stat3 is necessary for proliferation and survival in …...tumor and stem cell biology stat3 is...

Tumor and Stem Cell Biology

STAT3 Is Necessary for Proliferation and Survival in ColonCancer–Initiating Cells

Li Lin1,6, Aiguo Liu1,7, Zhengang Peng5, Huey-Jen Lin2,5, Pui-Kai Li3, Chenglong Li3, and Jiayuh Lin1,4

AbstractSTAT3 is constitutively activated in colon cancer but its contributions in cancer-initiating cells have not been

explored. In this study, we characterized STAT3 in aldehyde dehydrogenase (ALDH)-positive (ALDHþ) andCD133-positive (CD133þ) subpopulations of human colon tumor cells that exhibited more potent tumor-initiating ability than ALDH�/CD133� cells in tumor xenograft assays in mice. We found that ALDHþ/CD133þ

cells expressed higher levels of the active phosphorylated form of STAT3 than either ALDH�/CD133� orunfractionated colon cancer cells. STAT3 inhibition by RNA interference–mediated knockdown or small-molecule inhibitors LLL12 or Stattic blocked downstream target gene expression, cell viability, and tumor-sphere-forming capacity in cancer-initiating cells. Similarly, treatment of mouse tumor xenografts with STAT3short hairpin RNA (shRNA), interleukin 6 shRNA, or LLL12 inhibited tumor growth. Our results establish thatSTAT3 is constitutively activated in colon cancer–initiating cells and that these cells are sensitive to STAT3inhibition. These findings establish a powerful rationale to develop STAT3 inhibitory strategies for treatingadvanced colorectal cancers. Cancer Res; 71(23); 7226–37. �2011 AACR.

Introduction

Colorectal cancer is a tumor caused by abnormal division ofthe cells lining the large intestine. According to the AmericanCancer Society, there were an estimated 102,900 new cases and51,370 deaths due to colorectal cancer in the United States in2010. As such, there is a need for better treatment approachesfor colorectal cancer. The cellular mechanisms contributing tocolorectal cancer are still not well understood but involvesignaling protein dysregulationwhich includes the constitutiveactivation of STAT3 (1–3). The constitutive activation ofSTAT3 is frequently detected in primary human colorectalcarcinoma cells and established human colorectal cancer cell

lines (1–3), and elevated levels of STAT3 phosphorylation werecorrelated with the tumor invasion, nodal metastasis, and thestage (P < 0.05; refs. 1, 3). Constitutive STAT3 activation incolorectal cancer cells is associatedwith invasion, survival, andgrowth of colorectal cancer cells and colorectal tumormodel inmice in vivo (2, 4–6). These reports indicate that STAT3 is oneof themajor oncogenic pathways activated in colorectal cancerand can serve as an attractive therapeutic target for colorectalcarcinoma. To date, however, whether STAT3 is activated incolorectal cancer stem cells is unknown.

The concept of the cancer stem cells or cancer-initiatingcells holds that only a minority of cells within a tumor have theability to generate a new tumor. Cancer stem cells werereported to show pluripotency and self-renewal (7). Cancerstem cells were first identified in leukemias and more recentlyin solid tumors. Increasing evidence suggests that the cancerstem cells concept is also relevant to colorectal cancer (8).CD133, a transmembrane protein (Prominin-1 or AC133) wasused to isolate stem cells from a host of other normal andcancerous tissues, including colorectal cancer. However, thespecificity of CD133 alone as a marker for colonic stem cells isuncertain (9–11).

A promising new marker for cancer stem cells is aldehydedehydrogenase 1 (ALDH1). ALDH is a detoxifying enzyme thatoxidizes intracellular aldehydes and thereby confers resistanceto alkylating agents (12). Corti and colleagues (13) showed thatALDHþ cells isolated from murine brain were capable of self-renewal and of differentiating into multiple lineages. Furtherstudies showed that ALDH1 is a specific marker for breastcancer stem cells (14, 15). ALDH was also investigated as aspecificmarker for identifying and isolating normal andmalig-nant human colonic stem cells and as a way to quantify thenumber of stem cells over the course of colon cancer

Authors' Affiliations: 1Center for Childhood Cancer, The Research Insti-tute at Nationwide Children's Hospital, Department of Pediatrics, CollegeofMedicine; 2Molecular Biology andCancerGeneticsProgram, 3DivisionofMedicinal Chemistry and Pharmacognosy, College of Pharmacy; 4Exper-imental Therapeutics Program, 5Medical Technology Division, School ofAllied Medical Professions, The Ohio State University ComprehensiveCancer Center, The Ohio State University, Columbus, Ohio; and 6Divisionof Cardiology, Department of Internal Medicine, 7Department of Pediatrics,Tongji Hospital, Tongji Medical College, Huazhong University of Scienceand Technology, Wuhan, Hubei, PR China

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

Corresponding Authors: Jiayuh Lin, Center for Childhood Cancer, TheResearch Institute at Nationwide Children's Hospital, Department of Pedi-atrics, College ofMedicine, TheOhioStateUniversity, 700Children'sDrive,Columbus, OH 43205. Phone: 614-722-5086; Fax: 614-722-5895; E-mail:[email protected]; and Li Lin, Division of Cardiology, Department of InternalMedicine, Tongji Hospital, Tongji Medical College, Huazhong University ofScience and Technology, 1095 JiefangAve.,Wuhan 430030, China; E-mail:[email protected]

doi: 10.1158/0008-5472.CAN-10-4660

�2011 American Association for Cancer Research.

