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Tumor and Stem Cell Biology

LIN28B Promotes Colon Cancer Progression andMetastasis

Catrina E. King1,4, Miriam Cuatrecasas5, Antoni Castells6, Antonia R. Sepulveda2, Ju-Seog Lee7, and Anil K. Rustgi1,3,4

AbstractLIN28B is a homologue of LIN28 that induces pluripotency when expressed in conjunction with OCT4, SOX2,

and KLF4 in somatic fibroblasts. LIN28B represses biogenesis of let-7 microRNAs and is implicated in bothdevelopment and tumorigenesis. Recently, we have determined that LIN28B overexpression occurs in colontumors. We conducted a comprehensive analysis of LIN28B protein expression in human colon adenocarci-nomas. We found that LIN28B overexpression correlates with reduced patient survival and increased probabilityof tumor recurrence. To elucidate tumorigenic functions of LIN28B, we constitutively expressed LIN28B in coloncancer cells and evaluated tumor formation in vivo. Tumors with constitutive LIN28B expression exhibitincreased expression of colonic stem cell markers LGR5 and PROM1, mucinous differentiation, and metastasis.Together, our findings point to a function for LIN28B in promoting colon tumor pathogenesis, especiallymetastasis. Cancer Res; 71(12); 4260–8. �2011 AACR.

Introduction

LIN28B is a homologue of LIN28 (1), a heterochronic geneinitially described in Caenorhabditis elegans as a regulator ofdevelopmental timing (2–5). It induces pluripotency whenexpressed in somatic fibroblasts in conjunction with OCT4,SOX2, and KLF4 (6). Both LIN28 and LIN28B contain a coldshock domain and retroviral-type (CCHC) zinc fingers thatconfer RNA-binding ability (2, 7) and inhibit biogenesis oftumor-suppressive microRNAs of the let-7 family (8–11).

LIN28 and LIN28B are implicated in multiple developmentalprocesses, largely as a consequence of their ability to represslet-7 biogenesis. In C. elegans, let-7 is critical to the larval fatetransition and vulval specification (12); LIN28 mutants reiter-ate larval fates (2). In human embryonic stem cells, relief oflet-7 suppression via LIN28 downregulation occurs duringdifferentiation (13). During neuronal differentiation, LIN28is also specifically downregulated and constitutive expressionblocks gliogenesis in favor of neurogenesis (14). LIN28Bmuta-tions are associated with delayed onset of puberty in humans(15), which can be modeled via constitutive expression of

LIN28 in mice (16). Induction of LIN28 overexpression intransgenic mice expands transit-amplifying cells in the intest-inal crypt compartment causing gut pathology and death (16),thus suggesting potential roles for LIN28 and LIN28B inintestinal and colonic development and disease.

LIN28 and LIN28B are implicated in tumorigenesis indifferent cancers, but LIN28B overexpression in human can-cers has been shown more frequently (17). For example,LIN28B is overexpressed in hepatocellular carcinomas (1).

In addition, an analysis of LIN28 and LIN28B expression invarious cancers pointed to LIN28B as perhaps the morerelevant homologue in tumorigenesis (17). This study impli-cates a role for LIN28B in Wilms’ tumors, where the c-mycbinding site on LIN28B promoter is frequently demethylated,and chronic myeloid leukemia, where expression of LIN28Boccurs more commonly in blast crisis. Moreover, LIN28Boverexpression occurs in non–small-cell lung cancer, breastcancer, melanoma, whereas aberrations in LIN28 expressionwere not as readily identifiable (17).

LIN28B may be upregulated as a consequence of adenoma-tous polyposis coli (APC) mutation, which occurs in the vastmajority of colon tumors, resulting in sustained b-cateninstabilization and upregulation of the Wnt pathway targets (18,19). The oncogenic transcription factor c-myc is an estab-lished target of the Wnt pathway that functions in part viatranscriptional activation of LIN28B (18, 20). Accordingly,increased LIN28B expression in colon tumors may occur asa consequence of elevated c-myc levels and Wnt pathwayderegulation (2, 18, 20). Yet, the role of LIN28B in tumorigen-esis in vivo remains to be determined.

