mir-196b-5p regulates colorectal cancer cell migration and … · ments were conducted in a panel...

Biology of Human Tumors

miR-196b-5p Regulates Colorectal CancerCell Migration and Metastases throughInteraction with HOXB7 and GALNT5Verena Stiegelbauer1,2, Petra Vychytilova-Faltejskova3,4, Michael Karbiener5,Anna-Maria Pehserl1,2, Andreas Reicher1,2, Margit Resel1, Ellen Heitzer6,Cristina Ivan7,8, Marc Bullock9, Hui Ling7, Alexander Deutsch10,Annika Wulf-Goldenberg11, Jan Basri Adiprasito1,2, Herbert Stoeger1,Johannes Haybaeck12,13, Marek Svoboda4, Michael Stotz1,2, Gerald Hoefler12,Ondrej Slaby3,4, George Adrian Calin7,8, Armin Gerger1,14, and Martin Pichler1,2,7

Abstract

Purpose: miR-196b-5p has been previously implicated inmalignant transformation; however, its role in colorectal cancerhas not been fully explored. In this study, we examine the clinicaland biological relevance of miR-196b-5p, and the molecularpathways regulated by miR-196b-5p in colorectal cancer.

Experimental Design:miR-196b-5p expression was quantitat-ed by qRT-PCR in 2 independent cohorts composed of 292patients with colorectal cancer in total, to explore its biomarkerpotential. Transient and stable gain- and loss-of-function experi-ments were conducted in a panel of colorectal cancer cell linesandmice, to evaluate the impact ofmiR-196b-5ponproliferation,chemosensitivity, migration/invasion, and metastases formationin vitro and in vivo. The molecular pathways influenced by miR-196b-5p were characterized using whole transcriptome profiling,in silico target prediction tools, luciferase interaction assays, andphenocopy/rescue gene knockdown experiments.

Results: Low miR-196b-5p expression was significantly asso-ciated with metastases and poor outcomes in 2 independentcolorectal cancer patient cohorts (P < 0.05, log-rank test).miR-196b-5p inhibition led to significantly increased colorectalcancer cell migration/invasion and metastases formation inmice, whereas ectopic overexpression showed the opposite phe-notype. Molecular profiling and target confirmation identifiedan interaction between miR-196b-5p and HOXB7 and GALNT5,which in turn regulated colorectal cancer cell migration.

Conclusions: The association of low levels of miR-196b-5pand poor prognosis in patients with colorectal cancer can beexplained by its influence on cancer cell migration and metas-tases formation. miR-196b-5p has an impact on colorectalcancer progression pathways through direct interaction withgenes involved in cancer cell migration. Clin Cancer Res; 23(17);5255–66. �2017 AACR.

IntroductionColorectal cancer is the third most common cancer in males

and the second most common cancer in females. In 2015,about 69,090 men and 63,600 women were diagnosed withcolorectal cancer in the United States (1). The introduction ofnew drugs including inhibitors of angiogenesis and EGFRsignaling have improved survival time in metastatic colorectalcancer (2), but despite these improvements, metastatic colo-rectal cancer still remains an incurable disease in mostpatients. This has prompted research to uncover the molecularmechanisms, which drive metastatic colorectal cancer, to helpidentify biomarkers better able to predict clinical outcome,and to inform novel therapeutic strategies in the context ofincreasingly personalized cancer care (3–6).

The discovery of the miRNAs in human cancer has added newinsights into understanding cancer biology and has openedthe door for novel diagnosis and treatment tools in patientswith cancer (7, 8). miRNAs are a class of small, endogenous,noncoding, single-stranded RNAs that play a crucial role asposttranscriptional regulators for their larger counterpartsmRNA expression (9). These small RNA molecules are knownimportant regulators of carcinogenesis and in general, oneparticular miRNA can act as a tumor suppressor or a tumor

1Division of Oncology, Department of Internal Medicine, Medical University ofGraz, Graz, Austria. 2Research Unit for Non-Coding RNAs and Genome Editing,Medical University of Graz, Graz, Austria. 3Molecular Oncology II - Solid Cancers,Molecular Medicine, Central European Institute of Technology, Masaryk Univer-sity, Brno, Czech Republic. 4Department of Comprehensive Cancer Care, MasarykMemorial Cancer Institute, Czech Republic. 5Department of Phoniatrics, ENTUniversity Hospital, Medical University of Graz, Graz, Austria. 6Institute of HumanGenetics, Medical University of Graz, Graz, Austria. 7Department of ExperimentalTherapeutics, The University of Texas MD Anderson Cancer Center, Houston,Texas. 8The Center for RNA Interference andNon-coding RNAs, The University ofTexas, MD Anderson Cancer Center, Houston, Texas. 9Academic Surgery, Uni-versity of Southampton, Southampton, United Kingdom. 10Division of Haematol-ogy, Department of Internal Medicine, Medical University of Graz, Austria.11Experimental Pharmacology & Oncology GmbH, EPO, Berlin, Germany. 12Insti-tute of Pathology, Medical University of Graz, Graz, Austria. 13Department ofPathology, Otto von Guericke University Magdeburg, Leipziger Str. 44, D-39120,Magdeburg, Germany. 14Center for Biomarker Research inMedicine, Graz, Austria.

Note: Supplementary data for this article are available at Clinical CancerResearch Online (http://clincancerres.aacrjournals.org/).

Corresponding Authors: Martin Pichler, Division of Oncology, Medical Univer-sity of Graz, Auenbruggerplatz 25, 8010 Graz, Austria. Phone:þ4331638581320;Fax: þ4331638513355; E-mail: [email protected]; and ArminGerger, [email protected]

doi: 10.1158/1078-0432.CCR-17-0023

�2017 American Association for Cancer Research.