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development (16). Xenograft tumors were successfully gener-ated using ALDHþ cells from 7 primary colon cancer cells andALDH� cells did not generate tumor xenografts (16). Whenusing ALDH and CD133 together to form tumor xenografts,ALDHþ/CD133þ cells showed an increased ability to generatetumor xenografts compared with ALDHþ/CD133� or ALDHþ

alone (16). Taken together, these data suggest that ALDH is abetter marker than CD133 for colorectal cancer stem cells.However, using both ALDH and CD133 seem to be better thanto enrich the cancer stemcell populationusingALDHorCD133alone.This study extends thatwork by using bothALDHandCD133

together as markers for cancer-initiating cells or colorectalstem cells and examines the STAT3 phosphorylation and inter-leukin 6 (IL)-6 expression in these cancer-initiating cells. Ourresults showed that colorectal cancer–initiating cells, charac-terized by ALDHþ/CD133þ subpopulation of colorectal cancercells expressing higher levels of STAT3 phosphorylationand IL-6, compared with unseparated and ALDH�/CD133�

subpopulations. These results suggest that STAT3 is a noveltherapeutic target in colorectal cancer–initiating cells.

Materials and Methods

Colorectal cancer cell linesHuman colorectal cancer cell lines (SW480, HCT116, DLD-1,

and HT29) were purchased from the American Type CultureCollection (ATCC) and maintained in Dulbecco's ModifiedEagleMedium supplementedwith 10%FBS (Invitrogen). Thesecancer cell lines have been routinely tested and authenticatedby the ATCC and Asterand, respectively. The known genotyperelative to adenomatous polyposis coli, beta-catenin, and DNAmismatch repair enzymes (MLH1, MSH2) genes were shown inSupplementary Table S1. ALDHþ/CD133þ cancer-initiatingcells were grown in a serum-free mammary epithelial basalmedium (MEBM; Clonetics division of Cambrex BioScience),supplemented with B27 (Invitrogen), 20 ng/mL EGF (BDBiosciences), 4 mg/mL Gentamycin (Invitrogen), 1 ng/mLHydrocortisone (Sigma-Aldrich), 5 mg/mL Insulin, and 100mmol/L beta-mercaptoethanol (Sigma-Aldrich).

STAT3 inhibitors, LLL12, and StatticThe laboratory of Dr. Pui-Kai Li's at theOhio State University

College of Pharmacy synthesized small-molecule LLL12 thatselectively targets STAT3 (17). Stattic, a previously reportedSTAT3 inhibitor (18), was purchased from Calbiochem.

Isolation of colon cancer–initiating cellsThe population with high ALDH enzymatic activity was

isolated by using the ALDEFLUORKit (StemCell Technologies)as previously described (14). Briefly, cells were trypsinized tosingle cells and subsequently suspended in ALDEFLUOR assaybuffer containing ALDH substrate and then incubated for 40minutes at 37�C. In all experiments, the ALDEFLUOR-stainedcells treated with diethylaminobenzaldehyde, a specific ALDHinhibitor, served as ALDH-negative controls. Anti-humanPE-CD133 antibody were purchased from Miltenyi Biotec.ALDHþ/CD133þ and ALDH�/CD133� subpopulations were

separated from SW480, HCT116, DLD-1, and HT29 coloncancer cells by a fluorescence-activated cell sorting WantageSE (Becton Dickinson) Flow Cytometry. After sorting, ALDHþ/CD133þ cells were cultured in serum-free stem cell medium(MEBM) to maintain cancer stem cell characteristics. ALDH�/CD133� and unseperated cells were cultured in regular medi-um and replaced with stem cell medium (MEBM) for 3 daysbefore harvesting.

Tissue microarray slides, immunohistochemistry, andimmunofluorescence staining

Human colon cancer tissue microarray slides were obtainedfrom the Biochain Institute, Inc. and AccuMax ISU ABXIS Co.containing 109 colon cancer cases. After baking and depar-affinization, the slides were boiled in a pressure cookerfilled with 10 mmol/L sodium citrate (pH 6.0) and then sub-jected to immunohistochemistry or immunofluorescencestaining. Phospho-STAT3 (Tyr705) antibody (1:25; SignalingTechnology) and or ALDH1 (1:100; BD Pharmingen), CD133(1:50; Miltenyi Biotec) were used. Alexa Fluor 488–conjugatedanti-rabbit IgG and Alexa Fluor 594–conjugated anti-mouseIgG (Cell Signaling Technology) and the Histostain-Plus Kits(Invitrogen) were used in immunofluorescence or immuno-histochemistry staining as described by the manufacturer.Immunostained slides were scored under microscope by usingthe criteria of percentage and intensity positive as describedpreviously by Ginestier (14). Significance of correlationbetween phospho-STAT3 and ALDH1 or CD133 was deter-mined, respectively, using 2-sided Pearson x2 test. P < 0.05 wasconsidered as statistical significance.

Cell viability assayColon cancer–initiating cells maintained serum-free MEBM

(3,000 per well in 96-well plates) were incubated with desiredconcentrations of compounds in triplicate at 37�C for 72 hours.MTT viability assay was done according to manufacturer'sprotocol (Roche Diagnostics). The absorbance was read at595 nm.

Western blot analysisCells were lysed in cold radioimmunoprecipitation assay

lysis buffer containing protease inhibitors and subjected toSDS-PAGE. Proteins were transferred on to polyvinylidenedifluoride membrane and were probed with a 1:1,000 dilutionof antibodies (Cell Signaling Technology) against phospho-specific STAT3 (Tyrosine 705; P-STAT3), phospho-indepen-dent STAT3, phospho-specific ERK1/2 (Threonine 202/Tyro-sine 204), cleaved PARP, cleaved caspase-3, cyclin D, survivin,and glyceraldehyde-3-phosphate dehydrogenase (GAPDH).The degree of changes in P-STAT3 was determined usingdensitometry and normalized to GAPDH.

Reverse transcriptase PCRTotal cell RNAwas collected from cells by using RNeasy Kits

(Qiagen). cDNA was generated from 500 ng sample RNA usingOmniscript RT (Qiagen). Two microliter of cDNA was subse-quently used for PCR using Taq PCR Master Mix Kit (Qiagen)according to themanufacturer's instruction. Primer sequences

STAT3 in Colorectal Cancer–Initiating Cells

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and source information can be found in SupplementaryTable S2.