We examined LIN28B expression in human colon tumors intissue microarrays and found that its overexpression corre-lates with reduced patient survival and increased probabilityof tumor recurrence. To elucidate further roles of LIN28B incolon tumorigenesis, we constitutively expressed LIN28Bin colon cancer cell lines and subsequently evaluatedtumor-promoting properties of LIN28B-expressing cells in

Authors' Affiliations: 1Gastroenterology Division and Department ofMedicine, Departments of 2Pathology and 3Genetics, and 4AbramsonCancer Center, University of Pennsylvania, Philadelphia, Pennsylvania;5Pathology Department, Hospital Cínic, CDB, University of Barcelona;6Gastroenterology Department, Hospital Clínic, CIBERehd, IDIBAPS, Bar-celona, Catalonia, Spain; and 7Department of Systems Biology and Divi-sion of Cancer Medicine, The University of Texas MD Anderson CancerCenter, Houston, Texas

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

Corresponding Author: Anil K. Rustgi, Gastroenterology Division andDepartment of Medicine, 600 CRB, University of Pennsylvania, 415 CurieBlvd., Philadelphia, PA 19104. Phone: 215-898-0154; Fax: 215-573-5412;E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-10-4637

�2011 American Association for Cancer Research.

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xenografted nude athymic mice. Surprisingly, we observedincreased differentiation with glandular formation in LIN28B-expressing tumors. However, although smaller than vector-only controls, the LIN28B-expressing tumors have a greatertendency to metastasize. In addition, we conducted geneexpression profile analyses of colon cancer cell lines, primaryxenograft tumors, and metastases constitutively expressingLIN28B. We found stem cell–related genes, including thecolonic stem cell markers LGR5 and PROM1, to be upregulatedas a function of constitutive LIN28B expression. Takentogether, our findings support a role for LIN28B in theprogression of colon cancer.

Materials and Methods

Tumor tissue microarray analysisA uniform cohort of 228 (133 men and 95 women) patients

with colon carcinoma (88 in stage 2, and 140 in stage 3)diagnosed between November 1993 and October 2006 wasselected. Rectal tumors were excluded. The age of the patientsranged from 35 to 88 years (mean: 66.4 years). The majority ofthe tumors (156) were located on the left colon; that is, 132sigmoid, 13 splenic flexure, and 11 in the descendent colon,whereas 72 tumors were located on the right colon; that is, 26cecum, 24 ascendent, 14 transverse, and 8 in the hepaticflexure. Seven patients had synchronous tumors. Tumor sizevaried from 1.6 to 14 cm (mean: 3.8 cm). Six tumors were ofgrade 1, 212 of grade 2, and 10 of grade 3. Tissue samples wereobtained from patients under Institutional Review Boardapproval. Two cores of normal mucosa and 2 cores of tumortissue for each patient were paraffin embedded in orderedmicroarrays. Tumor microarrays were sectioned in prepara-tion for immunohistochemical staining. LIN28B stainingintensity was scored by a pathologist: 1, low LIN28B intensity;2, intermediate intensity; and 3, high intensity. Log-rank tests(Mantel–Cox and Breslow) were conducted to compare sur-vival and recurrence distributions of intensity groups (1–2 vs.3). Correlation of staining intensity with survival and recur-rence was determined via c2 analysis; 95% CIs were calculatedfor confirmation of statistical significance.

ImmunohistochemistryParaffin-embedded tissue microarrays and xenograft

tumors were incubated at 60�C prior to dewaxing and rehy-dration. Antigen retrieval was done by placing sections in 10mmol/L citric acid (pH 6) and microwaving for 15 minutes.Endogenous peroxidases were quenched in 15-mL hydrogenperoxide and 185 mL of water. Tissues were washed with PBSprior to treatment with Avidin D and Biotin, using theVectastain ABC Kit (Vector Labs). Samples were washed againwith PBS prior to treatment with Starting Block (ThermoScientific) for 10 minutes. Tissues were incubated with LIN28B(1:200; Cell Signaling) or green fluorescent protein (GFP; 1:100;Abcam) primary antibodies diluted in PBT (10� PBS, 10%bovine serum albumin, 10% Triton X-100 in ddH2O) at 4�Covernight. Samples were washed with PBS on the followingday, incubated in secondary antibody (1:1,000), washed andthen treated with ABC Peroxidase Staining Kit (Pierce) as per

the manufacturer's protocol. For detection, a 3,30-diamino-benzidine (DAB) Peroxidase Detection kit (Vector Labs) wasused and color development was monitored using a lightmicroscope. The development process was terminated byremoving DAB and rinsing the sections with ddH2O for 1minute before counterstaining with hematoxylin.

