Differential Regulation of LET-7 by LIN28B Isoform Speciﬁc … · repertoire of mRNA transcripts (6, 7). Notably, LIN28B is a posttranscriptional repressor of LET-7 miRNA biogenesis

Chromatin, Epigenetics and RNA Regulation

Differential Regulation of LET-7 by LIN28BIsoform–Specific FunctionsRei Mizuno1,2,3,4, Priya Chatterji1,2,3,4, Sarah Andres1,2,3,4, Kathryn Hamilton1,2,3,4,5,Lauren Simon1,2,3,4,5, Shawn W. Foley6, Arjun Jeganathan1,4, Brian D. Gregory6,Blair Madison7, and Anil K. Rustgi1,2,3,4

Abstract

The RNA-binding protein LIN28B plays an important role indevelopment, stem cell biology, and tumorigenesis. LIN28Bhas two isoforms: the LIN28B-long and -short isoforms.Although studies have revealed the functions of the LIN28B-long isoform in tumorigenesis, the role of the LIN28B-shortisoform remains unclear and represents a major gap in the field.The LIN28B-long and -short isoforms are expressed in a subsetof human colorectal cancers and adjacent normal colonicmucosa, respectively. To elucidate the functional and mecha-nistic aspects of these isoforms, colorectal cancer cells (Caco-2and LoVo) were generated to either express no LIN28B or the-short or -long isoform. Interestingly, the long isoform sup-pressed LET-7 expression and activated canonical RAS/ERKsignaling, whereas the short isoform did not. The LIN28B-longisoform–expressing cells demonstrated increased drug resis-tance to 5-fluorouracil and cisplatin through the upregulation

of ERCC1, a DNA repair gene, in a LET-7–dependent manner.The LIN28B-short isoform preserved its ability to bind pre-let-7,without inhibiting the maturation of LET-7, and competed withthe LIN28B-long isoform for binding to pre-let-7. Coexpressionof the short isoform in the LIN28B-long isoform–expressingcells rescued the phenotypes induced by the LIN28B-longisoform.

Implications: This study demonstrates the differential antagonis-tic functions of the LIN28B-short isoform against the LIN28B-long isoform through an inability to degrade LET-7, which leadsto the novel premise that the short isoform may serve to coun-terbalance the long isoform during normal colonic epithelialhomeostasis, but its downregulation during colonic carcinogen-esis may reveal the protumorigenic effects of the long isoform.Mol Cancer Res; 16(3); 403–16. �2018 AACR.

IntroductionLIN28, an RNA-binding protein, is expressed at high levels in

mouse and human embryonic stem cells and in early embryo-genesis (1, 2). It is a key contributor to the formation of inducedpluripotent stem (iPS) cells (3). Two paralogs, LIN28A andLIN28B, are present in vertebrates (4). They bind to their targetmRNAs or miRNA and play a major role in posttranscriptionalcontrol, such as splicing, polyadenylation, mRNA stabilization,mRNA localization, and translation (5). Structurally, LIN28B has

two RNA-binding domains, a cold shock domain (CSD), and twoCCHC type zinc finger domains (ZFD) that facilitate binding to arepertoire of mRNA transcripts (6, 7). Notably, LIN28B is aposttranscriptional repressor of LET-7 miRNA biogenesis (8, 9,10, 11, 12). The CSD binds to the terminal loop of LET-7precursor, pre-let-7, and ZFDs bind to the GGAG motif in pre-let-7 (13). Lin28A induces uridylation of pre-let-7 at its 30 end,which escapes Dicer processing, resulting in degradation. Morespecifically, Lin28A recruits a TUTase (Zcchs11/TUT4) to pre-let-7to inhibit processing by Dicer (11, 14, 15). However, Lin28Brepresses LET-7 through a different mechanism and does so in thenucleus through the sequestration of LET-7 transcripts and block-ing their processing by the Microprocessor (16). Overall, Lin28-mediated regulation of LET-7 is critical in development, stem cellbiology, and tumorigenesis.

LIN28A and LIN28B are upregulated during embryonic devel-opment but downregulated in adult somatic tissues (17). They areoverexpressed in diverse cancers such as chronic myelogenousleukemia, hepatocellular carcinoma (HCC), neuroblastoma, lungcancer, breast cancer, ovarian cancer, and cervical cancer (18, 19,20). LIN28B is also overexpressed in a subset of colorectal cancers(21, 22). We showed that LIN28B overexpression in colorectalcancers is associated with poor prognosis and cancer recurrenceand that LIN28B promotes migration, invasion, andmetastasis ofcolorectal cancer cell lines in mouse xenograft models (21, 23).We have demonstrated that LIN28B has oncogenic properties inthe initiation and progression of colon cancer in geneticallyengineered mouse models, and that the LIN28B-Let-7 axis iscritical as LIN28B overexpression and Let-7 (a3-b2) deletion

1Division of Gastroenterology, Perelman School of Medicine, University ofPennsylvania, Philadelphia, Pennsylvania. 2Department of Medicine, PerelmanSchool of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania.3Department of Genetics, Perelman School of Medicine, University ofPennsylvania, Philadelphia, Pennsylvania. 4Abramson Cancer Center, PerelmanSchool of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania.5Division of Gastroenterology, Hepatology, and Nutrition, Department ofPediatrics, Children's Hospital of Philadelphia, Philadelphia, Pennsylvania.6Department of Biology, University of Pennsylvania, Philadelphia, Pennsylvania.7Division of Gastroenterology, Department of Medicine, Washington University,St. Louis, Missouri.

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

CorrespondingAuthor:A.K. Rustgi, University of Pennsylvania Perelman Schoolof Medicine, 951 BRB, 421 Curie Blvd., Philadelphia, PA 19104. Phone: 215-898-0154; Fax: 215-573-5412; E-mail: [email protected]

doi: 10.1158/1541-7786.MCR-17-0514

�2018 American Association for Cancer Research.

MolecularCancerResearch

www.aacrjournals.org 403

on April 24, 2021. © 2018 American Association for Cancer Research. mcr.aacrjournals.org Downloaded from

Published OnlineFirst January 12, 2018; DOI: 10.1158/1541-7786.MCR-17-0514

http://crossmark.crossref.org/dialog/?doi=10.1158/1541-7786.MCR-17-0514&domain=pdf&date_stamp=2018-2-13

http://mcr.aacrjournals.org/

accelerate colon cancer development and progression (24, 25).The upregulation of LIN28B or downregulation of LET-7 has beenreported to contribute to the acquisition of chemoresistance invarious types of cancer such as breast cancer (26), esophagealcancer (27), acute myeloid leukemia (28), and pancreatic cancer(29).

LIN28B's actions to promote tumorigenesis are not restricted toone specific mechanism. For example, the LIN28/LET-7 axis canmodulate glucose homeostasis by augmenting insulin-PI3K–mTOR signaling (30) and can regulate aerobic glycolysis topromote cancer cell progression (31). Other protumorigenicfunctionsmay bemediated via LET-7–independent effects. LIN28also functions through posttranscriptional regulation by directbinding to specificmRNAs thatmaypromote a stem cell–like stateor tumorigenesis, such as Insulin-like growth factor 2 (IGF2),LGR5, and PROM1 (23, 24, 32).

Guo and colleagues demonstrated that LIN28B has two iso-forms, so-called LIN28B-long isoform and LIN28B-short isoform(33). There is partial deletion of the CSD in the short isoformwithpreservation of the ZFDs. This suggests possible differences in thetargetmRNAs thatmay be bound by either isoform. There is a gapin our mechanistic understanding of the functional role ofLIN28B-short isoform or the relationship between LIN28B-shortand -long isoforms. Therefore, in this study,we aimed to reveal therole of the LIN28B-short isoform in colonic tumorigenesis. Wefound differential regulation of LET-7miRNAs between LIN28B-long and -short isoforms. Specifically, the LIN28B-long isoformsuppressed mature LET-7 expression, whereas LIN28B-short iso-form did not have this inhibitory effect. This differential regula-tion of LET-7miRNAs affected the downstream signaling of RAS/ERK signaling and potential chemoresistance. We also revealedthat LIN28B-short isoform functions as an antagonist againstLIN28B-long isoform, suggesting a model of dysequilibriumwhere the short isoform promotes differentiation in normalintestinal homeostasis through the inability to degrade LET-7,and the long isoform is predominant during colon cancer initi-ation and progression.

Materials and MethodsCell lines

All human cell lines used in this study (Caco-2, LoVo, HCT116,SW480, Colo205, T84, HepG2, and Huh7.5) were obtained fromthe American Type Culture Collection. These cell lines wereauthenticated by the STR locus. Caco-2, LoVo, HCT116, T84,HepG2, and Huh7.5 were maintained in DMEM (Thermo FisherScientific), and SW480 and Colo205 were maintained in RPMI1640 (Thermo Fisher Scientific) supplementedwith 10%FBS (GEHealthcare Life Sciences) and 1% penicillin–streptomycin (P/S;Thermo Fisher Scientific) in a 37�C incubator with 5%CO2. Cellswere tested formycoplasma every 2months andwere cultured forno more than 15 passages from the validated stocks.