Tumorsphere cultureThe ALDHþ/CD133þ cells was plated as single cells in ultra-

lowattachment 6-well plates (Corning) andplated at a density of25,000 viable cells per well. Cells were grown in a serum-freeMEBMasdescribed above in a humidified incubator (5%CO2) at37�C. At the second day after seeding, the ALDHþ/CD133þ cellswere treated with 2.5 to 5 mmol/L of LLL12 or 5 to 10 mmol/L ofStattic. Tumorspheres were observed undermicroscope 15 dayslater.

IL-6 ELISA assayAfter sorting, ALDHþ/CD133þ and ALDH�/CD133� cells

were cultured in 96-well plates at a density of 12,000 viable cellsper well. Twenty-four hours later, the medium was collectedand the IL-6 concentrations were detected by using theHumanIL-6 ELISA Development Kit (Peprotech) as described by themanufacturer.

Lentiviral infectionsLentivirus short hairpin RNA (shRNA) that specifically tar-

gets human STAT3 (19) and control lentivirus that expressesgreen fluorescent protein (GFP) were provided by Dr. AntonioIavarone at the Columbia University. IL-6 shRNA lentivirus waspurchased from Santa Cruz Biotechnology, Inc. STAT3, IL-6, orcontrol GFP shRNA lentivirus (CTL shRNA) was introducedinto SW480 and HCT116 colon cancer–initiating cells for 48hours, followed by selection with puromycin (0.2 mg/mL) for 72hours. Western blot assay was used to detect the expression ofP-STAT3 and STAT3 in colon cancer–initiating cells. MTT cellviability and reverse transcriptase PCR (RT-PCR) assay wereconducted and in vivo cancer-initiating cell growth wasdetermined.

Mouse xenograft tumor modelAll animal studies were conducted in accordance with the

principles and standard procedures approved by IACUC at theResearch Institute at Nationwide Children's Hospital. Fortumor-initiation study, the ALDHþ/CD133þ or ALDH�/CD133� cells (1 � 102, 1 � 103, or 1 � 104) from SW480,HCT116, DLD-1, and HT29 were mixed with 50% Matrigel(Invitrogen) in a total of 100 mL and were injected subcuta-neously into the right flank area of 4- to 5-week-old femalenonobese diabetic/severe combined immunodeficiency(NOD/SCID) mice, which were purchased from Jackson Lab-oratory. The tumor incidence ALDHþ/CD133þ and ALDH�/CD133� cells are the numbers of tumor detected/numbers ofmice inoculated and were determined 50 days after the inoc-ulation of cells in mice.

For shRNA lentivirus study, after sorting ALDHþ/CD133þ

HCT116, colon cancer–initiating cells (1 � 105) were infectedwith STAT3, IL-6, or GFP shRNA lentivirus (CTL shRNA) for 48hours. After 72 hours of selection with puromycin, cells weremixed with an equal volume of Matrigel and injected subcu-taneously into the flanks area of 4- to 5-week-old female NOD/SCID mice. Tumor growth was determined by measured the

length (L) andwidth (W) of the tumor every week with a caliperand tumor volume was calculated on the basis of the followingformula: volume ¼ (p/6) LW2.

To detect the effects of STAT3 inhibitor LLL12 in vivo,ALDHþ/CD133þ SW480 and HCT116 colon cancer-initiatingcells (1 � 105) were mixed with 50% Matrigel (Invitrogen) in atotal of 100 mL andwere injected subcutaneously into the flankarea of female NOD/SCID mice. After 17 and 19 days, SW480and HCT116 mice were divided into 2 treatment groupsconsisting of 5 and 6 mice per group, respectively: (a) dimethylsulfoxide (DMSO) control vehicle and (b) 5 mg/kg of LLL12(dissolved in 10% DMSO, 18% Cremophor EL and 72% sterile5% dextrose). Tumor growth and body weights of mice weremeasured every other day during 14 days period treatments. Atthe end of treatments, tumors were harvested from euthanizedmice. A portion of tumor tissues was snap-frozen in liquidnitrogen and stored in �80�C to examine the expression ofSTAT3 phosphorylation by Western blot. The rest of tumorstissues were dissociated mechanically and enzymatically toobtain a single-cell suspension. The single-cell suspension wasused for ALDEFLUOR and CD133-PE staining and followedflow cytometry assay as previously described (20).

Results

LLL12 is a potent agent to inhibit the STAT3phosphorylation in colorectal cancer cells

STAT3 is frequently activated in many types of human solidand blood cancers and contributes to progression of thosecancers (21, 22). The STAT3 pathway is also frequently con-stitutively activated in colorectal cancer and is consideredto play an important role in colorectal cancer carcinogenesis(1–6). To confirm the important role of STAT3 in colon cancercells, the novel STAT3 inhibitor LLL12 (17) was used to targetSTAT3 in 3 independent colon cancer cell lines using phospho-specific STAT3 antibody. Our results showed that LLL12significantly inhibited STAT3 phosphorylation at tyrosine res-idue 705 (Y705, P-STAT3) in SW480, HCT116, and DLD-1human colon cancer cell lines (Supplementary Fig. S1). Phos-phorylation at Y705 is important to activate STAT3 (23–25).The inhibition of P-STAT3 by LLL12 is consistent with thedecrease of STAT3 downstream target genes and the inductionof apoptosis, as evidenced by the cleavages of caspase-3(Supplementary Fig. S1).