Generation of constitutive LIN28B-expressing cell linesDLD-1 and LoVo cells were obtained from American Type

Culture Collection (ATCC; a recognized vendor) in 2000 andstored in liquid nitrogen prior to resuscitation in 2009 (celllines were authenticated by ATCC via DNA fingerprinting andcytogenetic analyses). Stable LIN28B expression in DLD-1 andLoVo cells was achieved using MSCV-PIG-LIN28B and emptyvector control plasmids (gifts from Dr. Joshua Mendell). Wetransfected Phoenix A cells with 30 mg plasmid DNA andmonitored transfection efficiency via detection of GFP expres-sion by light microscopy prior to virus collection. Viral con-taining supernatant was collected 48 hours posttransfection,filtered through a 0.45-mm membrane, immediately placed inliquid nitrogen, and stored at �80�C for later use. DLD-1 andLoVo cells were infected by applying virus-containing mediaplus polybrene (4 mg/mL) to cells and then subjecting them tocentrifugation at 1,000 � g for 90 minutes. Inoculated cellswere selected in puromycin, expanded, and subsequentlysorted for high GFP intensity and corresponding LIN28Bexpression.

Xenograft tumorsCrTac:NCr-Foxn1nu nude athymic mice (Taconic) were irra-

diated at 5 Gy 2 to 3 hours prior to injection of cells. Emptyvector and LIN28B-expressing DLD-1 and LoVo cells weretrypsinized and washed in PBS and resuspended at a concen-tration of 2� 107 cells/mL. Fifty microliters of cell suspensionswas mixed with 50 mL of BD Matrigel Basement MembraneMatrix (BDBiosciences) to achieve a volume of 100mL contain-ing 1 � 106 cells per injection. Cells were injected subcuta-neously into the rear flanks ofmice sedatedwith ketamine (100mg/kg) and xylazine (10 mg/kg). Following injection, micewere monitored periodically and sacrificed after 6 weeks. Allmice were cared for in accordance with University LaboratoryAnimal Resources (ULAR) requirements.

Detection of GFP fluorescence by image analysisXenografted nude mice were anesthetized with ketamine

(100 mg/kg) and xylazine (10 mg/kg) and fluorescence inten-sity was measured using a small animal imaging system(Kodak). Net tumor intensity was calculated over a fixedregion of interest that encompasses the dimensions of thelargest tumor. Statistical significance of comparisons betweenempty vector and LIN28B tumors was determined by applyingStudent's t test, where P < 0.05 is statistically significant.

Gene expression analysis in xenograft tumorsMice were euthanized in accordance with ULAR standards

prior to excision of xenograft tumors. Extraneous tissues weredissected away and discarded, tumors were then immediatelyplaced into RNAlater (Qiagen) and maintained on ice until

LIN28B in Colon Cancer Progression and Metastasis

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storage at �80�C. Tumor tissue was homogenized mechani-cally in RNAlater (Qiagen) and homogenized particulates werecollected via centrifugation at 4�C. RNA was isolated fromhomogenized tissues by using a mirVana RNA isolation kit(Ambion). For detection of mature let-7a and let-7b micro-RNAs, TaqMan MicroRNA Assay kits (Applied Biosystems)were employed to synthesize probe-specific cDNA from 10 ngof total RNA per sample. Probe-specific cDNA was amplifiedusing proprietary primers (Applied Biosystems) and TaqManUniversal PCR Master Mix, No AmpErase UNG (AppliedBiosystems). PCR amplifications were conducted in triplicateand normalized to levels of endogenous U47. The statisticalsignificance of comparisons between empty vector DLD-1 andLoVo versus LIN28B-DLD-1 and LIN28B-LoVo cells was eval-uated by applying Student's t test, with P < 0.05 consideredstatistically significant. For detection of mRNA transcripts,cDNA was synthesized from 3 mg isolated RNA per sample,using random oligomers and SuperScript II Reverse Tran-scriptase (Invitrogen). Synthesized cDNA was then subjectedto gene expression analysis via quantitative real-time PCR(qRT-PCR), using the ABI Prism 7000 Sequence DetectionSystem (Applied Biosystems). Transcripts were amplified anddetected using specific probes, with b-actin serving as anendogenous control (Ambion). Fold change for each transcriptwas determined by normalization to empty vector controls.