ImmunoblottingProteins from cells or tissues were isolated using NP40 lysis

buffer, and Western blots were performed with the NovexNuPAGE SDS-PAGE gel system (Invitrogen) in MOPS-SDS,according to the manufacturer's instructions as described previ-ously (24). To isolate proteins from three-dimensional (3D)cultured Caco-2 cells, Matrigel inserts were recovered prior to theprotein isolation with Matrisperse (BD Bioscience), according to

the manufacturer's instructions. Proteins were visualized with anOdyssey Infrared Imager (LI-COR Biosciences) for near-infrared(near-IR) fluorophore-conjugated antibodies. Near-IR fluores-cence was quantified using LI-COR Image Studio Software. Pri-mary antibodies used for Western blot analysis are listed inSupplementary Table S1.

Quantitative RT-PCR (real-time PCR)Total RNA from cells was isolated with the GeneJet RNA

purification Kit (#K0732; Thermo Fisher). For assaying mRNAlevels, RT reactions were performed with oligo-dT primers usingSuperScript III (Invitrogen). For miRNAs, RT reactions wereperformed using the miRNA RT Kit (#4366596; Life Technolo-gies), according to the manufacturer's instructions. QuantitativePCR utilized the Fast SYBR (Invitrogen) or TaqMan FastUniversal (Invitrogen) master mixes. Taqman probes for matureLET-7 miRNAs were obtained from Life Technologies(Cat. # 4427975, assay numbers 000377, 002406, 000382, and002282). LET-7 levels were normalized to U6 snRNA(Cat. # 4427975, assay numbers 001973; Life Technologies), andmRNA levels were normalized to GAPDH or PPIA. Primersequences are listed in Supplementary Table S2. Gene expressiondata are expressed as a fold change normalized to themean valuesfor controls. All experiments were conducted at least in threeindependent settingswith technical replicates (duplicates) in eachexperiment. To analyze themRNA expression levels of the LIN28Bisoforms, we designed RT-PCR primer sets (SupplementaryFig. S1A). Primer set 1 can measure relative mRNA expression ofLIN28B-long isoform; primer set 2 can measure relative mRNAexpression of overall LIN28B. The relative mRNA expression ofLIN28B-short isoform can be calculated by subtracting relativeexpression of LIN28B-long isoform from that of overall LIN28B.The primer sets were designed to match the efficiency (ref. 34;Supplementary Fig. S1B).

LIN28B shRNAknockdown and generation of LIN28B-long and-short isoform–expressing cells

LIN28B shRNA was cloned into the BII-mirT3G2B shRNAvector, which is a piggyBac (PB)-based vector that we generatedfor achieving inducible, stable shRNAexpression. LIN28B shRNAswas inserted at unique BamHI and SalI sites in the BII-mirT3G2Bvectors. Oligonucleotides for the LIN28B shRNA were obtainedfrom Invitrogen (Supplementary Table S3), annealed as per themanufacturer's instructions, and then ligated 1:1 along withmirBXL adapters (Supplementary Table S3) into unique BamHIand SalI sites in the BII-mirT3G2B vector. The BII-mirT3GB vectoris a tet-inducible vector containing the rtTA-M2 reverse tetracy-cline transactivator (35). Approximately 2.5 � 105 Caco-2 wereseeded in 6-well plates and 16 to 24 hours later were transfectedwith 500 ng of the pCMV-hyPBase transposase (36) and 1,500 ngof the respective PB transposon vector using 6mLof Lipofectamine2000 (Life Technologies) in 1 mL of antibiotic-free DMEM con-taining 10% FBS. Fresh medium was exchanged after 16 to 24hours, and 48hours after transfection, and then cells were selectedwith 10 mg/mL blasticidin (B-800; Gold Biotechnology).

To generate LIN28B-short or -long isoform–expressing cells,LIN28B-short or -long isoform plasmids were cloned from theMSCV-PIG-LIN28B plasmid (21). To prevent the knockdown oftransferred LIN28B isoforms by shLIN28B, we induced mutationsin LIN28B-coding sequences (LIN28B-mutant) to be resistant to

Mizuno et al.

Mol Cancer Res; 16(3) March 2018 Molecular Cancer Research404




shLIN28B using the QuikChange Site-Directed Mutagenesis kit(#200518; Agilent), according to the manufacturer's instruction.The mutagenic oligonucleotides that are resistant to shLIN28Bused are shown in Supplementary Table S3. The resulting mutantplasmids were verified by DNA sequencing.

To generate the LIN28B-short isoform plasmid, the MSCV-PIG-LIN28B–mutant plasmid was digested and inserted intothe PB-EF1-MCS-IRES-NEO vector (PB533A-2; System Biosci-ence) at the NheI site using the PiggyBac Transposon System.For the LIN28B-long isoform plasmid, the MSCV-PIG-LIN28B–mutant plasmid was digested with PstI and blunted with T4DNA polymerase. The plasmid was then digested and insertedinto PB-EF1-MCS-IRES-NEO vector at the Xbal and SwaI sites.Approximately 1 � 106 LIN28B-knockdown Caco-2 or wild-type LoVo cells were seeded in 6-well plates and 16 to 24 hourslater were transfected. After 48 hours of transfection, Caco-2 orLoVo cells were selected with 0.6 or 1 mg/mL neomycin(G-418; Gold Biotechnology), respectively.

To generate the LIN28B-short and -long isoform coexpressioncells, the LIN28B-short isoform plasmid (PB-EF1-LIN28B-short-IRES-NEO)was digested and inserted into the PB-CMV-MCS-EF1-GFP-Puro vector (PB513B-1; System Biosciences) at theNheI andEcoRI sites. The LIN28B-long isoform–expressing Caco-2 cells orLoVo cells were transfected using the PiggyBac Transposon Sys-tem. Caco-2 and LoVo cells were selected with 5 mg/mL or 0.5 mg/mL puromycin (P-600; Gold Biotechnology), respectively. In allexperiments, Caco-2 cells with shLIN28B were treated with 250ng/mLdoxycycline (Sigma-Aldrich) for at least 48 hours to induceshLIN28B expression.

3D culturesTo grow Caco-2 cells in 3D conditions, 5 � 103 cells were

embedded in 40 mL of Matrigel (#356234,BD Biosciences) andcultured in DMEMþ 10% FBSþ 1% P/S at 37�C in 5% CO2. Themedium was changed every 2 days. In the MEK inhibitor treat-ment experiment, U0126 (#662005, Millipore) was added in the

A

C

LIN28B-Long isoform (250 amino acids)

LIN28B-Short isoform (180 amino acids)

ZFDCSD

ZFD

LIN28B-Long

LIN28B-Short

GAPDH

Caco-2

HCT116

SW48

0

LoVo

Colo20

5

HepG2

Huh7.5

T84

B

LIN28B-long

LIN28B-short

GAPDH

TN TN TN TN TN TN TN TN

LIN28B-long

LIN28B-short

GAPDH

TN TN TN TN TN TN TN

#1 #2 #3 #4 #5 #6 #7 #8

#9 #10 #11 #12 #13 #14 #15

Figure 1.

Expression of LIN28B isoforms in human colorectal cancer cell lines and tissues. A, Schematic representation of LIN28B-long and -short isoforms. The LIN28B-longisoform is 250 amino acids with two RNA-binding domains: CSD and two CCHC-type ZFD. LIN28B-short isoform is 180 amino acids long and lacks the CSD domain.B, Distribution of LIN28B isoforms in human cancer cell lines byWestern blotting (WB) analysis. C,WB analysis of LIN28B isoforms in 15 pairs of deidentified humancolorectal cancers and adjacent normal mucosa.

LIN28B-Short Isoform Antagonizes the Long Isoform

www.aacrjournals.org Mol Cancer Res; 16(3) March 2018 405




medium at a concentration of 10 mmol/L from day 2. In thecontrol group, the same amount of dimethyl sulfoxide (DMSO)was added. To quantify the morphology of Caco-2 cells in 3Dculture, cells were stained with Hoechst 33342 (#62249; ThermoFisher Scientific). Hoechst 33342 was added in the medium at aconcentration of 40 mmol/L and cultured for 30 minutes in thetissue culture incubator. After PBS washes (x3), cells were imagedusing a spinning-disk confocal microscope (ECLIPSE Ti; Nikon)with an ORCA-ER camera (C4742-95-12ERG; Hamamatsu Pho-tonics). 3D Caco-2 cells were imaged using a 20� objective(S Plan Fluor, NA 0.45; Nikon). Images were acquired andanalyzed with MetaMorph software (Molecular Devices). 3DCaco-2 structures with a single epithelial cell layer were classifiedas empty cysts; 3D Caco-2 structures with multiple epithelial celllayers or with invading cells into lumen were classified as luminalaccumulating cysts. The quantification of the morphology of 3DCaco-2 cells was performed in a blinded manner.

