystal violet for 1 h. Visiblecolonies were counted with the aid of a microscope.

In vitro cell migration assay

The motility of transduced CR-CSCs was evaluated in 24-welltranswell chambers (Costar; Corning), as directed by themanufacturer. Briefly, the lower chambers of the 24-well platewere filled with 500 �l of stem cell medium containing EGFand FGF2; 1 � 104 cells in 500 �l of the same medium wereplaced into the upper compartment of the wells. The trans-well chambers were incubated at 37°C in 5% CO2 humidifiedatmosphere for 24 h. The cells that had invaded the lowersurface of the polycarbonate membranes (8-�m pore size)were fixed, stained with Coomassie blue, and quantified bycounting 5 microscopic fields/filter (at �100).

Gene array

Total RNA was extracted from cells stably transduced witheither T�4 antisense vector or empty vector, and from cellsgrowing either in proliferation medium or in differentiationmedium. RNA was labeled and hybridized to AffymetrixGeneChip1.0ST arrays (Affymetrix, Santa Clara, CA, USA) fol-lowing the manufacturer’s instructions. Hybridization valueswere normalized by the RMA method, and transcripts displayingdifferential expression on T�4 knockdown or differentiationwere selected when the fold modulation exceeded the value of2. Transcripts displaying the same kind of regulation in bothinstances are shown in Supplemental Tables 2 and 3.

RESULTS

T�4 is overexpressed in human CR-CSCs

To determine whether the increased levels of T�4 inCRCs involves the population of tumorigenic cells, weevaluated its expression in CR-CSCs from differentpatients. Real-time PCR showed that T�4 mRNA washighly expressed in CR-CSCs as compared to BerEP4-positive epithelial cells isolated from normal mucosae(Fig. 1A). Such T�4 overexpression was confirmed byNorthern and Western blot analysis, which showed aconsiderable up-regulation in the majority of CR-CSCsamples as compared with the human colon cancer celllines SW480, HT29, and CaCo2, normal mucosa, andcolon carcinoma cells (data not shown and Fig. 1B).Immunofluorescence studies of T�4 expression togetherwith cytoplasmic and DNA markers indicated that T�4was mainly localized in the cytoplasm of CR-CSCs (Fig.1C). Real-time PCR and Western blot analyses showed aconsiderable variation of T�4 levels on differentiation ofCR-CSCs (Fig. 1D, E), indicating that T�4 expression maynot be uniform in CR-CSCs and their progeny.

Down-regulation of T�4 attenuates the in vitro growthof CR-CSCs

To investigate the contribution of T�4 up-regulation inCR-CSC growth and proliferation, CR-CSCs from twodifferent patients were transduced with lentiviral vectorscarrying an HA sequence-tagged antisense cDNA for T�4

4293THYMOSIN �4 AND COLON CANCER STEM CELL

(T�4-As) and the EGFP reporter under a second consti-tutive promoter. An empty lentiviral vector containingonly a constitutively active EGFP sequence was used ascontrol. After flow cytometry sorting of EGFP� cells,knockdown of T�4 was confirmed by Western blot anal-ysis and real-time PCR. Infection with the antisense lenti-viral construct significantly reduced endogenous humanT�4 expression in CR-CSCs, whereas the control vectordid not (Fig. 2A, B). Real-time PCR using oligonucleotidesannealing in the HA tag confirmed the proper expressionof the antisense construct (Fig. 2B).

Alterations in the growth properties have been de-scribed in cells overexpressing T�4 (25). Therefore, wemeasured CR-CSC growth after flow cytometry sorting ofEGFP� cells. We found that T�4-As CR-CSC cells hadsignificantly reduced growth, �50% lower than in empty-vector-transduced cells (Fig. 2C). To determine the mech-anisms underlying growth inhibition after T�4 down-regulation, we examined DNA content and cell cycledistribution of empty-vector and T�4-As CR-CSCs. Al-though cell cycle distribution appeared minimally affectedby T�4 targeting (Fig. 2D), when we treated the T�4-As

CR-CSCs with nocodazole to block the exit from the Mphase, we noted a decrease in the percentage of cellsaccumulating in G2/M phase, indicating that a significantlylower number of CR-CSCs progressed through the cell cycle(Fig. 2E). Thus, T�4 has a role in CR-CSC growth byaccelerating the progression to the S phase of cell cycle.