Microarray analysisRNAwas isolated using a mirVana kit (Ambion) from empty

vector and LIN28B-expressing DLD-1 and LoVo colon cancercell lines, primary xenograft tumors, and LIN28B-expressingmesenteric metastases. Triplicate RNA isolates were sub-mitted to the Penn Microarray Facility. Quality control testsof the total RNA samples were carried out using an Agilentbioanalyzer and a Nanodrop spectrophotometry. One hun-dred nanograms of total RNA was then converted to first-strand cDNA by using reverse transcriptase primed by a poly(T) oligomer that incorporated a synthetic RNA sequence.Second-strand cDNA synthesis was followed by Ribo-SPIA(Single Primer Isothermal Amplification; NuGEN Technolo-gies Inc.) for linear amplification of each transcript. Theresulting cDNA was fragmented, assessed by bioanalyzer,and biotinylated. cDNA yields were added to Affymetrixhybridization cocktails, heated at 99�C for 2 minutes, andhybridized for 16 hours at 45�C to Affymetrix Human Gene 1.0ST Array GeneChips (Affymetrix Inc.). The microarrays werethen washed at low (6� saline sodium phosphate EDTA) andhigh (100 mmol/L MES, 0.1 mol/L NaCl) stringency andstained with streptavidin–phycoerythrin. Fluorescence wasamplified by adding biotinylated anti-streptavidin and anadditional aliquot of streptavidin–phycoerythrin stain. A con-focal scanner was used to collect fluorescence signal afterexcitation at 570 nm. All protocols were conducted asdescribed in the NuGEN Ovation manual and the AffymetrixGeneChip Expression Analysis Technical Manual.

Bioinformatics analysisThree independent biological replicates for each condition

were assayed on microarrays. Unsupervised hierarchical clus-

tering by sample was conducted to confirm that replicateswithin each condition grouped with most similarity and toidentify any outlier samples. Significance Analysis of Micro-arrays (SAM v3.0; www-stat.stanford.edu/�tibs/SAM/) wasused to generate lists of statistically significant differentiallyexpressed genes in pairwise comparisons of replicate averagesbetween conditions. Constitutive LIN28B expression in vitroresulted in 13,156 genes with higher RNA abundance and15,771 genes with lower RNA abundance when compared withempty vector controls. Candidate genes from the in vitroanalysis were filtered by fold change (threshold = 5-fold),and the resulting gene lists were tested for overrepresentation.Constitutive LIN28B expression in vivo resulted in 11,242 geneswith higher RNA abundance in LIN28B metastases than incontrol tumors and 17,579 genes with lower RNA abundancein LIN28Bmetastases than in control tumors. Candidate geneswere further filtered by fold change (threshold = 4-fold), andthe resulting gene lists were tested for overrepresentation ofGene Ontology annotation categories by using the DAVIDBioinformatics Resources (david.abcc.ncifcrf.gov).

To stratify patients with colon cancer according to thesimilarity of their gene expression patterns to LIN28B-specificgene expression signature, we adopted a previously developedmodel (21–23). Briefly, we first identified genes whose expres-sion was significantly associated with expression of LIN28Bfrom cell culture. A highly stringent cutoff (P < 0.001) wasapplied to avoid inclusion of potential false-positive genesduring training of prediction models. Expression data fromselected genes (704 genes) were then combined to form aclassifier according to compound covariate predictor algo-rithm, and the robustness of the classifier was estimated bymisclassification rate determined during the leave-one-outcross-validation (LOOCV) in the training set (data from cellculture). When applied to gene expression data from thepatients (n = 290; GSE14333), prognostic significance wasestimated by Kaplan–Meier plots and log-rank tests between2 predicted subgroups of patients. After LOOCV, sensitivityand specificity of prediction models were estimated by thefraction of samples correctly predicted.