Cell viability assaysCell viability was quantified using the colorimetric dye

WST-1 (#5015944001; Sigma-Aldrich). Caco-2 or LoVo cells

were seeded at a density of 1 � 104 cells/well in 96-well plates.After 6 hours of cell seeding, the WST-1 assay was performed toobtain time 0 values. Absorbance was measured at 450 nmusing a Microplate reader. Cells were incubated for 24 hours in96-well plates. 5-Fluorouracil (5-FU, Sigma-Aldrich) or cisplat-in (Sigma-Aldrich) was then added at the final concentration of0, 5, 10, 20, 40, or 80 mg/mL. After 24-hour incubation, theWST-1 assay was performed. The cell viability was determinedas percent viability compared with the vehicle control. Allexperiments were performed independently at least 3 timeswith 6 replicates.

LET-7 inhibition and transduction in Caco-2 cellsFor LET-7 inhibition experiments, LIN28B-null Caco-2 cells

were transfected in 6-well plates with 90 pmol of Anti-miR let-7(AM17000: hsa-let-7a-5p; Thermo Fisher Scientific) or negativecontrol 1 inhibitors (AM17010: Anti-miR miRNA Inhibitor Neg-ative Control #1; Thermo Fisher Scientific) using LipofectamineRNAiMAX (Thermo Fisher Scientific) according to the manufac-ture's instruction. Two days after transfection, LET-7 expressionwas checked by qPCR as described. For LET-7 mimetic transfection

A

GAPDH

Caco-2WT Null Short Long

LoVoNull Short Long

ns shRNA

LIN28B shRNAEmpty

LIN28B-ShortLIN28B-Long

+-+--

-++--

-+-+-

-+--+

LIN28B-Short

LIN28B-Long

--+--

---+-

----+

BCaco-2

****

0

0.5

1

1.5

LET-7a LET-7e LET-7g

Exp

ress

ion

rela

tive

to n

ull ***

*** NullShortLong

*

LoVo

0

0.5

1

1.5

2

2.5

3


Exp

ress

ion

rela

tive

to n

ull

** ** ***

WT-----

R² = 0.3986P = 0.0154

R² = 0.4698P = 0.0068

R² = 0.2839P = 0.0498

C

Figure 2.

LIN28B isoform differentially regulatesLET-7. A, Western blot of WT, LIN28B-null, LIN28B-short, and LIN28B-longisoform–expressing Caco-2 cells andWT, empty vector, LIN28B-shortisoform–, and LIN28B-long isoform–

expressing LoVo cells. GAPDH wasused as a loading control. Caco-2 cellswere treated with 250 ng/mL ofdoxycycline (DOX) for at least 3 days.B, Measurement of individual LET-7miRNAs by qPCR analysis. Results werenormalized to U6 snRNA. Data areshown as mean � SE. The experimentwas performed independently 3 timeseach with duplicates. � , P < 0.05;�� , P < 0.005; �� , P < 0.001. C, Theexpression ratio between the mRNAsof LIN28B-long and -short isoformnegatively correlates with LET-7a,LET-7e, and LET-7g expression.Fourteen samples were used in thisanalysis.

Mizuno et al.





experiments, LIN28B-long Caco2-cells were transfected in 6-wellplates with 90 pmol of mirVana miRNA mimic (#4464066,MC10050, hsa-let-7a-5p; Thermo Fisher Scientific) or mirVanamiRNA Mimic, Negative Control #1 (#4464058; Thermo

Fisher Scientific) using Lipofectamine RNAiMAX (Thermo FisherScientific) according to the manufacturer's instructions. Two daysafter transfection, LET-7a expression was checked by qPCR asdescribed.

LIN28B-LongDMSO U0126

BF

Hoe

chst

Null Short Long

BF

Hoe

chst

**

B

C

D

Null Short Long

Caco-2Null Short Long

LoVo

RAS

GAPDH

***

% o

f Tot

al s

truct

ures

Arb

itrar

y un

it (R

AS

/GA

PD

H)

Caco-2 **

0

0.5

1.5

1.0

Null Short Long

NS NS

Arb

itrar

y un

it (R

AS

/GA

PD

H)

LoVo

0

0.5

1.0

1.5

Null Short Long

***

****

Phospho-ERK

ERK

GAPDH

Caco-2Null Short Long

A

NS ***

Figure 3.

LIN28B-long isoform upregulates RAS/ERK signaling through LET-7suppression resulting in luminal cellaccumulation in 3DCaco-2 cysts.A, Theexpression of RAS in Caco-2 or LoVocells was analyzed byWestern blot. Thegraphs show the densitometry. Resultswere normalized to GAPDH. Data areexpressed as the mean � SE of threeindependent experiments. � , P < 0.05and �� , P < 0.005. B, Caco-2 cells werecultured in Matrigel and imaged at day10. Representative images of brightfield (BF) and nuclear staining byHoechst 33342 are shown. Bar, 100 mm.The graph shows the quantification ofthe morphology of 3D Caco-2structures. The mid-plane of >30structures was imaged for eachcondition in three independentexperiments and classified as havingempty lumen (Empty) or luminal cellaccumulation. Data are shown as mean�SE. �� ,P<0.001.C,The expressionofPhospho-ERK and ERK of Caco-2 cellswas analyzed by Western blot. Thegraph shows the densitometry. Resultswere normalized to GAPDH. Data areexpressed as the mean � SE of fiveindependent experiments. �� ,P<0.005.D,3DCaco-2 cells expressingLIN28B-long isoforms were treatedwith 10 mmol/L of U0126 or DMSO fromday 2 and imaged at day 10. Left plotsshow the representative brightfield andHoechst 33342 staining images. Scalebar, 200 mm. The mid-plane of >30structures was imaged for eachcondition in three independentexperiments. Data representmean � SE. �� , P < 0.005.






0

0.51.01.52.0

Null Short Long

Arb

itrar

y un

it (E

RC

C1/

GA

PD

H)

****

0

0.5

1.0

1.5

Null Short Long

Arb

itrar

y U

nit

(ER

CC

1/G

AP

DH

) **

A

B

D

Caco-2 LoVoCaco-2

LongShortNull

ERCC1

GAPDH

C

ERCC1

GAPDH

LoVoNull LongShort

Control inhibitor

Arb

itrar

y un

it (E

RC

C1/

GA

PD

H) *

012345

LET-7a00.20.40.60.81.01.2

LET-7a

LET-

7a E

xp.R

el.

to c

ontro

l

*

ERCC1

GAPDH

ControlLET-7aInhibitor

ControlERCC1

LET-7amimics

GAPDHLET-

7a E

xp.R

el.

to c

ontro

l

1.5

0

2.5

1.0

2.0

0.5

LET-7a

Arb

itrar

y un

it (E

RC

C1/

GA

PD

H)

0

0.40.2

0.60.81.01.2

Control mimicLET-7a

FE

0

20

40

60

80

100

200 40 60 80

Cisplatin

***

**

020

40

60

80

100

Cel

l via

bilit

y (%

of c

ontro

l)5-FU ( g/mL)

5-FU

**

**

0

204060

80100

200 40 60 80Cel

l via

bilit

y (%

of c

ontro

l)

5-FU

****** ************

*

0

20

40

60

80

100

200 40 60 80Cel

l via

bilit

y (%

of c

ontro

l)

Cisplatin

*****

*

***

5-FU ( g/mL)

Cel

l via

bilit

y (%

of c

ontro

l)

Caco-2

LoVo

200 40 60 80

Cisplatin (µg/mL)

Cisplatin (µg/mL)

Mizuno et al.





Electrophoretic mobility shift assayThe preparation of cell extracts was performed based upon a

previous study (37). Briefly, cultured cells were washed twice withice-cold PBS and scraped with a plastic cell scraper in 1 mL of ice-cold PBS. Cells were spun in a microfuge for 5 minutes at 700 x g,at 4�C. Then, 100 mL of ice-cold lysis buffer containing 1% Tritonx100, 25mmol/L Tris-HCl (pH 7.4), and 40mmol/L KCL per 107

cells was added and incubated for 20 minutes on ice. Sampleswere spun at full speed in amicrofuge at 4�C, and supernatantwascollected. Pre-let-7 (PM12304, PM10050; Thermo Fisher Scien-tific) was labeled with Texas-Red fluorescence using the 50 EndTagLabeling DNA/RNA Kit and Texas Red Maleimide (Vector Labo-ratory), according to the manufacturer's instructions. Bindingreactions were conducted with 1 ng of Texas-Red–labeled pre-let-7 in a total volume of 20 mL together with 50 mg of yeast tRNAand indicated amounts of protein extracts. The labeled RNA wasincubated at 37�C for 2 minutes and cooled on ice for 3 minutes.The binding buffer contained 20 mmol/L Tris/HCL, pH 7.5, 60mmol/L KCL, 20 U of RNase inhibitor (Thermo Fisher Scientific;#10777019), and 1mmol/L DTT. After 30minutes of incubationat room temperature, 4 mL of loading buffer was added to theelectrophoretic mobility shift assay (EMSA) sample and resolvedonaDNAretardation gel (EC6365BOX; ThermoFisher Scientific).RNA bands were visualized using ChemiDoc MP Imaging System(Bio-Rad).