T�4 promotes migration and anchorage-independentgrowth in CR-CSCs

Once the effect on cell growth and proliferation wasdetermined, we examined whether T�4 could alter othermalignant features of CR-CSCs, such as enhanced migra-tion and anchorage-dependent growth. Migration is a keyproperty of cancer cells, required for tumor invasion, andis directly correlated with tumor aggressiveness. The mo-tility of T�4-As-transduced CR-CSCs was examined using atranswell chamber assay. Cells that were able to reach thelower chamber after 48 h of incubation were stained andcounted. The loss of T�4 in the CR-CSCs was sufficient toconsiderably impair cell migration, which was �50–80%lower than control cells (Fig. 3A).

Figure 1. A) Real-time PCR of T�4 transcript in CR-CSCs from different patients. 18S rRNA expression was used fornormalization. T�4 mRNA up-regulation is expressed as logarithm (base 2) of fold changes vs. the calibrator sample (normalmucosae). B) Western blot analysis of T�4 in CR-CSCs and in the human colon carcinoma cell line SW480 (top panel), innormal mucosa (NM), and mixed colorectal carcinoma cells (T), both derived from the same patient (bottom panel). A purifiedT�4 peptide (top panel) or CR-CSC 11 lysate was run to confirm the specificity of antibody recognition. �-Tubulin was used asloading control. C) Three-color confocal microscopy analysis of CR-CSCs labeled for T�4 (green) and the cytoskeleton markersphalloidin or �-tubulin (red). DNA was stained by DAPI (blue). Single fluorescences are in gray scale. Scale bar � 10 �m.D) Real-time PCR of T�4 transcript in nondifferentiated and differentiated CR-CSCs from 5 patients. 18S rRNA expression wasused for normalization. T�4 mRNA in differentiated CR-CSCs is expressed as logarithm (base2) of fold changes vs. the calibratorsample (nondifferentiated cells). E) Densitometric analysis of Western blot for T�4 in CR-CSCs. T�4 protein expression is shownas percentage variation of T�4 protein in differentiated cells vs. nondifferentiated cells; �-actin was used to normalize samples.

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Anchorage-independent growth characterizes the ag-gressive cancer cells and is a surrogate assay for detectionof tumorigenic activity. Nontransformed epithelial cellsare dependent on anchorage via integrin signaling. Inthese cells, the attachment to the extracellular matrixfacilitates cell cycle progression through the integrin-mediated induction of cyclin D1 and inhibition of p21and p27KIP1 expression (36). Oncogenic activation andinhibition of tumor suppressors involved in cell cyclecontrol enable tumor cell growth in the absence ofanchorage-dependent integrin signaling.

To determine the contribution of enhanced T�4expression on anchorage-independent growth, CR-CSCs transduced with empty vector or T�4-As wereplated in soft agar and allowed to grow for 2 wk. T�4-Asclones formed a considerably lower number of colonies(�48% inhibition) as compared to empty-vector-trans-duced CR-CSCs (Fig. 3B), indicating that high levels ofT�4 generate a signal that contribute to anchorage-independent growth of CR-CSCs. Thus, targeting T�4expression results in a considerable inhibition of pro-liferation, migration, and colony formation of CR-CSCs, suggesting that high T�4 levels contribute topromote the tumorigenic activity of CR-CSCs.

Down-regulation of T�4 sustains CR-CSCdifferentiation and decreases their in vivo growth

Gene expression profiling of CR-CSCs showed thatT�4 targeting results in down-regulation of aldehyde

dehydrogenase 1 and Lgr5 (Supplemental Table 2),two key colon stem cell genes (32, 37). In contrast,the differentiation genes cytokeratin 20 and trefoilfactor 1 (38, 39) were inversely up-regulated (Sup-plemental Table 3), suggesting that T�4 contributeto maintain an undifferentiated phenotype in CR-CSCs.

On injection in immunocompromised mice, CR-CSCs generate tumor xenograft phenocopies of theoriginal human tumor, from which the cells werederived (31). To determine the in vivo effect of T�4targeting in CR-CSCs, we simultaneously injected anidentical number (5�105) of CR-CSCs transducedwith empty vector (on one flank) or T�4-As (contro-lateral flank) in SCID mice (n�9) and followed thegrowth of the tumors for several weeks. Targeting ofT�4 considerably affected tumor growth in all thexenografts examined, regardless of whether tumorformation was slower (CR-CSC11) or faster (CR-CSC18) (Fig. 4A).