ALDHþ/CD133þ cells exhibit more potent tumor-initiating ability than ALDH�/CD133� cells in mousetumor xenografts

It has been shown that ALDHþ and CD133þ subpopulationsin colorectal cancer cells exhibit colorectal cancer–initiatingcells properties in vitro and in vivo (16). To verify whetherALDHþ/CD133þ cells contain tumor-initiating ability, we sep-arated ALDHþ/CD133þ and ALDH�/CD133� subpopulationsfrom SW480, HCT116, DLD-1, and HT29 colorectal cancer cells(Supplementary Fig. S2A). The percentages of ALDHþ/CD133þ

in the 4 colon cancer cell lines were shown in SupplementaryFig. S2B. Our results showed that although 5 to 8 of 7 to 8 miceof 102 to 104 ALDHþ/CD133þ cells injected formed tumors,

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only 1 to 2 of 7 to 8 mice of 103 ALDH�/CD133� cells formtumor, and none of the 102 ALDH�/CD133� cells injectedformed tumor (Table 1). The volume of tumor formed byALDHþ/CD133þ are also larger than tumors formed byALDH�/CD133� cells (Supplementary Fig. S3). Taking togeth-er, these results suggest that using both ALDHþ and CD133þ

markers can enrich colorectal cancer–initiating cells. Theseresults increase our confidence to isolate colorectal cancer–initiating cells using both markers to examine the STAT3phosphorylation.

The ALDHþ/CD133þ subpopulation of colorectal cancercells express higher levels of STAT3 phosphorylationcompared with the ALDH�/CD133� subpopulationThe STAT3 phosphorylation of ALDHþ/CD133þ and

ALDH�/CD133� subpopulations in SW480, HCT116, DLD-1,and HT29 colorectal cancer cell lines were examined. Inter-estingly, our results showed that the ALDHþ/CD133þ subpop-ulation of SW480, HCT116, DLD-1 (Fig. 1A), and HT29 (Sup-plementary Fig. S4A) colorectal cancer cells expressed higherlevels of P-STAT3 compared with unseparated cells and theALDH�/CD133� subpopulation. ERK1/2 phosphorylation(Threonine 202/Tyrosine 204) was not at consistently highlevels in theALDHþ/CD133þ cells in all 4 colon cancer cell lines(Fig. 1A; Supplementary Fig. S4A). These results suggested thatSTAT3 pathway seems to serve a more important purpose incolorectal cancer–initiating cells, but ERK may not play a keyrole in colorectal cancer–initiating cells, at least in these 4colorectal cancer cell lines. These results show that colorectalcancer–initiating cells in the ALDHþ/CD133þ subpopulationexpresses phosphorylated or activated STAT3.To further investigate the STAT3 activation in clinical colon

cancer samples, P-STAT3 and ALDH1, CD133 protein expres-sion in human colon cancer tissues were also examined usingtissue microarray slides. We observed that there was a signif-icant association (P < 0.01) between staining of P-STAT3 andstaining of ALDH1 (Table 2). In addition, the tumor samplesexpressed elevated levels of phosphorylated STAT3 also asso-ciated with CD133 (P < 0.01; Table 2). In the 109 cases, therewere 32 samples (29.36%) in which P-STAT3 (Y705), ALDH1,and CD133 were all positive, and 38 samples (34.86%) withnegative P-STAT3 (Y705), ALDH1, and CD133. Taken together,

there were 64.22% samples which had the similar expression,muchmore than the samples with different expression of these3 proteins (P < 0.05; Table 2). So there was a significantassociation between P-STAT3, ALDH1 and CD133. The repre-sentative examples of immunohistochemistry/immunofluo-rescence staining of P-STAT3 and ALDH1/CD133 are shownin Fig. 1B and C. These results from human colon cancer tissuefurther showed that the elevated levels of P-STAT3 is expressedin colon cancer–initiating cells. These results indicated thatconstitutive STAT3 signalingmay be a novel therapeutic targetin colorectal cancer–initiating cells.

STAT3 inhibitors, LLL12 and Stattic, inhibited STAT3phosphorylation, and STAT3 downstream targets inALDHþ/CD133þ cells

To confirm the important role of STAT3 in colon cancer–initiating cells, we next examined the effect of STAT3 inhibi-tors, LLL12 and Stattic in SW480, HCT116, DLD-1, and HT29colorectal cancer–initiating cells. The results showed thatLLL12 and Stattic inhibited P-STAT3 (Y705) but not ERK1/2phosphorylation (Fig. 2A and B and Supplementary Fig. S4B) inALDHþ/CD133þ subpopulation of colorectal cancer cell lines.In Fig. 2B, Stattic seems to decrease STAT3 expression, whichcould explain, in part, the observed decrease in level ofP-STAT3. In addition, STAT3 shRNA also inhibited P-STAT3in ALDHþ/CD133þ subpopulation of colorectal cancer celllines compared with shRNA control (shRNA CTL; Fig. 2C).The inhibition of STAT3 by LLL12 also downregulates theexpression of many known or putative STAT3-regulatedgenes in colorectal cancer–initiating cells such as cyclinD1 (26), survivin (27), Bcl-2, Bcl-XL (26), Notch1, and Notch3(28, 29; Fig. 2D-a, Supplementary Fig. S4C). These genes arerelated to cancer cell proliferation, survival, and angiogen-esis. Moreover, the Notch pathway was reported to beinvolved in self-renewal of human cancer stem cells andtumorigenicity (28, 30). These results indicate that the LLL12is also potent in terms of inhibiting P-STAT3, downregulat-ing STAT3 downstream genes, and induces apoptosis inthese colorectal cancer–initiating cells. Furthermore, theexpression of STAT3 downstream genes, such as cyclinD1, survivin, Bcl-XL, Notch1, and Notch3 were also reducedby STAT3 shRNA (Fig. 2D-b).