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promoter ("oncomiR"), depending on the cellular and molec-ular context (10, 11). Besides their potential as diagnostic andprognostic biomarkers, one of the most promising themes inrecent years has been the development of therapeutic strategieswhich target miRNA differentially expressed in cancer versusnormal tissue (12). Several miRNAs have been previouslyidentified relevant for colorectal cancer pathogenesis (13–18).In this study we focused on miR-196b-5p, a miRNA that hasbeen involved in carcinogenesis of other epithelial types ofcancer. In colorectal cancer, previously published data suggestedthat high miR-196b-5p levels are associated with response tochemoradiotherapy (19, 20). In colorectal cancer, the influenceof miR-196-5p is more controversial, as one study including 126patients with colorectal cancer reported that high miR-196-5plevels were associated with short disease and overall survival(21), whereas a second study comprising more than 400patients from 3 independent cohorts suggested the oppositewas true (22).

This study aims to clarify the biological role and clinicalsignificance of miR-196b-5p in patients with colorectal cancerby quantitating expression in 2 large independent patientcohorts, linked to a database containing comprehensive patientdemographic and outcome data. The impact of miR-196b-5pin vitro and in vivo on the hallmark processes of cancer pro-gression was also analyzed to gain deeper insight into thecolorectal cancer relevant biological mechanisms influencedby miR-196b-5p.

Materials and MethodsPatients

To explore the prognostic value of miR-196b-5p, we mea-sured the expression levels by qRT-PCR in two independentcohorts of central European patients. The cohorts compriserandomly selected cancerous tissue of histologically confirmed

patients with colorectal cancer between 30 and 90 years of agewho were diagnosed between the years 2005 and 2012. Thepatients' tissue was derived from either the Institute of Pathol-ogy, Medical University of Graz, Austria (cohort 1, n ¼ 110;ref. 13) or the Department of Comprehensive Cancer Care,Masaryk Memorial Cancer Institute, Czech Republic (cohort 2,n ¼ 182). The patients' clinicopathologic data were retrievedfrom medical records at the same institutions. This study hasbeen approved by the Institutional Review Boards (No. 23-545ex 10/11, No. 25-324 ex 12/13, and 2015/1690/MOU). Allcases were reviewed based on pathology reports and histologicslides for pTNM categories. Patients were treated by standardsurgical procedures and received adjuvant treatment whenappropriate (stage II with risk factors or stage III). If advanceddisease at the date of diagnosis, the patients then receivedmedical treatment according to a physician-choice followingthe recommendation of the last version of European Society ofMedical Oncology (ESMO) guidelines (23). Dates of deathwere obtained from the medical history, central registry of theAustrian or Czech Bureau of Statistics, or by telephone calls totheir relatives as previously reported. miRNA measurement byqRT-PCR from cancerous tissue was performed as follows: Twoto 8 10-mm-thick tissue sections were used for microdissectionto obtain areas with at least 70% tumor cell content. miRNAswere isolated using the miRNeasy FFPE Kit 50 (Qiagen) accord-ing to the manufacturer's instructions. cDNA was synthesizedfrom 500 ng of total RNA using a miScript Reverse Transcrip-tion Kit (Qiagen). Quantification of miRNAs was performedusing the miScript SYBR Green PCR Kit (Qiagen) and thespecific miScript Primer Assays for miR-196b-5p (hsa-miR-196b-5p, MIMAT0001080, assay name: Hs_miR-196b_1) andRNU6b (assay name: Hs_RNU6-2_11; all miScript PrimerAssays from Qiagen) according to the manufacturer's recom-mendations on a Light Cycler 480 real-time PCR device(Roche). Expression values were calculated using normaliza-tion to RNU6B [after the formula 2^-(target gene – RNU6B)],log2-transformed, and further used for statistical analysis.

Cell cultureThe human colorectal cancer cell lines HRT18, RKO, DLD1,

SW480, and HCT116 were purchased from ATCC. The colorectalcancer cell lines were authenticated at the Cell bank of the CoreFacility of the Medical University of Graz (Graz, Austria), byperforming a short tandem repeat (STR) profiling analysis (Kit:Promega, PowerPlex 16HS System; catalog no. DC2101, last dateof testing: March 3, 2016). Mycoplasma testing was performedusing the Venor GeM Mycoplasma Detection Kit (Minerva Bio-labs). Cells were cultured as described in detail in the Supple-mentary Materials section.

Transient transfection of miR-196b-5p, siRNA treatment, andlentiviral transduction

For functional studies, we transfected the colorectal cancercell lines either withmiR-196b-5pmimics (10 nmol/L), inhibitor(50 nmol/L), respectively), or GALNT5 and HOXB7 siRNAs(20 nmol/L) and for confirmation of results obtained fromtransient approaches and studying in vivo effects, we generatedstable miR-196b-5p–overexpressing or inhibitor-expressing celllines by lentiviral transduction, as it is described in all details inthe Supplementary Materials section.

Translational Relevance

Colorectal cancer is a common type of cancer and the spread(metastases) from primary tumor to distant sites mainly relieson cancer cell migration and invasion. miRNAs have beenidentified as major regulators of this process in all types ofcancer cells, and the first clinical trials using miRNA-basedtherapeutics are underway. Before a particular miRNA can beconsidered as a potential biomarker or therapeutic target, itsrole as a cancer driver or inhibitor has to be clarified withoutany doubt. In this study, we add profound data that one ofthese miRNAs, miR-196b-5p, is negatively associated withsurvival in patients with colorectal cancer. On the basis ofthis clinical observation, we systematically characterized theunderlying cellular mechanisms and identified for the firsttime miR-196b-5p as a colorectal cancer cell migration andmetastases formation regulating factor as well as novel under-lying molecular mechanisms. Our study provides first evi-dence that miR-196b-5p plays a yet unrecognized role incolorectal cancer progression and delivery of miR-196b-5pmight represent a novel treatment strategy, which should befurther evaluated in preclinical colorectal cancer models for itstherapeutic potential.