Results

Aberrant LIN28B expression correlates with reducedpatient survival

To determine potential roles of LIN28B in colon cancerpathogenesis, we examined LIN28B expression in humancolon tumors and matched adjacent normal mucosa. Tumorand adjacent normal tissue samples resected from patientsreceiving adjuvant 5-fluorouracil were paraffin-embedded induplicate on to tissue microarrays. Immunohistochemistry(IHC) was done for LIN28B, and staining intensity wasscored by a pathologist blinded to clinical information.LIN28B staining in normal colon is faint; in contrast, intenseLIN28B staining was observed in colon tumors (Fig. 1A–D;Supplementary Fig. S1). Colon tumor samples werefurther analyzed via log-rank tests (Mantel–Cox and Bre-slow) comparing overall survival and recurrence distribu-tions of intensity groups. This revealed a correlation

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between low-intensity LIN28B staining in stage I and II colontumors with higher patient survival (Mantel–Cox, P value =0.046; Breslow, P value = 0.013; Fig. 1E) and lower probabilityof tumor recurrence (Mantel–Cox, P value = 0.036; Breslow,P value = 0.108; Fig. 1F).

LIN28B tumors exhibit reduced size and glandulardifferentiationGiven that high LIN28B levels in colon tumors correlate

with increased probability of tumor recurrence in patients,we hypothesized a biological role for LIN28B in colon cancerpathogenesis. To elucidate this further, we constitutivelyexpressed LIN28B in DLD-1 and LoVo colon cancer cells. Wethen established xenograft tumors by injecting 1 � 106 cellssubcutaneously into the rear flanks of nude athymic mice.We initially monitored tumor development via detection ofGFP fluorescence (expressed from the MSCV-PIG retroviralvector) in a total of 83 xengografted mice: 41 empty vectorcontrols and 42 LIN28B-overexpressing tumors. At 2 and

4 weeks postinjection, we found that LIN28B tumors emittedless fluorescence (Fig. 2A and B). This observation corre-sponds to a smaller tumor mass observed in LIN28B-expres-sing tumors (Fig. 2C and D). We next confirmed LIN28Boverexpression in tumors via IHC (Fig. 3A and B). In addi-tion, mature let-7 microRNAs, which are repressed byLIN28B, are reduced in the presence of constitutive LIN28Bexpression in xenograft tumors (Supplementary Fig. S2).Surprisingly, LIN28B tumors exhibited increased areas ofmoderate differentiation, increased glandular formation,and increased mucin production, in contrast to emptyvectors tumors that are poorly differentiated and rarelyexhibit mucinous, gland-like structures (Fig. 3C–F). Viabletumor areas were scored for poor and moderate differentia-tion, as well as mucin production revealing statisticallysignificant differences between empty vector and LIN28B-expressing xenograft tumors (Fig. 4). However, Ki-67 andcaspase-3 staining did not reveal significant differences(data not shown).

Figure 1. LIN28B overexpressionin colon carcinomas correlateswith reduced survival andincreased probability of tumorrecurrence. A–D, representativeH&E and IHC for LIN28B inmatched normal mucosa andcolon carcinomas in tissuemicroarrays. LIN28B staining isintense in a subset of colontumors. Magnification: 100� inA–D. E, LIN28B expression andpatient survival. High LIN28Bstaining intensity in stage I and IIcolon cancers correlates withreduced patient survival. F,LIN28B and tumor recurrence.High LIN28B staining intensity instage I and II tumors correlateswith increased probability oftumor recurrence.