Table 1. The tumor-initiating ability (tumor incidence: the numbers of tumor detected/numbers of miceinoculated) of ALDHþ/CD133þ and ALDH�/CD133� cells in NOD/SCID mice for 50 days

Cell numbers inoculated 1 � 104 1 � 103 1 � 102

HCT116 ALDHþ/CD133þ 7/7 6/7 5/7ALDH�/CD133� 3/7 2/7 0/7

SW480 ALDHþ/CD133þ 7/7 6/7 4/7ALDH�/CD133� 5/7 2/7 0/7

HT29 ALDHþ/CD133þ 8/8 8/8 5/8ALDH�/CD133� 6/8 2/8 0/8

DLD-1 ALDHþ/CD133þ 8/8 7/7 5/7ALDH�/CD133� 5/8 1/7 0/7






LLL12 and Stattic inhibit cell viability and tumorsphere-forming capacity of ALDHþ/CD133þ subpopulation ofcolorectal cancer cells

We next examined the inhibitory effects of LLL12, Stattic,and STAT3 shRNA on cell viability in colorectal cancer–initi-ating cells. Our results showed that LLL12, Stattic, and STAT3shRNA could inhibit cell viability of the ALDHþ/CD133þ cellsfrom SW480, HCT116, DLD-1 (Fig. 3A–C), and HT29 (Supple-mentary Fig. S4D) colorectal cancer cells, further supporting

the idea that colorectal cancer–initiating cells are sensitive tothe inhibition of STAT3. LLL12 and Stattic also inhibited thecell viability of ALDH�/CD133� subpopulation (Supplemen-tary Fig. S5A and B) and the bulk tumor cells (SupplementaryFig. 5C and D). These results may be explained by at least 2possibilities: Some (HCT116) or low (SW480 and DLD-1) levelsof P-STAT3 are expressed in ALDH�/CD133� cells; Stattic andLLL12 may also inhibit both P-STAT3 (Tyr705) and unpho-sphorylated STAT3 or nonnuclear function of STAT3 (31). The

A

B

C

Unseparated

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(T705)

Fold

changes

0.699

0.819

0.026

1.150

0.363

1.056

0.594

0.922

0.207

0.954

0.044

1.091

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STAT3

1

1

1

1

1

1

STAT3

GAPDH

P-ERK1/2

(T202/Y204)

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b

c

ALDH1 CD133 Negative control

HCT116 DLD-1

ALDH+/CD133+

ALDH–/CD133–

UnseparatedALDH+/CD133+

ALDH–/CD133–

UnseparatedALDH+/CD133+

ALDH–/CD133–

Figure 1. STAT3 phosphorylation ofALDHþ/CD133þ subpopulation ofcolon cancer cells is higherthan unseparated and theALDH�/CD133� subpopulations.A, ALDHþ/CD133þ andALDH�/CD133� subpopulationswere separated from SW480,HCT116, and DLD-1 colon cancercells by flow cytometry.Phosphorylation of STAT3 (Y705),ERK 1/2 (T202/Y204), phospho-independent STAT3, and GAPDHwere detected by Western blot. B,representative examples of theexpression of P-STAT3, ALDH1,and CD133 were shown byimmunohistochemistry (IHC) usingcolon cancer tissue microarrayslides. Negative/weak staining ofP-STAT3 (Y705)/ALDH1/CD133 (a)and positive staining of P-STAT3(Y705)/ALDH1/CD133 (b and c)tumor tissues were shown. Thespots for P-STAT3 (Y705), ALDH1and CD133 were from the matchedtissues section from the samepatient. The negative controls arestained with no antibody. C,coloncancer tissue microarray slideswere double-stained with P-STAT3(Y705) and ALDH1 usingimmunofluorescence (IF). ALDH1high expression tumor cells(cytoplasm, green) also expressedphosphylated-STAT3 in nuclei (red).Scale bar: 10 mm.

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inhibition of unphosphorylated STAT3 or nonnuclear functionof STAT3 in ALDH�/CD133� cells might contribute to thereduction of cell viability.Furthermore, we examined the efficacy of LLL12 and Stattic

in inhibiting colorectal cancer–initiating cells to survive andproliferate in anchorage-independent conditions and ability toform tumorspheres. Our results showed that LLL12 and Statticcan inhibit tumorsphere-forming capacity in the ALDHþ/CD133þ cells of SW480, HCT116, DLD-1 (Fig. 3D), and HT29(Supplementary Fig. S4E) colorectal cancer cells. The effects ofLLL12 aremore potent than Stattic. These results indicate thatLLL12 is an excellent drug candidate for targeting colorectalcancer–initiating cells for inhibiting phosphorylated or acti-vated STAT3 in human colorectal cell lines.

STAT3 inhibitors, LLL12 and Stattic, depleted ALDHþ/CD133þ subpopulation and the expression of ALDH1,CD133 in colon cancer cellsColon cancer stem cells are resistant to current chemother-

apy and radiation regimens available (8). To examine whetherSTAT3 inhibition might eliminate the ALDHþ/CD133þ sub-population, we treated cancer cells with 5 mmol/L of LLL12, 10mmol/L of Stattic, 10 mmol/L of doxorubicin, and 10 mmol/L of5-Fu for 24 hours, and sorted for the percentage of ALDHþ/CD133þ subpopulation by flow cytometry. Our results showedthat LLL12 could decrease the ALDHþ/CD133þ subpopulationin HCT116 and SW480 colon cancer cells (Fig. 4A; Supplemen-tary Fig. S6A), suggesting that this subpopulation of coloncancer–initiating cells is sensitive to STAT3-mediated inhibi-tion. We found that 10 mmol/L of Stattic also decreased thepercentage of ALDHþ/CD133þ subpopulation (Fig. 4A; Sup-plementary Fig. S6A). However, 10 mmol/L of doxorubicin or5-Fu increased the percentage of ALDHþ/CD133þ colorectalcancer–initiating cells (P < 0.05; Fig. 4A), which might indicatethat colon cancer–initiating cells are resistant to chemother-apy. We also detected the effects of Dox or 5-Fu (10 mmol/L) inSTAT3 activation in the bulk tumor cell population (Supple-mentary Fig. S6B). The results showed different responses toboth drugs onP-STAT3 in 2 independent colon cancer cell lines(SW480 and HCT116).To further investigate the role of IL-6/STAT3 pathway in