Statistical analysesUnless indicated otherwise in the text or figure legends,

unpaired, two-tailed Student t tests were performed to determinestatistical significance of comparisons between control and exper-imental cell lines with P < 0.05 as statistically significant. For allanalyses, unless noted otherwise, data from a minimum of threeindependent experiments were presented as mean � SEM.

ResultsLIN28B-long isoform is predominantly expressed in humancolorectal cancer tissues

LIN28B has two isoforms, namely the LIN28B-long andLIN28B-short isoforms. The LIN28B-long isoform consists of250 amino acids and has two RNA-binding domains: the CSDin its N-terminus and two CCHC type ZFDs in its C-terminus. TheLIN28B-short isoform lacks 70 amino acids in theN-terminus anddoes not have a complete CSD (ref. 33; Fig. 1A). To date, studieshave not evaluated the functional and mechanistic differencesbetween the isoforms.

To elucidate the role of the LIN28B-short isoform in thepathogenesis of colon cancer, we first examined the expression

of the LIN28B isoforms in human colorectal cancer cell lines.Western blotting revealed that some colorectal cancer cell lines,such as Caco-2 and HCT116, express endogenous LIN28B. Caco-2 cells express high LIN28B levels, including both isoforms,whereas HCT116 cells express a modest level of the LIN28B-longisoform. We could not detect any significant levels of theLIN28B isoforms in other colorectal cancer cell lines tested. Wealso evaluated LIN28B expression in HCC cell lines, HepG2 andHuh7.5. Both cell lines expressed high levels of the LIN28B-longisoform (Fig. 1B).

Next, we examined the distribution of LIN28B isoforms in15 pairs of deidentified human colorectal cancers and matchednormal colorectal tissues. Here, we used an anti-LIN28B anti-body whose antigen surrounds 240 amino acid position ofLIN28B, which can detect both of LIN28B isoforms. LIN28B-long isoform was predominantly expressed in 5 of 15 cancers(samples #1–5; Fig. 1C). A previous study of colorectal cancerdemonstrated that approximately 28% of these samples havehigh LIN28B expression (22), and our study is in concordance.We also found that the expression of LIN28B-short isoform wasobserved predominantly in the adjacent normal tissues (sam-ples #1–6; Fig. 1C). LIN28A proteins are reported to haveisoforms produced by alternative splicing in several animalspecies (1). Guo and colleagues identified the LIN28B isoformsfrom 50-RACE analysis using a cDNA library of fetal liver (33).This suggests that alternative splicing may produce the LIN28Bisoforms. Therefore, we designed RT-PCR primer sets to inves-tigate the mRNA expression levels of the LIN28B isoforms(Supplementary Fig. S1). We evaluated the mRNA expressionof each LIN28B isoform in human colorectal cancer tissues andmatched adjacent normal mucosa in an independent cohort of31 colorectal cancer patients (protein not available). Here, 7samples showed more than a 2-fold increase of overall LIN28Bexpression in tumor tissue. In these samples, the LIN28B-longand -short isoforms are predominantly expressed in cancer andnormal colon, respectively (Supplementary Fig. S2).

The LIN28B isoforms differentially regulate LET-7 expressionTo investigate the functional roles of each LIN28B isoform in

the pathogenesis of colon cancer, we generated specific LIN28Bisoform–expressing colorectal cancer cells. For this purpose, weused Caco-2 cells and LoVo cells. Because Caco-2 cells have highlevels of endogenous LIN28B-long and -short isoforms, we firstknocked out the endogenous LIN28B by shRNA and generatedLIN28B-null cells. Then, LIN28B-short or LIN28B-long isoformswere transduced stably into the LIN28B-null cells to generateLIN28B-short or -long–specific isoform-expressing Caco-2 cells.In LoVo cells, the specific LIN28B isoformswere transduced stably

Figure 4.LIN28B-long isoform contributes to the acquisition of drug resistance. A, Cell viability was analyzed by theWST-1 assay. 1� 104 Caco-2 or LoVo cells were seeded intriplicate. Various concentrations of 5-FU or cisplatin were added after 24 hours of cell incubation. The WST-1 assay was preformed 24 hours after drug treatment.Data are the mean � SE of three independent experiments. Black or blue asterisks show the statistical significances between LIN28B-long versus LIN28B-null andLIN28B-long versus LIN28B-short, respectively. � , P < 0.05; �� , P < 0.005; ��, P < 0.001. B, Left plots show the representative WB images of ERCC1 in Caco-2or LoVo cells. Right graphs show the densitometry. Results were normalized to GAPDH. Data are expressed as the mean � SE of three independent experiments.� , P <0.05 and �� , P <0.005.C,Measurement of LET-7amiRNAs in LIN28B-null Caco-2 cells transfectedwith LET-7a inhibitor or negative control precursormiRNAbyqPCR analysis. Results were normalized to U6 snRNA. Data are shown as mean� SE. The experiment was performed independently 3 times. � , P < 0.05. D, Left plotshows the representativeWB images of ERCC1 and GAPDH in LIN28B-null Caco-2 cells transfected with LET-7a inhibitor or negative control precursor miRNA. Rightgraphs show the densitometry. Results were normalized to GAPDH. Data are expressed as the mean � SE of three independent experiments. � , P < 0.05. E,Measurement of LET-7amiRNAs in LIN28B-long Caco-2 cells transfected with LET-7amimetic or negative control miRNA by qPCR analysis. Results were normalizedto U6 snRNA. Data are shown as mean � SE. The experiment was performed independently 3 times. ��, P < 0.0001. F, Left plots show the representative WBimages of ERCC1 and GAPDH in LIN28B-long Caco-2 cells transfected with LET-7amimetic or negative control miRNA. Right graphs show the densitometry. Resultswere normalized to GAPDH. Data are expressed as the mean � SE of three independent experiments. � , P < 0.05.






into wild-type LoVo cells because LoVo cells do not expressendogenous LIN28B as shown in Fig. 1B (Fig. 2A).

LIN28B binds to LET-7 miRNA precursors and inhibits theirprocessing, resulting in the decrease of mature LET-7miRNAs (8,9, 10, 11, 12). Because the LIN28B-short isoform lacks the CSD,

we predicted that the maturation of LET-7 miRNA might beaffected differentially between the LIN28B-short and -long iso-form–expressing cells. Therefore, we first evaluated the expressionlevels of the mature LET-7 miRNAs using the cells that weregenerated. To that end, LET-7 expression was decreased in

pre-LET-7a pre-LET-7eLIN28B-long

LIN28B-null

LIN28B-long-pre-let-7a

LIN28B-long-pre-let-7a

LIN28B-short-pre-let-7a

+ + + + + + + + + +

pre-LET-7a pre-LET-7eLIN28B-long

LIN28B-short+ + + + + + + + + +

LIN28B-longLIN28B-shortLIN28B-long-pre-let-7a

LIN28B-short-pre-let-7a

Free pre-let-7a Free pre-let-7a

pre-LET-7a pre-LET-7e

LIN28B-null

Free pre-let-7

LIN28B-longLIN28B-short

LIN28B-long-pre-let-7aLIN28B-short

-pre-let-7a

Free pre-let-7a Free pre-let-7a

µµ

A

B C

ED

GF

µ µ

Figure 5.