To assess the effect of T�4 targeting on tumormorphology, we next examined by histological andimmunohistochemical analysis the tumor xenograftsgenerated by T�4-As- and control-vector-transducedCR-CSCs. We found that tumors derived from theantisense clone displayed a less aggressive and moredifferentiated morphology (Fig. 4B). While the num-ber of proliferating cells was high in tumors gener-ated by the injection of control CR-CSCs, Ki67� cellswere significantly reduced in tumors arising from

Figure 2. T�4 protein down-regulation mediated by anti-sense lentiviral infection in CR-CSCs. A) Western blot analysisof CR-CSC 11 and CR-CSC 18 samples, untreated (mock) andtransduced with empty vector (vector) or T�4 antisensevector (T�4 As). �-Tubulin was used as loading control.B) Real-time PCR analysis of T�4, EGFP, and HA-tag se-quence on CR-CSCs 11 and 18 untreated or transduced withempty vector or T�4 antisense. Values are means sd of 3independent experiments with both lines plotted as log (base2) fold change of calibrator (untreated samples). 18S rRNA

expression was used for normalization. *P 0.01 vs. control values. C) Growth curves of CR-CSC 11 and CR-CSC 18transduced as above. D) Cell cycle analysis of CR-CSCs after a BrdU pulse of 24h. E) Cell cycle analysis of CR-CSCs exposedto nocodazole (100 ng/ml) for 24 h. One representative of 5 independent experiments with CR-CSC 11 and 18 is shown.

4295THYMOSIN �4 AND COLON CANCER STEM CELL

T�4-As CR-CSCs (Fig. 4B, middle panels). The mi-totic difference between the two types of tumors maybe due to a higher number of cells that underwentterminal differentiation in T�4-As tumors, as indi-cated by the higher expression of goblet cell markerMUC2 (Fig. 4B, bottom panels). In a normal colon,goblet cells are dispersed throughout the colonicepithelium and secrete mucus into the intestinallumen to trap and expel microorganisms. In theT�4-As-induced tumors, the higher number of gobletcells was associated with a strong number of depositsof mucin (Fig. 4B, bottom panels). Such increaseddifferentiation coupled with a decreased number ofmitotic cells is in line with reduced tumor growth andaggressiveness induced by T�4 targeting. Thus,down-regulation of T�4 seems to reduce in vivotumor growth by instructing cells toward a differen-tiative pathway that leads to a goblet cell phenotype.

T�4 promotes ILK expression, PTENdown-regulation, and Akt phosphorylationin CR-CSCs

To determine the molecular mechanisms responsible forthe protumor activity of T�4, we investigated the bio-chemical signaling potentially involved in the acquisitionof anchorage-independent growth and enhanced prolif-eration, migration, and tumorigenesis.

Previous studies have shown that T�4 can trigger anepithelial-mesenchymal transition in colorectal carci-noma (40, 41). Moreover, overexpression of T�4 in thehuman colon cancer cell line SW480 has been shown toincrease the expression of ILK and the phosphorylationstate of its downstream effector Akt. Accordingly, Westernblot analysis of the ILK/Akt pathway showed that T�4targeting reduced ILK expression and Akt phosphoryla-tion (Ser-473) in CR-CSCs (Fig. 5A). The analysis of

Figure 3. Reduced anchorage-independent growth and migration in T�4-As CR-CSCs. A) Number of migrating cells in CR-CSCstransduced with empty vector (vector) or T�4 antisense vector (T�4-As). B) Colony formation of cells transduced as above. Rightpanel: representative contrast images. Scale bar � 200 �m. Data are expressed as means sd of 4 independent experiments.

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upstream regulators of AKT pathway showed that PTEN isweakly expressed in CR-CSCs (Supplemental Fig. 1) butconsiderably up-regulated in CR-CSCs transduced withT�4-As (Fig. 5A, right panel). Thus, both ILK down-regulation and PTEN up-regulation may contribute to adecrease of AKT activation in T�4-targeted CR-CSCs. Incontrast, no significant modifications were found forthe expression of E-cadherin, cyclin-dependent ki-nase inhibitor p21 and p27KIP1, which are involved inthe regulation of cell cycle checkpoints and repair, or�-catenin whose genetic mutations have been corre-lated with several CRC (data not shown). Akt signal-ing plays a crucial role in many biological processes,including cell proliferation, survival, and differentia-tion (42). To investigate the role of decreased Aktactivity in the antitumor effects of T�4 targeting, weused a lentiviral mutant Akt with a myristoylatedsignal at the carboxyl terminus (Myr-Akt) to recon-stitute Akt activity in CR-CSCs previously transducedwith T�4-As (T�4-As/Myr-Akt). This mutation targetsAkt permanently to the cell membrane, where it iscontinuously susceptible to PDK phosphorylation(43, 44). Following lentiviral infection with Myr-Akt,Akt phosphorylation was again detectable in T�4-As-transduced CR-CSCs (Fig. 5B, left panel). On Myr-Akt expression, we found a significant rescue of cell