Stiegelbauer et al.

Clin Cancer Res; 23(17) September 1, 2017 Clinical Cancer Research5256




qRT-PCR, digital PCR, and Western blot analysisFor relativemRNAandmiRNAdetection, absolutemiRNA, and

protein quantification, we applied standard methods of quanti-tative PCR, digital PCR (dPCR), and Western blot analysis asdescribed in detail in the Supplementary Materials. Primersequences are listed in Supplementary Table S1.

Cellular assay, in vivo metastases formation, andbioluminescence imaging

To investigate whether miR-196b-5p expression has an influ-ence on cellular growth and chemosensitivity to commondrugs, we applied the WST-1 proliferation assay as describedin the Supplementary Materials section. We used 3 independentassays to monitor cell migration and invasion (xCELLigenceReal Time Assay, Transwell and Scratch Assay) of colorectalcancer cells after transient or stable miR-196b-5p overexpres-sion and inhibition. These assays and the in vivo experimentsare described in every detail in the Supplementary Materialssection.

Identification of putative miR-196b-5p target genesTo detect the most differentially expressed genes upon

forced miR-196b-5p–overexpressing colorectal cancer cells, amicroarray analysis was performed as described in the Sup-plementary Materials part. To identify putative mRNA targets,we used several prediction algorithms, qRT-PCR, Western blotanalysis, and luciferase assay as specified in the SupplementaryMaterials.

Statistical analysesAll statistical analyses were performed using SPSS version 20

software (SPSS Inc.) or MedCalc software (version 13.1.2.0). Inthe Supplementary Material, we are listing the Recommenda-tions for Tumor Marker Prognostic Studies (REMARK criteria)in relation to our reported results here (24). Unpaired or pairedStudent t test, Fisher exact test, c2 test, Spearman correlation,Mann–Whitney test, and Kruskal–Wallis test were appliedwhere appropriate to analyze the association between miR-196b-5p expression and clinicopathologic parameters. Data ofgene expression were log2-transformed. Overall survival wasdefined as the time from date of diagnosis to the date of deathby any cause, and it was assessed using the Kaplan–Meiermethod. For survival analysis, we separated patients intolow/high groups using cut-off value that separates the patientscalculated according to the P value in log-rank test between the25th and 75th of the cohort as was published previously (15).Univariate and multivariate Cox proportional hazards modelsincluding age, gender, tumor grade, tumor stage (according tothe AJCC/UICC 2010 TNM classification), and miRNA expres-sion levels. To test the proportional hazard assumption in Coxmodels, Schoenfeld residuals test was used. The reported resultsincluded HRs and 95% confidence intervals (CI). The log-ranktest was performed to compare the survival curves of individ-ual groups. A 2-sided P < 0.05 was considered statisticallysignificant.

ResultsClinical significance of miR-196b-5p in 2 independent cohorts

First of all, to explore the clinical significance of miR-196b-5pin patients with colorectal cancer, we measured miR-196b-5p

expression by qRT-PCR in 2 independent cohorts and furtheranalyzed its role as a potential prognostic factor. Patient char-acteristics for both cohorts are summarized in SupplementaryTable S2. In cohort 1, we found a significantly lower level of miR-196b-5p expression in stage III and stage IV patients (P < 0.05;Supplementary Fig. S1A), whereas no difference was found fortumor grade (Supplementary Fig. S1B). In cohort 2, we confirmeda significant lower expression for miR-196b-5p in stage III and IVpatients (P < 0.05; Supplementary Fig. S1C), and detected lowerexpression values in high-grade (G3/G4) tumors (P < 0.05;Supplementary Fig. S1D). For evaluating whether the levels ofmiR-196b-5p are associated with clinicopathologic parametersand survival of patients with colorectal cancer, we divided thecohorts into 2 groups (low and high miR-196b-5p expression)according to the criteria mentioned in the Material and Methodssection (15). In cohort 1, low levels of miR-196b-5p were signif-icantly associated with stage IV colorectal cancer at date ofdiagnosis (i.e., metastatic disease, P < 0.05, c2 test), whereas noassociation was observed for gender, age, or tumor grading (datanot shown). In cohort 2, low levels ofmiR-196-5pwere associatedwith high (G3 þ G4) tumor grade and also with stage IVcolorectal cancer (P < 0.05, c2 test). Figure 1 shows theKaplan–Meier curves for 5-year overall survival and revealsthat low miR-196b-5p expression is significantly associatedwith poor prognosis in both independent cohorts (P < 0.05,log-rank test). Univariate Cox proportional analysis identifiedhigh tumor grade (G3 vs. G1 þ G2, in cohort 1), advancedtumor stage (stage IV vs. stage II–III), and low expression ofmiR-196b-5p as poor prognosticators for OS (all P values<0.05), whereas age and gender were not significantly associ-ated with OS in both cohorts (Supplementary Table S3). To testwhether the prognostic value of low miR-196b-5p expressionprevailed after adjustment of other risk factors, multivariateanalyses were performed using a Cox proportional hazardmodel. Multivariate analyses including age, gender, tumorgrade, tumor stage, and miR-196b-5p expression, demonstrat-ed that low miR-196b-5p expression was an independentpredictor for poor survival in cohort 1 (HR 0.50; 95% CI,0.29–0.84; P ¼ 0.01; Supplementary Table S4).