LIN28B-short isoform competes with the LIN28B-long isoform for binding to pre-let-7. A, EMSA with Texas-Red–labeled pre-let-7a or pre-let-7e as a probe, mixedwith increasing concentration of whole-cell protein extracts from LIN28B-null LoVo cells. A total of 0, 3, 6, 12, or 24 mg of protein extracts were used. B, EMSA withpre-let-7a as a probe, mixedwith increasing concentration of whole cell protein extracts from LIN28B-short isoform–expressing LoVo cells. A total of 0, 0.75, 1.5, 3, 6,12, or 24 mg of protein extracts were used. Right graph shows the bound fraction of pre-let-7a against the total amount. C, EMSA with pre-let-7e as a probe,mixed with increasing concentration of whole cell protein extracts from LIN28B-short isoform–expressing LoVo cells. A total of 0, 0.75, 1.5, 3.6, 12, or 24 mg of proteinextracts were used. Right graph shows the bound fraction of pre-let-7 against the total amount. D, EMSA with pre-let-7a as a probe, mixed with increasingconcentration of whole cell protein extracts from LIN28B-long isoform–expressing LoVo cells. A total of 0, 0.75, 1.5, 3, 6, 12, or 24 mg of protein extracts were used.Right graph shows the bound fraction of RNA against the total amount. E, EMSAwith pre-let-7e as a probe, mixedwith increasing concentration of whole cell proteinextracts from LIN28B-long isoform–expressing LoVo cells. A total of 0, 0.75, 1.5, 3, 6, 12, or 24 mg of protein extracts were used. Right graph shows thebound fraction of RNA against the total amount. F, EMSA using the mixture of Texas-Red–labeled pre-let-7a or pre-let-7e and 12 mg of protein extract from LIN28B-long isoform–expressing LoVo cells with increasing concentration of protein from LIN28B-null LoVo cells (Lin28B-null cell extract: 0, 3, 6, 12, 24 mg). G, EMSAusing the mixture of Texas-Red–labeled pre-let-7a or pre-let-7e and 12 mg of protein extract from LIN28B-long isoform–expressing LoVo cells with increasingconcentration of protein from LIN28B-short isoform–expressing LoVo cells (Lin28B-short cell extract: 0, 3, 6, 12, 24 mg).

Mizuno et al.





LIN28B-long isoform–expressing cells compared with LIN28B-null cells. By contrast, the expression levels of LET-7 miRNAs inLIN28B-short isoform–expressing cells were not changed com-pared with LIN28B-null cells and significantly higher than thoseof LIN28B-long isoform–expressing cells (Fig. 2B). We evaluatedthe relationship between LET-7 expression and the expressionratio of the LIN28B-long and -short isoform. We found that theLET-7 expression correlates negatively with the increase in theLIN28B-long isoform/LIN28B-short isoform ratio (Fig. 2C).

The LIN28B-long isoform suppresses LET-7/RAS/ERK to induceluminal cell accumulation in 3D Caco-2 cysts

As there is a significant difference in the inhibitory effect on thelevels of mature LET-7miRNAs between the LIN28B isoforms, wenext investigated the roles of each LIN28B isoform on the RASsignalingpathway, known to involve LET-7 (38).We foundhigherRAS levels in LIN28B-long isoform–expressing cells comparedwith LIN28B-null or LIN28B-short isoform–expressing cells. Nosignificant difference was observed between LIN28B-null andLIN28B-short isoform–expressing cells (Fig. 3A). This expressionpattern of RASwas compatible with the expression pattern of LET-7 miRNAs as shown in Fig. 2b.

Caco-2 cells are known to form clear cystic structures when theyare cultured under 3D conditions (39). The activation of RAS/ERKsignaling is reported to induce cell accumulation in the lumens ofthe 3D cysts (40). Since the LIN28B-long isoform increases RASexpression, we examined the morphology of Caco-2 cells expres-sing each LIN28B isoform in 3D. LIN28B-null and LIN28B-shortisoform–expressing Caco-2 cells formed clear cystic structures. Bycontrast, LIN28B-long isoform–expressing Caco-2 cells showed asignificant increase of 3D cysts accumulating cells in the lumen(Fig. 3B). The phosphorylation of ERK was activated in LIN28B-long isoform–expressingCaco-2 cells in 3D (Fig. 3C). The luminalcell accumulation in LIN28B-long isoform–expressing cells wassignificantly inhibited by the MEK inhibitor, U0126 (Fig. 3D).Taken together, these results indicate that the LIN28B-long iso-form upregulates RAS/ERK signaling through the inhibition ofLET-7 miRNAs, thereby resulting in the accumulation of cells inthe lumens of Caco-2 3D cysts.

The LIN28B-long isoform induces chemoresistance through theupregulation of ERCC1 in a LET-7–dependent manner

It has been reported that increased LIN28B or decreased LET-7 expression contributes to the acquisition of chemotherapyresistance in cancers (26–29). To test this, we evaluated theviability of the cell lines expressing the LIN28B isoforms treatedwith either cisplatin or 5-FU, two standard chemotherapeuticagents. LIN28B-long isoform–expressing cells demonstrated asignificant increase in drug resistance compared with LIN28B-null cells. Notably, LIN28B-short isoform–expressing cells didnot confer any drug resistance either (Fig. 4A). Previous studieshave reported that excision repair cross-complementing group1 (ERCC1) expression is associated with drug resistance incolon cancer (41, 42). We found that ERCC1 expression wasupregulated only in LIN28B-long isoform–expressing cells (Fig.4B). As chemoresistance and ERCC1 expression showed anegative correlation pattern with LET-7 miRNAs (Figs.2B, 4A, and 4B), we examined whether ERCC1 expression isnegatively regulated by LET-7. We transfected a LET-7 inhibitoror negative control into LIN28B-null Caco-2 cells and com-pared ERCC1 expression (Fig. 4C). ERCC1 expression was

higher in LET-7–inhibited cells (Fig. 4D). We also transduceda LET-7 mimetic into LIN28B-long isoform–expressing Caco-2cells and found that ERCC1 expression was significantlydecreased in LET-7–transduced cells compared with controlcells (Fig. 4E and F). These results suggest that the LIN28B-long isoform may upregulate ERCC1 in a LET-7–dependentmanner.

The LIN28B-short isoform competes with the LIN28B-longisoform for binding to pre-let-7

Wenext examinedwhether thedifferential regulationofmatureLET-7 by the LIN28B isoforms is caused by the loss of the bindingability to LET-7 precursor due to the incomplete CSD in theLIN28B-short isoform. For this purpose, we performed EMSA tocheck the binding ability of each isoform to the LET-7 precursor,pre-let-7. Protein extract from LIN28B-null cells did not formprotein-pre-let-7 complex (Fig. 5A). Protein extracts from eitherLIN28B-long isoform–expressing cells or LIN28B-short isoform–

expressing cells formed the LIN28B protein-pre-let-7 complex in adose-dependent manner (Fig. 5B–E). These data suggested thatthe LIN28B-short isoform does not have the inhibitory effects onthe maturation of LET-7 miRNAs while preserving the bindingability to pre-let-7. These data prompted us to hypothesize that theLIN28B-short isoform could antagonize the LIN28B-long iso-form–mediated binding to pre-let-7. To test this hypothesis, weperformed EMSA using pre-let-7 and protein extracts fromLIN28B-long isoform–expressing cells at the concentration thatfree pre-let-7 completely diminishes with increasing concentra-tion of protein extract from LIN28B-null or LIN28B-short iso-form–expressing cells. Protein extracts from LIN28B-null cells didnot prevent the formation of the LIN28B-long isoform-pre-let-7complex (Fig. 5F). By contrast, as the concentration of proteinextract of LIN28B-short isoform–expressing cells increased, theLIN28B-long isoform-pre-let-7 complex decreased and theLIN28B-short isoform-pre-let-7 complex increased (Fig. 5G). Thisindicates that LIN28B-short isoform competes with the LIN28B-long isoform for binding to pre-let-7 and may function as anantagonist to the LIN28B-long isoform.

The LIN28B-short isoformantagonizes the phenotypes inducedby the LIN28B-long isoform

We next examined whether the LIN28B-short isoform antag-onizes the phenotypes induced by the LIN28B-long isoform in aLET-7-dependent manner. For this purpose, we generatedLIN28B-short isoform and -long isoform–coexpressing Caco-2or LoVo cells (Fig. 6A and B). Coexpression of LIN28B-shortisoform significantly increased the expression of mature LET-7miRNAs comparedwith the cells expressing only the LIN28B-longisoform (Fig. 6C).We also expressed the LIN28B-short isoform inHuh7.5 cells, known to have high endogenous LIN28B-longisoform expression (Fig. 1B; Supplementary Fig. S3A and S3B).The expression of mature LET-7a was significantly increased inLIN28B-short isoform–expressing Huh7.5 cells compared withwild-type Huh7.5 cells (Supplementary Fig. S3C). Coexpressionof the LIN28B-short isoform decreased the RAS expressioninduced by LIN28B-long isoform in Caco-2 or LoVo cells (Fig.6D). Coexpression of LIN28B-short isoform inhibited the lumi-nal cell accumulation in 3D-cultured Caco-2 cells and increasedthe formationof 3Dcystswith clear lumens (Fig. 6E). The LIN28B-short isoform also decreased ERCC1 expression and increased thesensitivity to chemotherapy (Fig. 6F and G).