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LIN28B tumors metastasize to multiple organsDuring necropsy, we observed mesenteric and liver metas-

tases in mice xenografted with LIN28B-expressing cells upongross examination (Fig. 5E), which prompted extensive exam-ination of internal organs. Although we did not observemetastases in any mice in the empty vector control group,we did note liver, lung, perisplenic adipose, lymph node, andmesenteric metastases in mice bearing tumors with consti-tutive LIN28B expression (Fig. 5A and B). We confirmed thatthese metastases were derived from xenografted cells viaimmunohistochemical GFP detection, which is unique toxenografted cells (Fig. 5C and D). Overall, we identifiedmetastases in approximately 17% of mice xenografted withLIN28B-expressing cells (7 of 42). Thus, constitutive LIN28Bexpression in colon cancer cells confers metastatic ability in atleast a subset of tumors (Fisher's exact one-tailed P = 0.0065).

Stem cell–like gene expression profile induced byconstitutive LIN28B expression

We next conducted microarray analysis on total RNA iso-lated from empty vector and LIN28B-expressing cell lines andprimary tumors, as well as LIN28B-LoVo metastases. Weidentified more than 184 transcripts (164 upregulated; 20downregulated) that are more than 5-fold changed with con-stitutive LIN28B expression in vitro (accession numbersGSM646687 and GSM646692). Some transcripts upregulatedby LIN28B, including the hematopoietic stem cell marker KIT(also called CD117 or c-KIT) and the intestinal stem cellmarkers LGR5 (leucine-rich repeat-containing G-protein–coupled receptor 5) and PROM1 (prominin 1), have reportedroles in stem cell biology (Table 1). Of note, LGR5 is a tran-scriptional target of canonical Wnt signaling (24, 25). Addi-tional Wnt targets including DKK1 (Dickkopf homolog 1;

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Figure 3. Histopathologic examination of empty vector and LIN28B-expressing primary tumors. A and B, confirmation of LIN28Boverexpression in tumors. Immunohistochemical detection of LIN28B invector-LoVo and LIN28B-LoVo xenografts; representative of 83 tumorstested (200� magnification). C–F, H&E staining of xenograft tumors.Empty vector tumors, which exhibit broad areas of necrosis (C) and poordifferentiation (E), whereas LIN28B-expressing tumors exhibit fewernecrotic areas (D), moderate differentiation, glandular formation, andincreased mucin production (F). Magnification: 100� in C and D; 200� inE and F.

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Figure 2. LIN28B tumors havereduced size. A, LIN28B tumorsemit less fluorescence thancontrols. GFP intensity ofxenograft tumors was assessed atbiweekly intervals; at eachinterval, fluorescence intensity forLIN28B tumors was less than thatof controls. Representative 2- and4-week analyses of a single cohortinjected with vector-LoVo orLIN28B-LoVo cells. B, LIN28Btumors are smaller than controls.Tumor weights 6 weekspostinjection and extraction.

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refs. 26, 27) and CCND2 (cyclin D2; refs. 28, 29) are upregulatedby LIN28B overexpression as well (Table 1). These results wereverified by qRT-PCR (data not shown).In addition, we identified more than 22 genes that are

more than 4-fold upregulated and 57 genes that are morethan 4-fold downregulated by constitutive LIN28B expres-sion in vivo (accession numbers GSM646700 andGSM646708). Many genes that are upregulated with consti-tutive LIN28B expression in vitro are also increased by

constitutive LIN28B expression in vivo, including the stemcell markers LGR5 (24) and PROM1 (30) and the let-7 targetsHMGA2 (high-mobility group AT-hook 2; refs. 31, 32) andIGF2BP1 (insulin-like growth factor 2 mRNA-binding protein1; ref. 33); these results were verified by qRT-PCR analysis(data not shown). Overall, the expression profile of emptyvector tumors varies dramatically from that of LIN28B-expressing primary tumors (Supplementary Fig. S3A). Nota-bly, the expression profile of LIN28B metastases also differs

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Figure 4. Differentiation and mucin production in LIN28B tumors. A, poor differentiation in xenograft tumors. Area exhibiting poor differentiation scored as apercentage of viable tumors. LIN28B-expressing tumors exhibit fewer areas of poor differentiation. B, moderate differentiation in xenograft tumors. Areaexhibiting moderate differentiation scored as a percentage of viable tumors. Moderate differentiation is increased in LIN28B-expressing tumors. C, mucinpositivity in poorly differentiated tumor areas. Mucin was detected via mucicarmine staining. Mucin positivity in poorly differentiated area scored as apercentage of viable tumor. D, mucin positivity in moderately differentiated tumor areas. Mucin positivity in poorly differentiated area scored as a percentage ofviable tumor. Differentiation is increased in LIN28B-expressing tumors. Mucin production is increased in LIN28B-expressing tumors.