Restricting our survival analyses on localized tumor stagesonly (stage II/III) and using the same cut off-value definedabove, we found again a statistically significant poor survivalfor patients with low miR-196b-5p expression (P < 0.05 forboth cohorts, log-rank test; Supplementary Fig. S2A and S2B).In cohort 1, we could also find similar and significant results instage IV metastatic patients (Supplementary Fig. S2C). Finally,checking data from level 3 Illumina miRNASeq (IlluminaSe-quencingTechnology:Genome Analyzer) of the Cancer GenomeAtlas (TCGA) dataset showed that low miR-196b-5p expressionwas associated with poor disease-free survival in univariate (HR0.67; 95% CI, 0.48–0.93; P ¼ 0.018348067) as well as mul-tivariate analysis after adjusting for stage (HR 0.6912; 95% CI,0.49–0.96; P ¼ 0.02843).

miR-196b-5p influences cell migration and invasionGiven the findings that low levels of miR-196b-5p were sig-

nificantly associated with metastatic disease and poor patientoutcome in both cohorts, we preceded our study to characterizeunderlying cellular and molecular mechanisms of this observa-tion. At first, we measured miR-196b-5p expression in 8 differentcolorectal cancer cell lines using relative qRT-PCR and validated

miR-196b-5p in Colorectal Cancer

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our findings by absolute digital PCR quantification in an inde-pendent laboratory. A strong and significant correlation for celllines (Spearman correlationR¼ 0.881; P¼ 0.004; SupplementaryFig. S3A and S3B) and 10 selected patient samples (Spearmancorrelation R ¼ 0.794; P ¼ 0.006; Supplementary Fig. S3C andS3D) was found. In addition, we divided the cell lines into amiR-196b-5p low/high category as well as the patient samples andcompared the expression values from the cell lines to patientsample to showmiR-196b-5p levels in the cell line model systemin relation to tissue samples (Supplementary Fig. S4A and S4B).To explore the biological role of miR-196b-5p, we started toperform gain (miR-196b-5p mimic) and loss (miR-196b-5pinhibitor) of function experiments by transiently manipulatingmiR-196b-5p levels in 3 independent colorectal cancer cell lines(HCT116, RKO, and HRT18; Supplementary Fig. S4C and S4D).To confirm the success of transfection ofmiR-196b-5pmimics, wevalidated decrease of known target genesHOXA9 andGATA6 in 2independent colorectal cancer cell lines (Supplementary Fig. S4Eand S4F; refs. 25, 26).

Cellular growth was not significantly influenced in theHCT116 and HRT18 cell line, whereas the RKO cells showeda slight but significant (P ¼ 0.0028) decrease of growth after 96hours of ectopic miR-196b-5p overexpression (SupplementaryFig. S5A–S5C). On the basis of previous reports about apossible association of miR-196b-5p expression and response tochemotherapy, we next explored the influence of miR-196b-5pexpression levels on drug sensitivity for the 3 most commonlyused colorectal cancer drugs (i.e., 5-fluorouracil, irinotecan, andoxaliplatin; refs. 19, 20). Testing several concentrations of thesedrugs, we could not detect any significant differences or trendswith regard to chemotherapy sensitivity in low or high miR-

196b-5p–expressing cells (Supplementary Figs. S6, S8A, andS8C). As miR-196b-5p expression levels were correlated withpatient survival and metastatic stage IV disease, we next ana-lyzed the migration behavior. For a screening approach, weapplied a real-time cell analysis migration measurement meth-od (xCELLigence system), which enabled us to measure themigratory process at multiple time points over 48 hours. Aftertreatment with the miR-196b-5p inhibitor (i.e., low miR-196b-5p levels), all 3 cell lines showed the earliest start and highestrate of migration in this assay system, whereas high miR-196b-5p expression levels (i.e., miR-196b-5p mimic) led to thelowest migration rates in colorectal cancer cells (Supplemen-tary Fig. S9A–S9C). To substantiate these findings in indepen-dent migration assays, we performed a scratch assay and atranswell assay for all 3 cell lines. We confirmed that low levels(i.e., inhibitor) of miR-196b-5p are associated with increasedmigration, whereas high levels of miR-196b-5p lead todecreased migration of colorectal cancer cells (Fig. 2). Inaddition to these 3 cell lines, we included DLD1 (with thehighest miR-196b-5p levels) and SW480 (with very low expres-sion levels) to perform a migration assay after inhibition(DLD1) or forced expression (SW480) of miR-196b-5p. Usingthe scratch wound healing assay, miR-196b-5p overexpressionled again to significantly decreased migration in SW480 cells,whereas inhibition of miR-196b-5p resulted in significantlyincreased migration in DLD1 cells (Supplementary Fig. S10Aand S10B). In addition, we found that low levels of miR-196b-5p increase HCT116 cancer cell invasion in 2 independentinvasion assays (Supplementary Fig. S11A–S11C). To furtherconfirm the results of transient transfections under differentconditions, we generated 3 cell lines with stable miR-196b-5p

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A, Kaplan–Meier curve for overall survival in cohort 1 of 110 patients with colorectal cancer. Patients were dichotomized into 2 groups according to miR-196b-5pexpression. Low miR-196b-5p expression was a significant factor for poor prognosis (P ¼ 0.002, log-rank test). B, Kaplan–Meier curve for overall survival incohort 2 of 182 patients with colorectal cancer. Low miR-196b-5p expression was confirmed as a significant factor for poor prognosis in this independentcohort (P ¼ 0.049, log-rank test).

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overexpression or inhibitor expression (Supplementary Fig. S12Aand S12B). Although we could successfully obtain a green fluo-rescent signal in all the transfected cells (indicating a successfullystable transfection), the miR-196b-5p levels could not be signif-icantly decreased by introduction of the inhibitor construct.Notably, this is in line with the mode of action of miRNAinhibitors, which after binding to the miRNA lead to a loss offunction rather than to reduce the expression levels (27). Conse-quently, we confirmed the same effects on cancer cellmigration inall stably manipulated cell lines using again 2 independentmigration assays (Fig. 3A–E).