D

LoVo

LIN28B Long

LIN28B Short

GAPDH

L+Emp L+S Caco-2

A

B

C

Ras

GAPDH

LoVoL+Emp L+S

Caco-2L+Emp L+S

L+Emp L+SCaco-2

ERCC1

GAPDH

L+Emp L+S

Long + Empty Long + Short

BF

Hoe

chst

Caco-2

LET-7a LET-7e LET-7g0

0.5

1

1.5

2

2.5

Exp

. Rel

. to

L+E

mp L+Emp

L+S

0

0.5

1

1.5

2


LoVo

Exp

. Rel

. to

L+E

mp

E

F

LIN28B-Long Caco-2/LoVo cell

PB-CMV-LIN28B-Short-EF1-GFP-Puro

LIN28B-Long + Empty (L+Emp)

LIN28B-Long + Short (L+S)

G

Arb

itrar

y un

it (R

AS

/GA

PD

H)

00.20.40.60.8

1

L+Emp L+S

Caco-2*

L+Emp L+SLoVo

020406080

100

Caco-2 LoVoCel

l via

bilit

y (%

of c

ontro

l)

Cisplatin* *

Cel

l via

bilit

y (%

of c

ontro

l)

5-FU* *

020406080

100

Caco-2 LoVo

** *

***

0

5

10

15

LIN28B-Long

LIN28B-Short

Arb

itrar

y un

it (L

IN28

B/G

AP

DH

)

*

NS

Caco-2

0

5

10

15

NS

**LoVo

Arb

itrar

y un

it (L

IN28

B/G

AP

DH

)

L+Emp L+S

Arb

itrar

y un

it (R

AS

/GA

PD

H)

**LoVo

00.20.40.60.8

1

L+Emp L+S

Arb

itrar

y un

it (E

RC

C1/

GA

PD

H)

Caco-2 *

L+EmpL+S

LIN28B-Long

LIN28B-Short

L+EmpL+S

Empty Luminal accumulation

% o

f Tot

al s

truct

ures

***

00.20.40.60.8

1

L+Emp L+S

Arb

itrar

y un

it (E

RC

C1/

GA

PD

H)

LoVo *

PB-CMV-MCS-EF1-GFP-Puro

PiggyBac

PB-CMV-LIN28B-Long-EF1-GFP-Neo

Mizuno et al.





DiscussionLIN28B is a critical regulator of genes in development, stem cell

biology, tissue regeneration, and tumorigenesis. LIN28B consistsof 250 amino acids with two RNA-binding domains, CSD andZFDs.During normal development, LIN28B is highly expressed instem cells or progenitor cells and regulates genes related toselfrenewal or proliferation. As progenitor cells differentiate,LIN28B expression is decreased (17). LIN28B is also known tobeupregulated in cancers, such as chronicmyelogenous leukemia,HCC, neuroblastoma, lung, breast, ovary, cervix, and colorectal(18, 19, 20, 21, 23). In human colorectal cancer, about 30%of thetumors overexpress LIN28B (22). LIN28B functions as an onco-gene because transgenic mice expressing LIN28B develop intes-tinal polyps and adenocarcinomas tumors (24, 25). Furthermore,LIN28B cooperates with WNT signaling to drive invasive intesti-nal and colorectal adenocarcinomas (22).

LIN28B has two isoforms, LIN28B-long and LIN28B-shortisoforms. The LIN28B-short isoform lacks 70 amino acids in theN-terminus with an incomplete CSDbut intact ZFDs. The LIN28Bisoforms were identified originally by 50-RACE analysis using acDNA library of fetal liver, which suggests that mRNAs corre-sponding to each LIN28B isoform could be generated by alter-native splicing (33). In fact, LIN28A proteins were reported tohave isoforms produced by alternatively spliced first exons inseveral animal species (1). In silico prediction of alternativesplicing sites using Human Splicing Finder (HSF; ref. 43) suggestspossible alternative splicing sites for the LIN28B-short isoform.The LIN28B-long isoform mRNA consists of 4 exons. The initi-ation of LIN28B-short isoform translation likely starts from thefirst ATG codon in exon 3 (33).The HSF predicts the existence ofseveral alternative donor splicing sites in the region upstream ofthe start codonof LIN28B-long isoform in exon1. Several possibleacceptor splicing sites, including wild-type splicing acceptor site,were found in the region upstream of the start codon of LIN28B-short isoform in exon 3. Therefore, alternative donor site splicingmight occur. Further investigation into the mechanisms throughwhich LIN28B isoforms are produced would be very interesting.

Here, we demonstrated that LET-7 expression was downregu-lated only by the LIN28B-long isoform but not by the LIN28B-short isoform. Consequently, RAS/ERK signaling, which is neg-atively regulated by LET-7 (38), was activated only in the LIN28B-long isoform–expressing cells. This activation of RAS/ERK signal-ing induced luminal cell accumulation of cells in the lumen of 3DCaco-2 cysts in the LIN28B-long isoform–expressing cells. Theupregulation of LIN28B or downregulation of LET-7 has beenreported to contribute to the acquisition of chemoresistance invarious types of cancer (26, 27, 28, 29). LIN28A/B is reported toconfer the emergence of cancer stem cells, which is considered to

contribute to the acquisition of drug resistance (17). In this study,we demonstrated that LIN28B-long isoform induced drug resis-tance in Caco-2 or LoVo cells. By contrast, the LIN28B-shortisoform did not change drug response compared with LIN28B-null cells. ERCC1, which is associated with DNA repair, has beenlinked with poor responses to chemotherapy in several typesof cancers (44, 45, 46, 47, 48). In colorectal cancer, the upregula-tion of ERCC1 also contributes to the acquisition of drug resis-tance (41, 42). We demonstrated that ERCC1 expression wasnegatively regulated by LET-7 using LET-7 inhibition and trans-duction experiments. Although we checked whether the 30UTR inErcc1 contains the conserved LET-7 site using TargetScan.orgprediction, no conserved sites were detected (data not shown).Therefore, we postulate the upregulation of ERCC1 in LIN28B-overexpressing cells may be the result of active RAS/EKR signalingby LET-7 (49, 50, 51).

Although the LIN28B-short isoform loses the ability to inhibitthe maturation of LET-7 miRNAs, EMSA demonstrated that theLIN28B-short isoform preserves the ability to bind the LET-7precursor, pre-let-7, as does the LIN28B-long isoform to pre-let-7 (Fig. 7A and B). The binding affinity to pre-let-7 seemed to besimilar between protein extracts from LIN28B-long and -shortisoform–expressing cells. It is difficult to measure the precisebinding affinity of each LIN28B isoform to pre-let-7 because wedid not use purified proteins but rather protein extracts from cellsin our experimental conditions. However, the binding affinity ofeach isoform to pre-let-7 can be assumed to be similar or at leastnot significantly different because they have the similar relativeexpression levels to the housekeeping gene, GAPDH,whichmightsuggest that the concentration of each isoform protein in the cellextractsmay be similar. A previous study revealed that the bindingaffinity between the ZFDs of LIN28 and pre-let-7g was similar tothe binding affinity between the whole LIN28 and pre-let-7g,whereas the binding affinity between CSD and pre-let-7g wassignificantly lower (52). Taken together, the LIN28B-short iso-form, which has ZFDs, could have similar or at least not signif-icantly lower affinity to ple-let-7 compared with the LIN28B-longisoform. Furthermore, it has been noted that both the CSD andZFDs in LIN28A are necessary for the inhibition of thematurationof LET-7 (9). The addition of the LIN28B-short isoform to themixture of the LIN28B-long isoform and pre-let-7 decreased theformation of LIN28B-long isoform-pre-let-7 complex in a dose-dependent manner. This suggests that the LIN28B-short isoformcould potentially antagonize the LIN28B-long isoform–mediatedbinding to the pre-let-7 in our experimental conditions (Fig. 7C).In fact, the coexpression of the LIN28B-short isoform in colorectalcancer cell lines rescued the phenotypes induced by LIN28B-longisoform in a LET-7–dependent manner. Coexpression of the

Figure 6.LIN28B-short isoform rescues the phenotypes induced by the LIN28B-long isoform. A, Scheme of experimental design. PB513B-1-empty or PB513B-1 LIN28B-shortvectors were transfected intoLIN28B-long isoform–expressing cells. Control cells (LþEmp) and coexpressing cells (LþS) are generated. B, The expression ofLIN28B-long or -short isoforms was confirmed by WB. The graphs show the densitometry. Results were normalized to GAPDH. Data are obtained from threeindependent experiments. � , P < 0.05 and �� , P < 0.005. C, Measurement of individual LET-7 by qPCR analysis. Results were normalized by U6 snRNA. � , P < 0.05.D, The expression levels of RASwere analyzed byWB. The graphs indicate the densitometry ofWB. Resultswere normalized toGAPDH.Data are obtained from threeindependent experiments. � , P < 0.05 and �� , P < 0.005. E, Caco-2 cells expressing LIN28B-long (LþEmp) or LIN28B-long and short isoforms (LþS) are culturedin 3Dcondition. Representative images of brightfield andHoechst 33342 staining are shown. Scale bar, 200mm. Themid-plane of>40 structureswas imaged for eachcondition in three independent experiments and classified as having empty lumen (Empty) or luminal cell accumulation (LA). Data are shown as mean � SE.�� , P < 0.001. F, The expression level of ERCC1 was analyzed byWestern blot. The graphs indicate the densitometry. Results were normalized to GAPDH. Data wereobtained from three independent experiments. � , P < 0.05. G, The cell viability after drug treatment was analyzed by the WST-1 assay. Caco-2 cells were treatedwith 10 mg/mL 5-FU or 40 mg/mL of Cisplatin for 24 hours. LoVo cells were treatedwith 20 mg/mL 5-FU or 40 mg/mL of Cisplatin for 24 hours. Data are obtained fromthree independent experiments each with 5 replicates. All the graph data are the mean � SE. � , P < 0.05.






