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Chromatin, Epigenetics and RNA Regulation

Differential Regulation of LET-7 by LIN28B Isoform–Specific Functions

Rei Mizuno, Priya Chatterji, Sarah Andres, Kathryn Hamilton, Lauren Simon, Shawn W. Foley, Arjun Jeganathan, Brian D. Gregory, Blair Madison and Anil K. Rustgi
DOI: 10.1158/1541-7786.MCR-17-0514 Published March 2018

Abstract

The RNA-binding protein LIN28B plays an important role in development, stem cell biology, and tumorigenesis. LIN28B has 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-short isoform remains unclear and represents a major gap in the field. The LIN28B-long and -short isoforms are expressed in a subset of human colorectal cancers and adjacent normal colonic mucosa, respectively. To elucidate the functional and mechanistic aspects of these isoforms, colorectal cancer cells (Caco-2 and LoVo) were generated to either express no LIN28B or the -short or -long isoform. Interestingly, the long isoform suppressed LET-7 expression and activated canonical RAS/ERK signaling, whereas the short isoform did not. The LIN28B-long isoform–expressing cells demonstrated increased drug resistance 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 with the LIN28B-long isoform for binding to pre-let-7. Coexpression of the short isoform in the LIN28B-long isoform–expressing cells rescued the phenotypes induced by the LIN28B-long isoform.

Implications: This study demonstrates the differential antagonistic functions of the LIN28B-short isoform against the LIN28B-long isoform through an inability to degrade LET-7, which leads to the novel premise that the short isoform may serve to counterbalance the long isoform during normal colonic epithelial homeostasis, but its downregulation during colonic carcinogenesis may reveal the protumorigenic effects of the long isoform. Mol Cancer Res; 16(3); 403–16. ©2018 AACR.

Introduction

LIN28, an RNA-binding protein, is expressed at high levels in mouse and human embryonic stem cells and in early embryogenesis (1, 2). It is a key contributor to the formation of induced pluripotent stem (iPS) cells (3). Two paralogs, LIN28A and LIN28B, are present in vertebrates (4). They bind to their target mRNAs or miRNA and play a major role in posttranscriptional control, such as splicing, polyadenylation, mRNA stabilization, mRNA localization, and translation (5). Structurally, LIN28B has two RNA-binding domains, a cold shock domain (CSD), and two CCHC type zinc finger domains (ZFD) that facilitate binding to a repertoire of mRNA transcripts (6, 7). Notably, LIN28B is a posttranscriptional repressor of LET-7 miRNA biogenesis (8, 9, 10, 11, 12). The CSD binds to the terminal loop of LET-7 precursor, pre-let-7, and ZFDs bind to the GGAG motif in pre-let-7 (13). Lin28A induces uridylation of pre-let-7 at its 3′ end, which escapes Dicer processing, resulting in degradation. More specifically, Lin28A recruits a TUTase (Zcchs11/TUT4) to pre-let-7 to inhibit processing by Dicer (11, 14, 15). However, Lin28B represses LET-7 through a different mechanism and does so in the nucleus through the sequestration of LET-7 transcripts and blocking their processing by the Microprocessor (16). Overall, Lin28-mediated regulation of LET-7 is critical in development, stem cell biology, and tumorigenesis.

LIN28A and LIN28B are upregulated during embryonic development but downregulated in adult somatic tissues (17). They are overexpressed in diverse cancers such as chronic myelogenous leukemia, hepatocellular carcinoma (HCC), neuroblastoma, lung cancer, 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 colorectal cancers is associated with poor prognosis and cancer recurrence and that LIN28B promotes migration, invasion, and metastasis of colorectal cancer cell lines in mouse xenograft models (21, 23). We have demonstrated that LIN28B has oncogenic properties in the initiation and progression of colon cancer in genetically engineered mouse models, and that the LIN28B-Let-7 axis is critical as LIN28B overexpression and Let-7 (a3-b2) deletion accelerate colon cancer development and progression (24, 25). The upregulation of LIN28B or downregulation of LET-7 has been reported to contribute to the acquisition of chemoresistance in various types of cancer such as breast cancer (26), esophageal cancer (27), acute myeloid leukemia (28), and pancreatic cancer (29).