Figure 5. A subset of LIN28B-expressing primary tumorsmetastasize. A, lung metastasisfrom a LIN28B-DLD-1subcutaneous tumor. H&Estaining. B, liver metastasis from aLIN28B-LoVo subcutaneoustumor. H&E staining; note invasionof metastasis into normal liver(C and D) metastases are GFP-positive. IHC for GFP expressedfrom MSCV-PIG retroviral vectorconfirms that these tissuesoriginated from xenograftinjections. E, metastases visibleupon gross examination. Arrowsindicate liver and mesentericmetastases from LIN28B-LoVotumors. Magnification: 100� inA and B; 200� in C and D.

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from that of LIN28B primary xenograft tumors (Supplemen-tary Fig. S3A). Some genes, such as TNS4 (tensin 4), CHI3L1(chitinase 3-like 1), and KLK6 (kallikrein-related peptidase6), specifically modulated in LIN28B metastases versusLIN28B primary tumors have described roles in migration,invasion, and metastasis (refs. 34–36; Table 1).

Because we observed metastases with constitutive LIN28Bexpression in cells, we sought to determine whether the geneexpression profile induced by LIN28B resembled thatof naturally occurring metastatic human colon tumors.Therefore, we cross-compared the LIN28B-specific geneexpression signature with that of patients with colon ade-nocarcinomas. For this analysis, we used publicly availablegene expression data from 290 patients with colon adeno-carcinomas (37). When patients were stratified according tothe LIN28B-specific gene expression signature (Supplemen-tary Fig. S3B), disease-free survival of patients with tumorsbearing gene expression patterns similar to the LIN28Bsignature is significantly shorter (P = 0.02, by log-rank test,Supplementary Fig. S3B), indicating that LIN28B in coloncancer may influence clinical outcome in patients with coloncancer by promoting early metastasis. Specificity and sensi-tivity for correctly predicting LIN28B overexpression duringLOOCV were both 1.0.

Discussion

We found that increased LIN28B staining intensity in non-metastatic colon tumors correlates with reduced overall sur-vival and increased probability of tumor recurrence in patientsreceiving adjuvant 5-fluorouracil. We then examined tumor-promoting properties of LIN28B-expressing colon cancer cellsvia tumorigenesis studies in nude athymic mice. Primaryxenograft tumors that constitutively express LIN28B display

decreased size and increased differentiation characterized byglandular formation relative to empty vector controls. Yet,LIN28B-expressing xenograft tumors are capable of metasta-sis, which is not observed with empty vector xenograft tumorsdo not.

We have shown increased LIN28B expression in primarycolon tumors, which may be the result of increased LIN28Btranscriptional activity mediated by c-myc. Because c-myc is atranscriptional target of canonical Wnt signaling, it is possiblethat LIN28B is upregulated in colon tumors as a consequenceof APC mutation (or other changes that deregulate Wntsignaling), which occurs in the majority of sporadic colontumors. The ability of c-myc to transactivate LIN28 has notbeen described and may account for differential properties ofLIN28 and LIN28B in colon cancer. However, it is important tonote that distinct functions have not been described for eitherhomologue to date and it is possible that the 2 are completelyredundant.

Although our findings reveal potential roles for LIN28B incolon cancer metastasis, it is surprising for genes thatpromote tumorigenesis and/or metastasis to simultaneouslyenhance differentiation. Thus, our finding that primarytumors constitutively expressing LIN28B are smaller andmore differentiated than their empty vector counterpartsis intriguing. Given that constitutive LIN28 expression inundifferentiated cells blocks gliogenesis in favor of neuro-genesis (14), it is possible that LIN28B overexpression incolonic epithelial cells results in restriction of cell fate asobserved previously in neuronal cells. Similar phenotypeshave previously been described in the intestine. For example,loss of the Math1 (also called Atoh1) transcription factorleads to depletion of goblet, enteroendocrine, and Panethcells, while permitting enterocyte differentiation in theintestinal epithelium (38).