LIN28B-short isoform increased LET-7 expression, decreased RASexpression with a decrease in luminal cell accumulation in 3DCaco-2, and decreased ERCC1 expression with increased chemo-therapy sensitivity.

In summary, we have elucidated a novel role of the LIN28B-short isoform in the context of colorectal cancer. The LIN28B-short isoformdoes not suppress LET-7. The LIN28B-short isoformmay function as an antagonist against the LIN28B-long isoform innormal colonic epithelial homeostasis, which is abrogated duringcolonic tumorigenesis with the upregulation of the LIN28B-longisoform and the downregulation of the LIN28B-short isoform.These results suggest that a drug could be designed against theCSD of LIN28B that might mimic the LIN28B-short isoform inLIN28B-positive colon cancers.

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

Authors' ContributionsConception and design: R. Mizuno, A. Jeganathan, A.K. RustgiDevelopment of methodology: R. Mizuno, P. Chatterji, A. Jeganathan, B.Madison, A.K. RustgiAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): R. Mizuno, S. Andres, A. Jeganathan, A.K. Rustgi

Analysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): R. Mizuno, P. Chatterji, S. Andres, K. Hamilton,S.W. Foley, B.D. Gregory, A.K. RustgiWriting, review, and/or revision of the manuscript: R. Mizuno, P. Chatterji,S. Andres, K. Hamilton, B.D. Gregory, B. Madison, A.K. RustgiAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): A. Jeganathan, A.K. RustgiStudy supervision: B.D. Gregory, A.K. Rustgi

AcknowledgmentsThis work was supported by the NIH/NIDDK P30DK050306,

R01DK056645, K01DK093885, NIH K01DK100485 (KEH), Hansen Founda-tion, Lustgarten Family colon cancer grants, and the Penn Colon CancerTranslational Center of Excellence.

The authors thank the Molecular Pathology and Imaging Core (J. Katz,A. Bedenbaugh, and D. Budo), the Human Microbial and Analytic RepositoryCore (G. Wu and L. Chau), and the Cell Culture and iPS Core (E. Morrisey,W. Yang, and H. Nakagawa).

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

Received September 18, 2017; revised November 7, 2017; accepted Novem-ber 29, 2017; published OnlineFirst January 12, 2018.

References1. Moss EG, Tang L. Conservation of the heterochronic regulator Lin-28, its

developmental expression and microRNA complementary sites. Dev Biol2003;258:432–42.

2. Yang D, Moss EG. Temporally regulated expression of Lin-28 in diversetissues of the developing mouse. Gene Expr Patterns 2003;3:719–26.

3. Yu J, Vodyanik MA, Smuga-otto K, Antosiewicz-bourget J, Frane JL, Tian S,et al. Induced pluripotent stem cell lines derived fromhuman somatic cells.Science 2007;318:1917–20.

4. Shyh-chang N, Daley GQ. Review lin28 : primal regulator of growth andmetabolism in stem cells. Stem Cell 2013;12:395–406.

LIN28B-short isoform competes with -long isoform and prevents its binding to pre-let-7

LIN28B-long isoform binds to pre-let-7 and inhibits its maturation

LIN28B-short isoform binds to pre-let-7 but does not inhibit its maturation

A

B

C

pre-let-7 LIN28B-longLET-7

Degradation

RAS/ERK Signaling ↑ERCC1 ↑Drug resistance ↑

CSDZFD

LIN28B-shortLET-7

Maturationpre-let-7

ZFD

CSDZFD

ZFD

LIN28B-long

LIN28B-shortLET-7

Maturation

Figure 7.

A model of the antagonistic function ofthe LIN28B-short isoform against theLIN28B-long isoform though LET-7regulation. A, The LIN28B-long isoformbinds to pre-let-7 and inhibits itsmaturation affecting the downstreamsignaling of LET-7. B, The LIN28B-shortisoform preserves binding ability topre-let-7 without inhibiting itsmaturation. C, The LIN28B-shortisoform antagonizes the LIN28B-longisoform by competing for binding topre-let-7.

Mizuno et al.





5. Mayr F, HeinemannU.Mechanisms of Lin28-mediatedmiRNA andmRNAregulation — a structural and functional perspective. Int J Mol Sci2013;16532–53.

6. Moss EG, Lee RC, Ambros V. The cold shock domain protein LIN-28controls developmental timing in C. elegans and is regulated by the lin-4RNA. Cell 1997;88:637–46.

7. Balzer E, Moss EG. Localization of the developmental timing regulatorLin28 to mRNP complexes, P-bodies and stress granules. RNA Biol2007;4:16–25.

8. Newman MA, Thomson JM, Hammond SM. Lin-28 interaction with theLet-7 precursor loop mediates regulated microRNA processing. RNA2008;1539–49.

9. Piskounova E, Viswanathan SR, JanasM, Lapierre RJ, DaleyGQ, Sliz P, et al.Determinants of microRNA processing inhibition by the developmentallyregulated. J Biol Chem 2008;283:21310–4.

10. RybakA, FuchsH, Smirnova L, BrandtC, Pohl EE,Nitsch R, et al. A feedbackloop comprising lin-28 and let-7 controls pre- let-7 maturation duringneural stem-cell commitment. Nat Cell Biol 2008;10.

11. Heo I, Joo C, Kim Y, HaM, YoonM, Cho J, et al. TUT4 in concert with lin28suppresses MicroRNA biogenesis through Pre-MicroRNA uridylation. Cell2009;138:696–708.

12. Lightfoot HL, Bugaut A, Armisen J, Lehrbach NJ, Miska EA,Balasubramanian S. A LIN28-dependent structural change in pre-let-7gdirectly inhibits dicer processing. Biochemistry 2011;50:7514–21.

13. Nam Y, Chen C, Gregory RI, Chou JJ, Sliz P. Molecular Basis forinteraction of let-7 microRNAs with Lin28. Cell 2011;147:1080–91.

14. Hagan JP, Piskounova E, Gregory RI. Lin28 recruits the TUTase Zcchc11 toinhibit let-7 maturation in mouse embryonic stem cells. Nat Struct MolBiol 2009;16:1021–5.

15. Thornton JE, Chang H, Piskounova E, Gregory RI. Lin28-mediated controlof let-7 microRNA expression by alternative TUTases Zcchc11 (TUT4) andZcchc6 (TUT7). RNA 2012;11:1875–85.

16. Piskounova E, Polytarchou C, Thornton JE, Lapierre RJ, Pothoulakis C,Hagan JP, et al. Lin28A and Lin28B inhibit let-7 MicroRNA biogenesis bydistinct mechanisms. Cell 2011;147:1066–79.

17. Viswanathan SR, Daley GQ. Lin28: a microRNA regulator with a macrorole. Cell 2010;140:445–9.

18. Viswanathan SR, Powers JT, Einhorn W, Hoshida Y, Toffanin S, SullivanMO, et al. Lin28 enhances tumorigenesis and is associated with advancedhuman malignancies. Nat Genet 2009;41:843–8.

19. Diskin SJ, Capasso M, Schnepp RW, Cole KA, Attiyeh EF, Hou C,et al. Common variation at 6q16 within HACE1 and LIN28Binfluences susceptibility to neuroblastoma. Nat Genet 2012;44:1126–30.

20. Molenaar JJ, Domingo-Fern�andez R, Ebus ME, Lindner S, Koster J, DrabekK, et al. LIN28B induces neuroblastoma and enhancesMYCN levels via let-7 suppression. Nat Genet 2012;44:1199–206.

21. King CE, Cuatrecasas M, Castells A, Sepulveda AR, Lee JS, Rustgi AK.LIN28B promotes colon cancer progression and metastasis. Cancer Res2011;71:4260–8.

22. Tu HC, Schwitalla S, Qian Z, LaPier GS, Yermalovich A, Ku YC, et al.LIN28 cooperates with WNT signaling to drive invasive intestinal andcolorectal adenocarcinoma in mice and humans. Genes Dev 2015;29:1074–86.

23. King CE, Wang L, Winograd R, Madison BB, Mongroo PS, Johnstone CN,et al. LIN28B fosters colon cancer migration, invasion and transformationthrough let-7-dependent and -independent mechanisms. Oncogene2011;30:4185–93.