growth in T�4-targeted CR-CSCs (Fig. 5B, middleand right panels). Moreover, we observed that theT�4-As/Myr-Akt CR-CSCs have cellular propertiessimilar to the empty vector CR-CSCs, in terms ofmigration, survival, and anchorage-independentgrowth (Fig. 5C). More important, the expression ofMyr-Akt was able to restore the rate of tumor growthin vivo, determining the formation of tumor massesthat were comparable or slightly larger than thoseproduced by control CR-CSCs (Fig. 6A, B). In addi-tion, morphological analysis of xenograft specimensshowed that injection of T�4-As/Myr-Akt CR-CSCsgenerates tumors with cellular and morphologicalfeatures similar to tumors generated by the injectionof control CR-CSCs (Fig. 6C). Similarly, the mitoticindex was restored to values comparable to thoseobserved in tumors induced by control CR-CSCs (Fig.6C, D). Thus, all the antitumoral effects mediated byT�4 loss can be rescued by the expression of an activeform of Akt.

DISCUSSION

Despite continuous efforts to improve prevention andtherapy, CRC is still a frequent cause of death in

Figure 4. T�4 protein down-regulation reduces the in vivo growth of tumors in SCID mice. A) Tumor growth curve in SCID micesubcutaneously injected on the right flank with CR-CSC 11 or CR-CSC 18 cells transduced with the empty vector (vector) andin the left flank with cells transduced with the antisense vector (T�4-As). Tumor growth was monitored and measured withcalipers. Measurements were then plotted. B) Hematoxylin-and-eosin (H&E) staining and immunohistochemical analysis ofhuman Ki67 and MUC2 in subcutaneous xenografts obtained by injection of vector or T�4-As CR-CSCs (�10). In T�4-Astumors, it is possible to see a higher number of goblet cells (black arrows) in comparison with vector tumors.

4297THYMOSIN �4 AND COLON CANCER STEM CELL

western countries. The discovery of CR-CSCs hasbrought new opportunities to improve the therapeuticresponse in advanced CRC. However, little data isavailable so far on the mechanisms that regulate thetumorigenic activity of CR-CSCs.

Here, we show that T�4 promotes several key malig-nant features of CR-CSCs, ultimately enhancing tumorgrowth and aggressiveness. T�4 is weakly expressed innormal colonic epithelial cells, but considerably up-regulated in CR-CSCs. Therefore, to investigate the roleof T�4 on CR-CSCs, we used an antisense construct thatpermanently reduced its expression by 60–70%. Wefound that T�4 down-regulation significantly impairsCR-CSC proliferation by slowing down the progressionthrough the cell cycle, with a consequent reduction ofcell growth. A similar effect was observed on migrationof CR-CSCs. Migration is a key property in tumor cellbiology, particularly when observed in cells endowed

with anchorage-independent growth, which allows themigrated cells to survive and proliferate in the extracel-lular matrix before invading the surrounding tissues.Thus, the ability of T�4 to enhance considerably CR-CSC survival, proliferation, migration, and anchorage-independent growth makes this molecule a relevantplayer in the tumorigenic process.

Our data are consistent with the observation thatoverexpression of T�4 in the colon carcinoma SW480line increased growth rate and colony formation in softagar, and it promotes tumor invasion (18, 28). Weobserved that down-regulation of T�4 largely reducesthe tumorigenic potential of CR-CSCs, which producesmall and slow-growing tumor xenografts after injec-tion in immunocompromised mice. Such tumors gen-erated by T�4-As-transduced CR-CSCs showed a lessaggressive phenotype combined with a reduced num-ber of mitosis and a higher number of goblet cells,

Figure 5. T�4 protein down-regulation leads to adecrease in ILK expression, a reduction of Akt phos-phorylation (Ser-473), and increase in PTEN expres-sion. A) Left panel: Western blot on transduced CR-CSC 11 and CR-CSC 18 for Ser-473 Akt, total Akt,�-cadherin, and �-tubulin. Middle panel: densitometricanalysis of Western blot for ILK protein (normalized to�-tubulin) and of Ser-473 phosphorylated Akt (normal-ized to the amount of the total Akt). Right panel:Western blot for PTEN and �-actin of untransduced(mock) and transduced CR-CSC 11 and CR-CSC 18.B) Left panel: Western blot analysis confirming theoverexpression of the exogenous Myr Akt. �-Tubulinwas used as loading control. Middle and right panels:growth curve of CR-CSC 11 and CR-CSC 18 samples

transduced with empty vector, T�4-As or T�4-As, and Myr-Akt (T�4-As/Myr-Akt) vectors. C) Inhibition of migration andcolony formation in semisolid medium of CR-CSCs transduced as above.