ALDHþ/CD133þ colorectal cancer–initiating cells, ALDHþ/CD133þ andALDH�/CD133� colon cancer cells were collected

after sorting. ELISA assay showed that ALDHþ/CD133þ cellssecreted higher levels of IL-6 than ALDH�/CD133� cells (Fig.4B). Interestingly, the expression of IL-6, IL-6R, GP130, and IL-8were higher in ALDHþ/CD133þ cells than ALDH�/CD133�

cells (Fig. 4C-a) as detected by RT-PCR assay. We also exam-ined the ALDH1 and CD133 expression after the LLL12 treat-ment. We found that the expression of ALDH1 and CD133 waslower after treatment with LLL12 (Fig. 4C-b). In addition,LLL12 inhibited the expression of IL-6, GP130, and IL-8 (Fig.4C-b). However, the expression of IL-6R was not changedconsistently in all 4 cell lines. Our data also observed thatIL-6 (25–50 ng/mL) induced the expression of IL-8 in SW480and HCT116 colon cancer cells, which could be blocked byLLL12 (Supplementary Fig. S6C). The results confirm that theIL-6/STAT3 pathway plays a central role in the maintenance ofthe ALDHþ/CD133þ subpopulation in colon cancer cells.

IL-6 shRNA decreased STAT3 phosphorylation ofcolorectal cancer–initiating cells in vitro and inhibitedcancer-initiating cell growth in vivo

To determine whether the increased levels of phosphory-lated or activated STAT3 in ALDHþ/CD133þ cells is dependenton upstream ligand, IL6, we treated ALDHþ/CD133þ cells withlentiviral IL-6 shRNA versus control lentivirus without encod-ing IL-6 shRNA. Our results in Fig. 4D-a shows that lentiviral IL-6 shRNA, but not control lentivirus, downregulated the phos-phorylated STAT3. These results provide evidence that elevat-ed levels of phosphorylated STAT3 in ALDHþ/CD133þ cells isIL-6–dependent and the inhibition of IL-6 downregulatesphosphorylated STAT3.

To further show the tumor dependence on STAT3 and itsupstream activation (IL-6), we used shRNA that specificallyknock down STAT3 and its upstream signaling protein, IL-6.Our results in Fig. 4D-b shows that STAT3 and IL-6 shRNAsignificantly suppressed colon cancer stem cell tumor growthcompared with lentivirus GFP (as a control). These datasupported tumor dependence on STAT3 and its upstreamactivation by IL-6.

LLL12 suppresses tumor growth of colorectal cancer–initiating cells in mouse tumor model in vivo

We have shown that LLL12 inhibited P-STAT3, cell viability,and tumorsphere growth in colorectal cancer–initiating cells

Table 2. The association of P-STAT3 (Y705) with the expression of ALDH1 and CD133 in colon carcinoma

Coloncarcinoma

Total numbersof cancer tissue Positive, n (%) Negative, n (%)

Association with P-STAT3 x2 Pa

Both positive, n (%) Both negative, n (%)

P-STAT3 109 60 (55.05) 49 (44.95)ALDH1 109 54 (48.54) 55 (50.45) 45 (41.28) 40 (36.70) 34.606 <0.01CD133 109 45 (41.28) 64 (59.6) 40 (36.70) 44 (40.37) 35.473 <0.01ALDH1/CD133 109 35 (32.11) 45 (41.28) 32 (29.36) 38 (34.86) 10.771 <0.05aThe correlation of P-STAT3 (Y705) with ALDH1or/and CD133 (both positive or negative) was assessed by x2 test. P < 0.05 isconsidered as statistically significant. Colon cancer tissues from a total numbers of 109 cancer patients were examined.






expressing elevated levels of STAT3 phosphorylation in vitro.We further tested LLL12 against colorectal cancer–initiatingcells isolated from the SW480 andHCT116 colon cancer cells inNOD/SCID mice xenograft model in vivo. The results showedthat LLL12 significantly suppresses (P< 0.01) the tumor growth(Fig. 5A-a) and tumor mass (Fig. 5A-b). The average reductionin tumor weight is 49.67% to 61.89% in LLL12-treated mice

compared with DMSO vehicle in xenograft mouse model(Fig. 5B). LLL12 also inhibited STAT3 but not ERK1/2 phos-phorylation of SW480 and HCT116 colon cancer–initiatingcells (Fig. 5C). We also used flow cytometry to determine thepercentage of ALDH1þ/CD133þ subpopulation in the tumorstreated with vehicle or LLL12. Our results in Fig. 5D-a, bshowed that LLL12 reduced the percentage of ALDHþ/CD133þ

A

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(T202/Y204)

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(T202/Y204)

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changes

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changes

0.554

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DMSO5 µmol/L

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LLL12 DMSO5 µmol/L

LLL12

Stattic Stattic

10 µmol/L 20 µmol/L

5 µmol/L 5 µmol/LDMSOLLL12 LLL12

Figure 2. STAT3 inhibitors LLL12,Stattic, and STAT3 shRNA inhibitedSTAT3 phosphorylation and STAT3downstream genes expression inALDHþ/CD133þ colon cancer–initiating cells. The ALDHþ/CD133þ

subpopulation was separated fromSW480, HCT116, and DLD-1 coloncancer cells by flow cytometry, andcultured in serum-free stem cellmedium (MEBM) tomaintain cancerstem cell characteristics. A and B,ALDHþ/CD133þ colon cancer–initiating cells were treated withDMSO, LLL12 (5 mmol/L) or Stattic(10–20mmol/L) for 24 to 48 hours. C,STAT3 or control GFP shRNAlentivirus (CTL shRNA) wasintroduced into colon cancer–initiating cells for 48 hours, followedby selection with puromycin for 72hours. Phosphorylation of STAT3and ERK1/2, STAT3 protein, andGAPDH in colon cancer–initiatingcells were detected byWestern blotas described in Materials andMethods. D, ALDHþ/CD133þ

subpopulation of SW480, HCT116,and DLD-1 colon cancer cellswere treated with DMSO, LLL12(5 mmol/L), CTL, or STAT3 shRNA.Reverse-transcriptase PCR revealsdecreased expression of STAT3downstream target genes in LLL12or STAT3 shRNA treated cells ascompared with control.