For in vivo confirmation of this potentially prometastaticphenotype, we used stable miR-196b-5p inhibitor (silencing)expressing HCT116 cells and their respective control andlabeled them with a luciferase-expressing vector for in vivoimaging purposes. As shown in Fig. 3F, mice intrasplenicinjected with miR-196b-5p silenced cells showed more fre-quently [6 mice (75%) vs. 2 mice (28.5%) and more intense(mean photons emission: 317814 vs. 59191; P ¼ 0.09; Fig. 3F;

Supplementary Fig. S13A] signals. Regarding the gold standardof pathologic examination, 7 out of 8 mice (87.5%) in thegroup of miR-196b-5p inhibitor showed intra-abdominalmetastases in the whole abdomen, whereas only 2 out of 7(28.5%) mice in the control group were positive for this feature(P < 0.05; Fig. 3G and H). Further macroscopic and histologicexamination of liver tissue revealed that 7 of 8 mice (87.5%) inthe group of miR196b-5p inhibitor versus 2 out of 7 mice in thecontrol cell group had detectable metastases (P ¼ 0.041; Fig. 3I;Supplementary Fig. S13B and S13C). The median number ofmetastases per liver was 2.5 in the group of miR-196b-5pinhibitor versus 0 in the control group (P ¼ 0.04; Supplemen-tary Fig. S13D). Finally, we used human specific primers todetect human gene expression of GAPDH and the putativetarget genes HOXB7 and GALNT5, and could confirm thefindings of histologic examination [i.e., human gene expressionwas detectable in 7 of 8 (87.5%) livers of miR-196b-5p–silenced cells versus 2 out of 7 (28.5%) in control livers, P <0.05]. Thus, the in vivo data support our in vitro findings, that

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A–C, Representative examples of scratch assays in HCT116, HRT18, and RKO cells after transient transfection of negative control, miR-196b-5p mimic orinhibitor. 0 hours, scratch at beginning; 24 hours, scratch after 24 hours; 48 hours, scratch after 48 hours. The bar charts below the scratch picturesshow 3 independent experiments each. The miR-196b-5p inhibitor–treated cells migrated into the scratch significantly faster than the control(� , significant, P < 0.05; �� , P < 0.01; �� , P < 0.001; Student t test), whereas the opposite effect was observed when miR-196b-5p mimics was used. D and E,Bar chart graphs show the results of transwell migration assays in RKO and HCT116 cells (the HRT18 cell line did not migrate under the conditions used).miR-196b-5p inhibition leads to an increased number of migrated cells through the transwell membrane in both colorectal cancer cell lines, whereasoverexpression of miR-196b-5p results in decreased migration (see also the representative pictures of crystal violet–stained membranes on the right sideof the bar charts; � ¼ significant, P < 0.05; �� , P < 0.01; �� , P < 0.001; Student t test).






colorectal cancer cells with low miR-196b-5p expressionshowed a higher degree of metastases formation in vivo.

Molecular mechanisms involved in miR-196b-5p cancer cellmigration

In search for possible molecular mechanisms that trigger thismetastatic behavior, we next measured changes of expressionlevels of classical epithelial–mesenchymal transition (EMT) mar-kers inmiR-196b-5p–overexpressing and silenced cells, but couldnot detect any significant differences (Supplementary Fig. S14Aand S14B). To detect potential molecular miRNA::mRNA inter-actions in an unbiased approach, we performed a microarray-based whole transcriptome profiling analysis in 3 independent

biological replicates comparing the HCT116 miR-196b-5p stablyoverexpressing cells against control cells. On the basis of themicroarray results, showing 38 significantly upregulated and 41significantly downregulated genes (see also the heatmap in Fig. 4;Supplementary Table S5), we performed an in silico target predic-tion approach for these mRNAs and selected potentially interact-ing mRNA for further analysis (see detailed description in theSupplementary Methods section and Supplementary Table S6).To confirm the reliability of the microarray results, we furtherselected potential interactors with previously published dataabout involvement in carcinogenesis (i.e., GALNT5, HOXB7,MUC13, DKK1, CPA4, KRTAP2-3, and KRTAP1). We could con-firm the downregulation of all these genes (100% concordance)

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A–C, Scratch assays in 3 different colorectal cancer cell lines after stable miR-196b-5p overexpression (OE) or silencing (inhibitor). Representative picturesof scratch assay in HCT116, HRT18, and RKO cells are shown. 0 h, scratch at beginning; 24 hours, scratch after 24 hours; 48 hours, scratch after 48 hours. Barchart graphs below are showing the results of measurement of scratch closure after 24 and 48 hours for the 3 tested cell lines in 3 independent replicates.The stable miR-196b-5p–silenced cells showed a significantly earlier scratch closure than the control cells (P < 0.05 considered as significant), whereas cells withstable miR-196b-5p overexpression showed decreased migration. D and E, Bar chart graphs show the results of transwell migration assays in RKO and HCT116cells. Again, miR-196b-5p inhibition leads to an increased number of migrated cells through the transwell membrane in both colorectal cancer celllines (panels below the bar charts show representative pictures of crystal violet stained membranes). Because HRT18 did not migrate through the transwellmembrane under the selected conditions, we could not analyze the migration rate for this cell line (� , significant; P < 0.05; �� , P < 0.01; �� , P < 0.001; Student t test).F, In vivo imaging monitoring shows more positive mice 3 weeks after intra-spleen injection of Luciferase-labeled stable miR-196b-5p–silenced HCT116 cells incomparison with control cells. G, Postmortem analysis shows a reliable correlation between in vivo imaging and intra-abdominal metastases (representativeexamples for control mouse #7 and inhibitor mouse #8).H and I, Bar charts indicate a significant proportion ofmice (control group: 28.5% vs. inhibitor group: 87.5%)in the group of miR-196b-5p inhibitor with more intra-abdominal metastases and histologically proven liver metastases (P < 0.05).