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suggesting a more pronounced propensity of these cellsto acquire a differentiated phenotype.

The effect of T�4 down-regulation in CR-CSC differenti-ation is somehow surprising on the basis of results previouslydescribed with cardiovascular and hair follicle stem cells (12,21). However, T�4 has been recently reported to inhibitosteogenic and enhance adipogenic differentiation of mes-enchymal cells (45). T�4 initiates cell fate determination ofmesenchymal cells through a biophysical mechanism involv-ing cytoskeleton reorganization and altered cell-cell adhe-sion rather than by direct regulation of lineage-determiningtranscriptional programs. Thus, our data confirm that T�4plays a variety of different roles depending on the cell typeand whether it acts extracellularly (i.e., exogenous peptideadministration) or intracellularly (46).

The involvement of actin filaments in oncogenictransformation has been suggested by the findingsthat the tumorigenicity of certain cancer cells wassuppressed by the enforced expression of genes encod-ing different structural components of the actin cy-

toskeleton (47). A highly regulated assembly and disas-sembly of the actin filaments appears crucial for cells torespond to the extracellular signals in terms of migra-tion, changing shape and division. T�4 plays a pivotalrole among the actin-binding proteins that regulate theorganization of the actin structures. Although the earlysignaling generated by T�4 at the cytoskeleton remainsto be defined, T�4 has been recently proposed topromote CRC epithelial-mesenchymal transitionthrough the up-regulation of ILK (22), whose expres-sion and activity are significantly increased in severaltypes of cancer (48, 49). ILK connects integrins andgrowth factor receptors to a variety of downstreamsignaling, such as cell adhesion, proliferation, migra-tion, differentiation, and survival (50).

PTEN is a major tumor suppressor gene thattargets the Akt pathway through a dual mechanisminvolving the direct inhibition of PI3K and theconsequent down-regulation of ILK activity (51). Ourbiochemical analysis of the T�4-targeted CR-CSCs

Figure 6. Expression of a constitutively active Akt rescues in vivo growth of T�4-As CR-CSCs. A) Representative tumor xenograftsobtained from injection of T�4-As/Myr Akt, T�4-As, or empty vector CR-CSCs, removed 7 wk after injection. B) Growth curvesof tumor xenografts in nude mice subcutaneously injected with T�4-As-, T�4-As/Myr-Akt-, or empty vector-transduced CR-CSCsamples. Tumor growth was measured by calipers. Measurements were then plotted in a graph. C) H&E-stained sections ofsubcutaneous tumors. Immunohistochemical analysis of intradermally induced tumors using the anti-human Ki67 to identifymitotic cells (�10). D) Mitotic index of the corresponding tumors. Data are means sd of 6 tumors/group.

4299THYMOSIN �4 AND COLON CANCER STEM CELL

has shown a reduction of ILK expression coupledwith a considerable increase of PTEN, which resultsin decreased Akt phosphorylation. Akt is involved ina variety of biological functions, including angiogen-esis, glycogen synthesis, gene expression, inhibitionof apoptosis, cell cycle arrest, and cell transformation(49). Our findings demonstrate that exogenous Aktexpression restores growth, migration, and tumori-genic activity lost by CR-CSCs after T�4 targeting.Although in vitro proliferation was not entirely recov-ered in our experiments, tumor growth in vivo wasvery pronounced on Akt reconstitution, sometimeseven higher than in tumors obtained from controlCR-CSCs. The different degree of recovery could bedue to the complex signaling pathways generated byT�4, which may go beyond the involvement of Akt. Ifthis is the case, it is likely that the signals from thetumor microenvironment can overcome the Akt-independent losses of T�4 pathway.

Despite the need of further studies aimed at eluci-dating upstream and downstream T�4 signaling, on thebasis of the data available so far, the Akt pathwayrepresents the most relevant mediator for T�4-inducedeffects on CR-CSC malignancy. Since T�4 up-regula-tion contributes to CR-CSC proliferation, migration,and aggressiveness, it is likely that targeting T�4 signal-ing may provide a valuable strategy for the treatment ofCRC.

The authors thank the Italian Ministry of Health, the ItalianMinistry for University and Research (FIRB_RBIP06ZJ78) andthe Italian Association for Cancer Research (AIRC) for support-ing the colon CSC research. The authors thank Dr. EnricoDuranti for technical assistance.

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Received for publication March 31, 2010.Accepted for publication June 10, 2010.

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