24. Madison BB, Liu Q, Zhong X, Hahn CM, Lin N, Emmett MJ, et al. LIN28Bpromotes growth and tumorigenesis of the intestinal epithelium via Let-7.Genes Dev 2013;27:2233–45.

25. Madison BB, Jeganathan AN, Mizuno R, Winslow MM, Castells A,Cuatrecasas M, et al. Let-7 represses carcinogenesis and a stem cellphenotype in the intestine via regulation of hmga2. PLoS Genet2015;11:1–21.

26. Lv K, Liu L, Wang L, Yu J, Liu X, Cheng Y, et al. Lin28 mediates paclitaxelresistance by modulating p21, Rb and Let-7a miRNA in breast cancer cells.PLoS One 2012;7:1–8.

27. Sugimura K, Miyata H, Tanaka K, Hamano R, Takahashi T, Kurokawa Y,et al. Let-7 expression is a significant determinant of response to chemo-

therapy through the regulation of IL-6/STAT3 pathway in esophagealsquamous cell carcinoma. Clin Cancer Res 2012;18:5144–53.

28. Chen Y, Jacamo R, Konopleva M, Garzon R, Croce C, Andreeff M. CXCR4downregulation of let-7a drives chemoresistance in acute myeloid leuke-mia. J Clin Invest 2013;123:2395–407.

29. Bhutia YD, Hung SW, Krentz M, Patel D, Lovin D, Manoharan R, et al.Differential processing of let-7a precursors influences RRM2 expressionand chemosensitivity in pancreatic cancer: role of LIN-28 and SET onco-protein. PLoS One 2013;8.

30. Zhu H, Shyh-Chang N, Segre AV, Shinoda G, Shah SP, Einhorn WS,et al. The Lin28/let-7 axis regulates glucose metabolism. Cell 2011;147:81–94.

31. Ma X, Li C, Sun L, Huang D, Li T, He X, et al. Lin28/let-7 axis regulatesaerobic glycolysis and cancer progression via PDK1. Nat Commun2014;5:1–13.

32. Polesskaya A, Cuvellier S, Naguibneva I, Duquet A, Moss EG, Harel-BellanA. Lin-28 binds IGF-2 mRNA and participates in skeletal myogenesis byincreasing translation efficiency. Genes Dev 2007;1125–38.

33. GuoY, ChenY, ItoH,Watanabe A,GeX, KodamaT, et al. Identification andcharacterization of lin-28 homolog B (LIN28B) in human hepatocellularcarcinoma. Gene 2006;384:51–61.

34. Fakhraldeen SA, Clark RJ, Roopra A, Chin EN, Huang W, Castorino J, et al.Two isoforms of the RNA binding protein, coding region determinant-binding protein (CRD-BP/IGF2BP1), are expressed in breast epitheliumand support clonogenic growth of breast tumor cells. J Biol Chem2015;290:13386–400.

35. Urlinger S, Baron U, Thellmann M, Hasan MT, Bujard H, Hillen W.Exploring the sequence space for tetracycline-dependent transcriptionalactivators: novel mutations yield expanded range and sensitivity. Proc NatlAcad Sci U S A 2000;97:7963–8.

36. Yusa K, Zhou L, Li MA, Bradley A, Craig NL. A hyperactive piggyBactransposase for mammalian applications. Proc Natl Acad Sci U S A2011;108:1531–6.

37. Fillebeen C, Wilkinson N, Pantopoulos K. Electrophoretic mobility shiftassay (EMSA) for the study of RNA-protein interactions: the IRE/IRPexample. J Vis Exp 2014;7:1–9.

38. Johnson SM, Grosshans H, Shingara J, Byrom M, Jarvis R, Cheng A, et al.RAS is regulated by the let-7 microRNA family. Cell 2005;120:635–47.

39. Jaffe AB, Kaji N, Durgan J, Hall A. Cdc42 controls spindle orientation toposition the apical surface during epithelial morphogenesis. J Cell Biol2008;183:625–33.

40. Magudia K, Lahoz A, Hall A. K-Ras and B-Raf oncogenes inhibit colonepithelial polarity establishment through up-regulation of c-myc. J CellBiol 2012;198:185–94.

41. Li P, Fang YJ, Li F, OuQJ, ChenG,MaG. ERCC1, defectivemismatch repairstatus as predictive biomarkers of survival for stage III colon cancer patientsreceiving oxaliplatin- based adjuvant chemotherapy. Br J Cancer 2013;1238–44.

42. Bohanes P, Labonte MJ, Lenz H. Review a review of excision repaircross- complementation group 1 in colorectal cancer. CLCC 2011;10:157–64.

43. Desmet FO, Hamroun D, Lalande M, Collod-B€eroud G, Claustres M,B�eroud C. Human splicing finder: an online bioinformatics tool to predictsplicing signals. Nucleic Acids Res 2009;37:1–14.

44. Olaussen KA, Dunant A, Fouret P, Brambilla E, Andre F, Haddad V, et al.DNA repair by ERCC1 in non-small-cell lung cancer and cisplatin-basedadjuvant chemotherapy. N Engl J Med 2006;355:983–91.

45. KwonH, RohMS, Oh SY, Kim S, KimMC, Kim JS, et al. Prognostic value ofexpression of ERCC1, thymidylate synthase, and glutathione S-transferaseP1 for 5-fluorouracil/oxaliplatin chemotherapy in advanced gastric cancer.Ann Oncol 2007;18:504–9.

46. Bellmunt J, CuelloM,Cecere FL, Albiol S,GuillemV,Gallardo E, et al. Geneexpression of ERCC1 as a novel prognostic marker in advanced bladdercancer patients receiving cisplatin-based chemotherapy. Ann Oncol 2007;18:522–8.

47. Kim MK, Cho KJ, Kwon GY, Park SI, Kim YH, Kim JH, et al. ERCC1predicting chemoradiation resistance and poor outcome in oesophagealcancer. Eur J Cancer 2008;44:54–60.

48. Dabholker M, Vionnet J, Bostick-Bruton F, Yu JJ, Reed E. Messenger RNAlevels of XPAC and ERCC1 in ovarian cancer tissue correlate with repsonseto platimun-based chemotherapy. J Clin Invest 1994;94:703–8.






49. Youn CK, Kim MH, Cho HJ, Kim HB, Chang IY, Chung MH, et al.Oncogenic H-Ras up-regulates expression of ERCC1 to protect cells fromplatinum-based anticancer agents. Cancer Res 2004;64:4849–57.

50. Lee-Kwon W, Park D, Bernier M. Involvement of the Ras/extracellularsignal-regulated kinase signalling pathway in the regulation of ERCC-1mRNA levels by insulin. Biochem J 1998;331:591–7.

51. Li W, Melton DW. Cisplatin regulates the MAPK kinase pathway to induceincreased expression of DNA repair gene ERCC1 and increase melanomachemoresistance. Oncogene 2012;31:2412–22.

52. Desjardins A, Yang A, Bouvette J, Omichinski JG, Legault P. Importance ofthe NCp7-like domain in the recognition of pre-let-7g by the pluripotencyfactor Lin28. Nucleic Acids Res 2012;40:1767–77.


Mizuno et al.




2018;16:403-416. Published OnlineFirst January 12, 2018.Mol Cancer Res Rei Mizuno, Priya Chatterji, Sarah Andres, et al. Functions

Specific− by LIN28B IsoformLET-7Differential Regulation of

Updated version

10.1158/1541-7786.MCR-17-0514doi:

Access the most recent version of this article at:

Material

Supplementary

http://mcr.aacrjournals.org/content/suppl/2018/01/12/1541-7786.MCR-17-0514.DC1

Access the most recent supplemental material at:

Cited articles

http://mcr.aacrjournals.org/content/16/3/403.full#ref-list-1

This article cites 46 articles, 13 of which you can access for free at:

Citing articles

http://mcr.aacrjournals.org/content/16/3/403.full#related-urls

This article has been cited by 1 HighWire-hosted articles. Access the articles at:

E-mail alerts related to this article or journal.Sign up to receive free email-alerts

Subscriptions

Reprints and

[email protected]

To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at

Permissions

Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://mcr.aacrjournals.org/content/16/3/403To request permission to re-use all or part of this article, use this link



http://mcr.aacrjournals.org/lookup/doi/10.1158/1541-7786.MCR-17-0514

http://mcr.aacrjournals.org/content/suppl/2018/01/12/1541-7786.MCR-17-0514.DC1

http://mcr.aacrjournals.org/content/16/3/403.full#ref-list-1

http://mcr.aacrjournals.org/content/16/3/403.full#related-urls

http://mcr.aacrjournals.org/cgi/alerts

mailto:[email protected]

http://mcr.aacrjournals.org/content/16/3/403


Documents

Differential Regulation of LET-7 by LIN28B Isoform Speciﬁc … · repertoire of mRNA transcripts (6, 7). Notably, LIN28B is a posttranscriptional repressor of LET-7 miRNA biogenesis