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from themicroarray by quantitative RT-PCR (Supplementary Fig.S15A). To confirm these 7 downregulated candidates in anindependent cell line model, we measured all 7 genes in stablemiR-196b-5p overexpression HRT18 cells. In this independentcell line model, we could validate HOXB7 and GALNT5 assignificantly downregulated (Supplementary Fig. S15B). To fur-ther explorewhether these 2 genes are influenced bymiR-196b-5pin a different experimental setting, we transiently transfectedHCT116 and HRT18 cells with miR-196b-5p. Both genes weresignificantly downregulatedonmRNA(Fig. 5AandB) andproteinlevels (Fig. 5C) upon ectopically miR-196b-5p overexpression. Incontrast, introducing transiently miR-196b-5p inhibitor toHCT116 and HRT18 cells significantly increased the mRNA andprotein expression of GALNT5 and HOXB7 (Supplementary Fig.S15C–S15E). To test whether theHCT116 cells used for the in vivoexperiment (with stable miR-196b-5p silencing) have upregula-tion of GALNT5 and HOXB7 target genes, we performed mRNAand protein detection in comparison with the control cells. Bothtarget genes were significantly upregulated onmRNA and proteinlevel in miR-196b-5p–silenced cells (Supplementary Fig. S15Fand S15G). In an attempt to confirm this upregulation of targetgenes inmetastases of ourmouse study, a qRT-PCR analysis usinghuman-specific primers for detecting human RNA from livermetastases indicated human gene expression in 7 of 8 (87.5%)mice of miR-196b-5p–silenced cells versus 2 out of 7 (28.5%)

mice in control cells (P < 0.05). Overall, we observed higherexpression levels of the target genesHOXB7 and GALNT5 in miR-196b-5p–silenced liver metastases, but the number of affectedlivers in the control group (n ¼ 2) did not allow meaningfulstatistical conclusions (Supplementary Fig. S16A and S16B). Adirect interaction of miR-196b-5p and GALNT5 and HOXB7 wasfurther substantiated by cloning the predicted wild-type andmutated 30-UTR-binding site (Fig. 5D) in a luciferase-reporterplasmid. Reduced luciferase activity after miR-196b-5p cotrans-fection in HEK cells was observed in the GALNT5 and HOXB7wild-type sequences, whereas the mutated binding site restoredthe normal luciferase activity (Fig. 5E and F). To further prove theinteraction of miR-196b-5p with the putative binding site, weinhibited the endogenous miR-196b-5p expression in cells withhighmiR196b-5p expression (HCT116-stablemiR-196b-5p over-expression) and cotransfected thewild-type 30UTR-luciferase con-structs of HOXB7 or GALNT5. The inhibition of miR-196b-5presulted in increased luciferase activity for both genes, supportingour hypothesis (Supplementary Fig. S16C and S16D). Finally,we performed correlation analysis between miR-196b-5p andGALNT5/HOXB7 in tissue samples using publicly available dataof TCGA. A significantly negative correlation between miR196b-5p and both target genes was found (P < 0.05, Spearman correla-tion; Supplementary Fig. S17).To test the significance of HOXB7and GALNT5 in clinical samples, we analyzed the TCGA dataset

Figure 4.

Heatmap of statistically significant top 16 upregulated and 18 downregulated genes in stably overexpressing miR-196b-5p HCT116 cells (OE) comparedwith control (OE control) cells. Genes clustered using hierarchical clustering (Pearson dissimilarity, Ward method) on expression values present in picture(P < 0.05 considered as significant).






and found that high HOXB7 expression is associated with poorsurvival (Supplementary Fig. S18). Using GEO microarray data-sets (GSE12945 andGSE14333; refs. 28, 29), we identified a poorprognostic value of highHOXB7 andGALNT5 expression levels in2 additional datasets (Supplementary Figs. S19 and S20).

Finally, to test the hypothesis whether miR-196b-5p exerts itseffect on cancer cell migration by influencing HOXB7 andGALNT5 expression, we first performed siRNA-mediated knock-down experiments for both genes (Supplementary Fig. S21A andS21B) to prove the phenocopy of the miR-196b-5p phenotype.Reducing the expression levels of each of the mRNAs led to areduced migration of cancer cells (Fig. 6A and B), and this effectwas evenmore pronouncedwhenboth geneswere knocked-downin parallel (Fig. 6C). Next, we explored whether expression ofpreviously published downstream effector genes of HOXB7,including CCND1 and CDKN1B (30, 31) are indeed influencedby overexpression of miR-196b-5p in colorectal cancer cell lines.

For both effector genes, we observed differential expression ofmRNAs after forcedmiR-196b-5p expression,which is in linewithpreviously published studies (Supplementary Fig. S21C andS21D). Finally, we did a rescue experiment to show that knock-down of GALNT5 andHOXB7 reduces the migration in cells withtransient inhibition of miR-196b-5p. As shown in Fig. 6, siRNA-mediated knockdown of GALNT5 and HOXB7 expression inHCT116 cells with transient miR-196b-5p inhibitor expression(those cells exhibiting the promigratory/prometastatic pheno-type above) resulted in a significant reduction of cellular migra-tion in Transwell assay (Fig. 6D).