Lin et al.





subpopulation in tumor. In addition, the body weights of micehave no obvious reduction in both DMSO vehicle- and LLL12-treated mice (Supplementary Fig. S7). These results showedthat LLL12 is potent in suppressing tumor growth from thecolorectal cancer–initiating cells in vivo.

Discussion

STAT3 is frequently activated in many types of humansolid and blood cancers, including colon cancer (1–3, 22).

Blocking signaling to STAT3 inhibits cancer cell growth,showing that STAT3 is crucial to the survival and growth oftumor cells (21, 22, 32) and is an attractive therapeutic targetfor cancer. At the present time, the main effort to targetconstitutive STAT3 signaling is only focused on the bulk ofcancer cells. No published literatures have been reportedabout whether STAT3 is activated in colon cancer–initiatingcells, and no approach has been initiated to explore theSTAT3 as a possible therapeutic target in colon cancer–initiating cells. In this article, we took a pilot study to explore

Figure 3. LLL12 (A), Stattic (B), andSTAT3 shRNA (C) inhibited cellviability of SW480, HCT116, andDLD-1 colon cancer–initiating cells.The ALDHþ/CD133þ subpo-pulation of colon cancer cells wasseeded in 96-well plates (3,000cellsper well) in triplicates in a serum-free MEBM. The following day,cancer-initiating cells were treatedwith LLL12, Stattic, CTL, or STAT3shRNA. MTT assay was used todetermine cell viability. D, Statticand LLL12 inhibited tumorsphereformation of ALDHþ/CD133þ

subpopulation of SW480, HCT116,and DLD-1 colon cancer cells. TheALDHþ/CD133þ cancer-initiatingcells were plated as single cells andtreated with Stattic (5–20 mmol/L) orLLL12 (2.5 and 5 mmol/L) at thesecond day. Tumorspheres wereobserved under microscope 15days posttreatments.

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CTL STAT3 shRNA CTL STAT3 shRNA CTL STAT3 shRNA

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the STAT3 in colon cancer–initiating cells characterizedby ALDHþ/CD133þ subpopulation. Our study confirmedthat ALDHþ/CD133þ colon cancer cells exhibited morepotent tumor-initiating ability than ALDH�/CD133� cells

in mouse tumor xenografts. We showed that elevated P-STAT3 is detected in colon cancer–initiating cells cell linesand in human colon cancer tissues derived from cancerpatients. These results suggest that activated STAT3 is

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shRN

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STAT3

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P-ERK1/2

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HCT116ALDH+/CD133+

HT29ALDH+/CD133+

Figure 4. A, LLL12 (5 mmol/L) and Stattic (10 mmol/L) decreased the percentage of ALDHþ/CD133þ subpopulation. However, 10 mmol/L doxorubicin or 5-Fuincreased the percentage of ALDHþ/CD133þ colorectal cancer–initiating cells (�, P < 0.05). B, ELISA assay showed that ALDHþ/CD133þ subpopulationsecreted more IL-6 than ALDH�/CD133� subpopulation of colon cancer cells. C, the expression of ALDH1, CD133, IL-6, IL-6R, GP130, IL-8 wasdetectedbyRT-PCR. (a) ALDHþ/CD133þ andALDH�/CD133� colon cancer cells were collected after sorting. The expression of IL-6, IL-6R,GP130, IL-8werehigher in ALDHþ/CD133þ cells than ALDH�/CD133� cells; (b) the expression of ALDH1, CD133, IL-6, GP130, and IL-8 were lower after treated with LLL12 inALDHþ/CD133þ colon cancers. D, after sorting, ALDHþ/CD133þ HCT116 and/or SW480 colon cancer–initiating cells were infected with CTL, IL-6,and/or STAT3 shRNA. (a) IL-6 shRNA decreased STAT3 phosphorylation of SW480 and HCT116 colorectal cancer–initiating cells in vitro; (b) IL-6 andSTAT3 shRNA inhibited ALDHþ/CD133þ HCT116 cancer-initiating cell growth in vivo.

Lin et al.





indeed a novel therapeutic target in colon cancer–initiatingcells.To explore the role thatSTAT3 plays in colon cancer–

initiating cells, we examined the effects of STAT3 inhibition.Two small molecular STAT3 inhibitors, LLL12 (17) andStattic (18) were employed. LLL12 is a novel and morepotent derivative of LLL3, a previously reported STAT3inhibitor from our laboratories (33, 34). Our results showedthat LLL12 was potent inhibitor to inhibit P-STAT3; STAT3

downstream targets expression and induces apoptosis innonseparated colon cancer cells. Importantly, STAT3 inhi-bitors, LLL12 and Stattic inhibited P-STAT3, STAT3 down-stream gene expression, cell viability, and the formation oftumorspheres in ALDHþ/CD133þ subpopulation of coloncancer–initiating cells. In addition, STAT3 shRNA was usedto inhibit the STAT3 expression and activity. Our resultsshowed that STAT3 shRNA also inhibited STAT3 phosphor-ylation, cell viability, and STAT3 downstream genes

Figure 5. LLL12 suppressed tumorgrowth in mouse xenografts withSW480 or HCT116 colon cancerstem cells. The mice were givendaily intraperitoneal dosages of 5mg/kg LLL12 or DMSO vehicle.Tumor volume (A-a), tumor mass(A-b), and tumor weight (B) werereduced in all LLL12-treated micecompared with DMSO vehiclegroup. C, one representativesample from tumor tissuesgenerated from SW480 or HCT116colon cancer–initiating cellsshowing STAT3 but not ERK1/2phosphorylation were also inhibitedby LLL12 treatment. D, at the end oftreatments, tumors tissues weredissociated to obtain a single-cellsuspension and analyzed by flowcytometry. LLL12 reduced thepercentage of ALDHþ/CD133þ

subpopulation in tumor.