Table 1. Transcripts upregulated by LIN28B

Gene symbol Gene name Fold changeb (vector vs. LIN28B)

Transcripts upregulated by constitutive LIN28B expression in vitroa

LGR5 Leucine-rich repeat-containing G-protein–coupled receptor 5 7.2PROM1 Prominin 1 22.6KIT v-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog 36.6HMGA2 High mobility group AT-hook 2 2.6IGF2BP1 Insulin-like growth factor 2 mRNA-binding protein 1 9.4CCND2 Cyclin D2 6.6DKK1 Dickkopf homolog 1 37.1

Fold changeb (tumor vs. metastases)

Transcripts upregulated in LIN28B-expressing metastases in vivoc

CHI3L1 Chitinase 3-like 1 4.4KLK6 Kallikrein-related peptidase 6 4.3TNS4 Tensin 4 4.6

aSelected transcripts upregulated by constitutive LIN28B expression in colon cancer cells in vitro.bFold changes indicated are the mean of 3 experimental replicates.cSelected transcripts upregulated in metastases derived from primary xenograft tumors constitutively expressing LIN28B in vivo.

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Furthermore, LIN28B upregulates the colonic epithelialstem cell markers LGR5 and PROM1. In the intestine, expres-sion of the cell surface protein PROM1 is restricted to the cryptand adjacent epithelial cells (30) whereas expression of theorphan receptor LGR5 occurs exclusively in cycling columnarcells within the crypt base (24). Because LGR5 and PROM1mark intestinal and colonic epithelial stem cells, upregulationof these factors by LIN28B suggests possible roles for LIN28B inintestinal stem cells.Our finding that high LIN28B staining intensity in primary

human colon tumors correlates with reduced patient survi-val and increased probability of tumor recurrence is espe-cially intriguing, given that LIN28B-expressing xenografttumors metastasize in the context of a more differentiatedprimary tumor. While low tumor grades usually correspondto early stage and better prognosis, metastasis and poorprognosis are observed in a subset of low-grade tumors (39,40). Given that we find both differentiation and metastasiswith constitutive LIN28B expression in primary tumors,LIN28B overexpression may mark low-grade tumors withpoor prognostic potential. Thus, LIN28B may serve as apotential diagnostic marker that may prove to be an invalu-able tool in colon cancer wherein early diagnosis is critical tosurvival.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

We thank Dr. JoshuaMendell for gifts of LIN28B expression vectors, as well asBen Rhoades and Louise Wang for technical assistance. We acknowledge helpfrom the NIH/NIDDK P30-DK050306 Center for Molecular Studies in Digestiveand Liver Diseases and its Morphology, Molecular Biology and Cell Culture CoreFacilities. We also thank the Penn Microarray Facility, including Drs. JohnTobias and Don Baldwin, Dr. Daqing Li of the Department of Otorhinolar-yngology/Head & Neck Surgery for the use of the Kodak small animal imagingsystem, and Colleen Bressinger for assistance with biostatistical analysis.

Grant Support

This work was supported by R01-DK056645 (A. Rustgi and C. King), TheHansen Foundation, National Colon Cancer Research Alliance (EIF), and grantsfrom the Ministerio de Ciencia e Innovaci�on (SAF2010-19273), Asociaci�onEspañola contra el C�ancer (Fundaci�on Científica y Junta de Barcelona), andAg�encia de Gesti�o d’Ajuts Universitaris i de Recerca (2009 SGR 849). C. King is aPfizer Animal Health scholarship recipient and a student of the University ofPennsylvania combined VMD/PhD program.

The costs of publication of this article were defrayed in part by the paymentof page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received January 10, 2011; revised March 29, 2011; accepted April 14, 2011;published OnlineFirst April 21, 2011.

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King et al.





Published OnlineFirst April 21, 2011.Cancer Res Catrina E. King, Miriam Cuatrecasas, Antoni Castells, et al.

Promotes Colon Cancer Progression and MetastasisLIN28B

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