DiscussionIn this study,we sought to identify an association betweenmiR-

196b-5p expression and clinical outcome in colorectal cancerpatients. miR-196b-5p has been implicated in several tumor

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scenarios including upregulation in acute lymphoblastic leuke-mia (32) and downregulation in glioblastoma (33), cervicalcancer (34), and B-cell lineage lymphoma (35). These discordantobservations highlight the fact thatmiR-196b-5pmay function ina context specific way as either an oncomiR or a tumor suppressorgene, as has beendescribed formanyothermiRNAs in an excellentrecently published review (11).

In respect to colorectal cancer, there is conflicting data about thebiological role and hence the potential prognostic utility of miR-196b-5p, which this study has attempted to resolve. By analyzing292 colorectal cancer specimens from patients in 2 independentcohorts, we confirmed that low miR-196b-5p expression is sig-nificantly associated with poor prognosis. Our findings are in linewith the data from Boisen and colleagues, who presented a large3 cohort study and demonstrated that higher miR-196b-5pexpression levels were independently associated with improvedoutcome in colorectal cancerwhether or not bevacizumab therapyhadbeenutilized (22). In addition, our results are underlined by arecently published study of Fantini and colleagues who found out

that miR-196b-5p is significantly upregulated in pretreatmentsurgical resection colorectal cancer samples and that the upregu-lation of miR-196b correlates with less severe clinicopathologiccharacteristics (25).

Given the finding that low expression of miR-196b-5p issignificantly associatedwith poor patient outcome, we performeda series of experiments to elucidate the cellular and molecularmechanisms underlying this clinical observation. Cancer cellgrowth and chemosensitivity were not consistently effected incolorectal cancer cell lines in which miR-196b-5p expression hadbeen manipulated, suggesting these hallmarks of cancer are notsignificantly impacted (19, 20).

Low miR-196b-5p expression was however associated withmetastatic stage IV disease in both of our cohorts suggestingmiR-196b-5p may be associated with metastatic rather than localtumor proliferation pathways. Hence, using 3 different colorectalcancer cell lines, and 2 methods of miR-196b-5p manipulation(transient and stable transfection), in a variety of complementaryin vitro and in vivo disease progression assays we were able to

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A and B, Scratch assays in 2 colorectal cancer cell lines after siRNA-mediated knockdown of GALNT5 or HOXB7. Representative pictures of scratchassays in HCT116 and HRT18 after transfection of control and siRNAs against GALNT5 or HOXB7. 0 hour, scratch at beginning; 24 hours, scratch after 24 hours;32 hours, scratch after 32 hours; 48 hours, scratch after 48 hours. Bar chart graphs below show the results of measurement of scratch closure over48 hours for the 2 tested cell lines in 5 biological replicates. Cells transfected with the siRNA control closed the scratch significantly faster than the GALNT5-and HOXB7-silenced cells (P < 0.05 considered as significant). C, Double knockdown of GALNT5 and HOXB7 led to a significantly reduced migration comparedto control cells as shown in a representative picture of the scratch assay in HCT116 (right). Bar chart graph (left) showing the results of the measurementof scratch at the beginning (0 hours) and scratch wound closure after 16 hours (16 hours). D, Transient inhibition of miR-196b-5p followed by siRNA-mediated knockdown of target genes HOXB7 and GALNT5 resulted in decreased migration of HCT116 cells as detected in transwell migration assay. Top, thebar chart of 6 replicates; bottom, representative pictures (�, significant, P < 0.05; �� , P < 0.01; �� , P < 0.001; Student t test).






identify a consistent phenotype whereby increased colorectalcancer migration/metastases appears to be associated withreduced cellular miR-196b-5p expression.

To elucidate a potential molecular link between miR-196b-5pexpression and cancer cell migration, we performed an unbiasedwhole transcriptome analysis using colorectal cancer cells inwhich miR-196b-5p was stably overexpressed. On the basis ofthe assumption that genes directly targeted by miR-196b-5p aresuppressed in miR-196b-5p–overexpressing cells, we identifiedseveral promising mRNA candidates. As we were primarily inter-ested in direct interaction partners, we performed an in silico targetprediction analysis of potential complementary sequence seedsand a complementary literature search to further identify miR-196b-5p targetswith a putative influence on cancer cellmigration.The process in its entirety identified 2 candidate genes, HOXB7and GALNT5 that have been previously implicated in gastric andcervical cancer cell migration and/or invasion (34, 36, 37). Incolorectal cancer, a study by Liao and colleagues reported thatHOXB7 is significantly correlated with invasive and aggressivecharacteristics and poor survival of patients. Liao and colleaguesalso revealed that HOXB7 overexpression promotes proliferationand tumorigenic growth of human colorectal cancer cells, both invitro and in vivo (30). These data prompted us to focus on these 2genes and we demonstrated that forced miR-196b-5p expressiondecrease GALNT5 and HOXB7 expression on mRNA and proteinlevels. As a previous study byHowand colleagues (34) has alreadyidentified direct interaction of miR-196b-5p and HOXB7 incervical cancer, we validate the identical interaction site of theHOXB7 30UTR (34). However, a role of GALNT5 in colorectalcancer has never been reported before, so we further focused ourstudy on the regulation of GALNT5.

In general, the family of N-acetylgalactosaminyltransferases(GALLNTs) are responsible for glycosylation of O-glycans linkedwith N-acetylgalactosamine (O-GalNAcs). A study by Zhang andcolleagues provides evidence of O-glycosylation influencing thecomposition of the extracellular matrix (ECM) and suggests a rolefor abundant protein modification in disease states where matrixcomposition and cell adhesion are altered (38). O-GalNAc gly-cosylation of ECM proteins is important for secretion and plays arole inmodulating ECM composition and affecting cell adhesion,growth, and embryogenesis. O-GalNAc glycans have been impli-cated in numerous molecular pathways with key roles in tumorformation, cell invasion, and cancer progression (39). Somemembers of the GalNTs enzyme family have already been asso-ciated with several types of human cancer. A study by Peng andcolleagues revealed that knockdown of GALNT7 markedly inhi-bits cervical cancer cell proliferation, migration, and invasion(40). For GALNT5 the only published study so far comes fromHe and colleagues, who demonstrated that low intratumoralGALNT5 expression is a significant negative prognostic predictorfor patients with gastric cancer (37).