A a

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expression in ALDHþ/CD133þ subpopulation of colon can-cer–initiating cells.

We compared the expression of IL-6/STAT3 signal path-way, such as IL-6, IL-6R, GP130, and IL-8 between ALDHþ/CD133þ and ALDH�/CD133� subpopulations. IL-6 has beenshown to activate STAT3 (35) and may play a role in coloncancer oncogenesis (36–38). Interestingly, our results showedthat ALDHþ/CD133þ cells expressed higher levels of IL-6,GP130, and IL-8 and secreted higher levels of IL-6 than thosein ALDH�/CD133� cells. In addition, introduction of the IL-6shRNA in ALDHþ/CD133þ cells downregulated the expres-sion of STAT3 phosphorylation. These results provide evi-dence that STAT3 activation in ALDHþ/CD133þ cells is IL-6dependent. The expression of IL-6 and IL-8 could be reducedby STAT3 inhibitor, LLL12. It was speculated that STAT3mayregulate the expression of IL-6 (28). Our data showed that IL-6is downregulated by STAT3 inhibitor, LLL12, supporting thatIL-6 may be regulated by STAT3. Furthermore, it has beenreported that activated STAT3 could selectively bind to IL-8promoter and induce IL-8 transcription (39). Our datashowed that IL-6 induced the expression of IL-8, which couldbe blocked by LLL12. These results suggest that IL-8 may be adownstream target of IL-6/STAT3 in colon cancer cells.Recent studies have suggested a role for interleukins, suchas IL-6 and IL-8, in breast cancer stem cells (15), which implythat inflammatory microenvironment is important in pro-moting the oncogenesis. Ginestier and colleagues reportedthat blockade of the IL-8 receptor CXCR1 selectively depleteshuman breast cancer stem cells (40). Our data suggested thatIL-6/STAT3/IL-8 activation in colon cancer–initiating cellsmight play an important role in the development of coloncancer.

We found that the expression of ALDH1 and CD133 wasreduced after treatment with LLL12, and this may be due tothe inhibition of their expression. It may also be an effect ofLLL12 on cellular heterogeneity, whereby it decreases theproportion of ALDHþ/CD133þ cells in the tumor cell pop-ulation which was shown by our in vitro and in vivo data. Inaddition, our data showed that STAT3 inhibitors, but notother cancer therapeutic drugs such as doxorubicin and 5-Fu, eliminated ALDHþ/CD133þ subpopulation of colon can-cer–initiating cells in colon cancer cell lines. These resultssuggested that colon cancer–initiating cells, which are moreresistant to conventional drugs, might be sensitive to STAT3inhibitors.

Furthermore, our results showed that STAT3, IL-6 shRNAand LLL12 exhibited suppressive activity on the tumor growthof human colon cancer–initiating cells derived frombulk colon

cancer cells. These results suggest that constitutive activeIL-6/STAT3 in these cancer-initiating cells enhanced prolifer-ation and survival, as well as tumor growth in mice, whereasSTAT3 blockade by STAT3, IL-6 shRNA and LLL12, suppressedtumor-initiating cell growth in vivo.

In summary, this study is the first report to show thatIL-6/STAT3 is activated in colon cancer–initiating cells.Targeting IL-6/STAT3 may be able to eliminate the coloncancer–initiating cells and provides a promising approach totreat advanced colon cancer. Our study also showed thatLLL12 is a potent STAT3 inhibitor for cancer-initiating cellsand is a promising drug candidate to target constitutiveSTAT3 signaling in colon cancer–initiating cells. Mostrecently, 2 literatures reported that IL-6/STAT3 pathwaymay be activated in glioblastoma stem cells (41, 42). Inaddition, targeting STAT3 by 2 small molecules, STA-21 andS31-201 or IL-6 shRNAs, respectively, can inhibit cell viabilityof these glioblastoma stem cells (41, 42). Furthermore, wealso observed that high levels of STAT3 phosphorylation aredetected in breast cancer–initiating cells compared withunseparated and nonbreast cancer–initiating cells (data notshown). These results are consistent with our observation incolon cancer–initiating cells that activated STAT3 seems toplay an important role in cancer-initiating cells. It is also ofinterest to investigate whether STAT3 is also activated incancer-initiating cells in other types of human cancer. IfSTAT3 is constitutively activated in other types of cancer-initiating cells or cancer stem cells, it may open a newtherapeutic opportunity to target STAT3 in cancer-initiatingcells of those cancer types.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

The authors thank Cynthia McAllister and Dave Dunaway at the FlowCytometry Core of Nationwide Children's Hospital.

Grant Support

This research was partly supported by start-up fund from the Department ofPediatrics to J. Lin and supported by National Natural Science Foundation ofChina (81001005) to L. Lin.

The costs of publication of this article were defrayed in part 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 December 29, 2010; revised July 15, 2011; accepted August 19, 2011;published OnlineFirst September 7, 2011.

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2011;71:7226-7237. Published OnlineFirst September 7, 2011.Cancer Res Li Lin, Aiguo Liu, Zhengang Peng, et al. Initiating Cells

−STAT3 Is Necessary for Proliferation and Survival in Colon Cancer

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