Finally, to test the hypothesis that these genes, HOXB7 andGALNT5, are factors whichmediate the effect ofmiR-196b-5p, weperformed migration assays following gene knockdown andrescue experiments inmiR-196b-5p–silenced cells for both genes.Reduced levels ofHOXB7 andGALNT5phenocopied the effects ofmiR-196-5p and importantly, this antimigratory effect became yetmore pronounced when both genes were knocked-down inparallel. These findings also highlight the fact that targetingmultiple mRNAs in parallel may amplify the biological impactin future the potential therapeutic benefits of miRNAs.

Our study is not without limitations. As with all retrospectivepatient cohorts, they are prone to selection bias and heterogeneityin terms of treatment schedule andmodalities. Although we usedseveral experimentalmodel systems,we cannot rule out that genesother than HOXB7 and GALNT5 might influence the cellularphenotype regulated by this miRNA. In our study, we did notperform in vivo knockdown experiments against HOXB7 andGALNT5 to diminish the prometastatic phenotype, but theseexperiments might be interesting future directions. In addition,further miR-196b-5p targeting strategies (with encapsulatedmimics or aptamers) should be performed in appropriate pre-clinicalmodels before testing this strategy in human patients withcolorectal cancer. Nevertheless, using independent cohorts andseveral experimental models and techniques, we sought to min-imize such effects and limitations.

In conclusion, the results of our study establishmiR-196b-5p asa prognostic factor in colorectal cancer. miR-196b-5p promotescolorectal cancer metastases, at least in part, through the regula-tion of HOXB7 and GALNT5. Further prospective studies andclinical trials are warranted to proof the role of miR-196b-5p as aprognostic marker and furthermore evaluate if miR-196b-5pmimics carry therapeutic value in patients with colorectal cancer.

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

DisclaimerThe sponsor of the study had no role in study design, data collection, data

analysis, data interpretation, writing of the report or the decision to submit thismanuscript for publication.

Authors' ContributionsConception and design: V. Stiegelbauer, M. Karbiener, J. Haybaeck, O. Slaby,M. PichlerDevelopment of methodology: V. Stiegelbauer, M. Resel, E. Heitzer, M. PichlerAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): V. Stiegelbauer, P. Vychytilova-Faltejskova, A. Reicher,M. Resel, A. Wulf-Goldenberg, H. Stoeger, J. Haybaeck, M. Svoboda, G. Hoefler,O. Slaby, M. PichlerAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): V. Stiegelbauer, M. Karbiener, A.-M. Pehserl,A. Reicher, M. Resel, E. Heitzer, C. Ivan, H. Ling, A. Deutsch, J.B. Adiprasito,J. Haybaeck, G. Hoefler, O. Slaby, A. Gerger, M. PichlerWriting, review, and/or revision of the manuscript: V. Stiegelbauer,M. Karbiener, A.-M. Pehserl, M. Bullock, H. Ling, J.B. Adiprasito, H. Stoeger,J. Haybaeck, M. Svoboda, M. Stotz, G.A. Calin, A. Gerger, M. PichlerAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): V. Stiegelbauer, A. Reicher, M. Resel, M. Bullock,H. Stoeger, M. StotzStudy supervision: A. Deutsch, A. Gerger, M. Pichler

Grant SupportM. Pichler research was supported by an Erwin-Schroedinger Scholarship of

the Austrian Science Funds (project no. J3389-B23) by a research grant of theVerein für Krebskranke of the Medical University of Graz, by the fund of theOesterreichische National bank no. 14869 (to M. Pichler), and by a researchgrant of the Styrian Krebshilfe. This study was supported in part by funds of theOesterreichische National bank (Anniversary Fund, project no. 15400 to A.Gerger). Furthermore, research activities fromM. Stotz are supported by a STARTgrant from the Medical University of Graz and a Hans und Blanca MoserFoundation for Early Stage Cancer Researcher. This work was supported bygrant IGA NT/13860-4/2012 of the Czech Ministry of Health, by the project"CEITEC" (Central European Institute of Technology (CZ.1.05/1.1.00/02.0068) and the project BBMRI CZ (LM2010004). G.A. Calin is The AlanM. Gewirtz Leukemia & Lymphoma Society Scholar. Work in G.A. Calin's

Stiegelbauer et al.





laboratory is supported in part by the NIH/NCI grants 1UH2TR00943-01 and 1R01 CA182905-01, the UT MD Anderson Cancer Center SPORE in Melanomagrant from NCI (P50 CA093459), Aim at Melanoma Foundation and theMiriam and Jim Mulva research funds, the UT MD Anderson Cancer CenterBrain SPORE (2P50CA127001), a Developmental Research award from Leu-kemia SPORE, a CLL Moonshot Flagship project, a 2015 Knowledge GAPMDACC grant, an Owens Foundation grant, and the Estate of C.G. Johnson Jr.

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received January 4, 2017; revised April 27, 2017; accepted May 16, 2017;published OnlineFirst May 22, 2017.

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Stiegelbauer et al.




2017;23:5255-5266. Published OnlineFirst May 22, 2017.Clin Cancer Res Verena Stiegelbauer, Petra Vychytilova-Faltejskova, Michael Karbiener, et al. Metastases through Interaction with HOXB7 and GALNT5miR-196b-5p Regulates Colorectal Cancer Cell Migration and

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