LIN28B's actions to promote tumorigenesis are not restricted to one specific mechanism. For example, the LIN28/LET-7 axis can modulate glucose homeostasis by augmenting insulin-PI3K–mTOR signaling (30) and can regulate aerobic glycolysis to promote cancer cell progression (31). Other protumorigenic functions may be mediated via LET-7–independent effects. LIN28 also functions through posttranscriptional regulation by direct binding to specific mRNAs that may promote a stem cell–like state or tumorigenesis, such as Insulin-like growth factor 2 (IGF2), LGR5, and PROM1 (23, 24, 32).

Guo and colleagues demonstrated that LIN28B has two isoforms, so-called LIN28B-long isoform and LIN28B-short isoform (33). There is partial deletion of the CSD in the short isoform with preservation of the ZFDs. This suggests possible differences in the target mRNAs that may be bound by either isoform. There is a gap in our mechanistic understanding of the functional role of LIN28B-short isoform or the relationship between LIN28B-short and -long isoforms. Therefore, in this study, we aimed to reveal the role of the LIN28B-short isoform in colonic tumorigenesis. We found differential regulation of LET-7 miRNAs between LIN28B-long and -short isoforms. Specifically, the LIN28B-long isoform suppressed mature LET-7 expression, whereas LIN28B-short isoform did not have this inhibitory effect. This differential regulation of LET-7 miRNAs affected the downstream signaling of RAS/ERK signaling and potential chemoresistance. We also revealed that LIN28B-short isoform functions as an antagonist against LIN28B-long isoform, suggesting a model of dysequilibrium where the short isoform promotes differentiation in normal intestinal homeostasis through the inability to degrade LET-7, and the long isoform is predominant during colon cancer initiation and progression.

Materials and Methods

Cell lines

All human cell lines used in this study (Caco-2, LoVo, HCT116, SW480, Colo205, T84, HepG2, and Huh7.5) were obtained from the American Type Culture Collection. These cell lines were authenticated by the STR locus. Caco-2, LoVo, HCT116, T84, HepG2, and Huh7.5 were maintained in DMEM (Thermo Fisher Scientific), and SW480 and Colo205 were maintained in RPMI 1640 (Thermo Fisher Scientific) supplemented with 10% FBS (GE Healthcare Life Sciences) and 1% penicillin–streptomycin (P/S; Thermo Fisher Scientific) in a 37°C incubator with 5% CO2. Cells were tested for mycoplasma every 2 months and were cultured for no more than 15 passages from the validated stocks.

Immunoblotting

Proteins from cells or tissues were isolated using NP40 lysis buffer, and Western blots were performed with the Novex NuPAGE SDS-PAGE gel system (Invitrogen) in MOPS-SDS, according to the manufacturer's instructions as described previously (24). To isolate proteins from three-dimensional (3D) cultured Caco-2 cells, Matrigel inserts were recovered prior to the protein isolation with Matrisperse (BD Bioscience), according to the manufacturer's instructions. Proteins were visualized with an Odyssey Infrared Imager (LI-COR Biosciences) for near-infrared (near-IR) fluorophore-conjugated antibodies. Near-IR fluorescence was quantified using LI-COR Image Studio Software. Primary antibodies used for Western blot analysis are listed in Supplementary 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 mRNA levels, RT reactions were performed with oligo-dT primers using SuperScript III (Invitrogen). For miRNAs, RT reactions were performed using the miRNA RT Kit (#4366596; Life Technologies), according to the manufacturer's instructions. Quantitative PCR utilized the Fast SYBR (Invitrogen) or TaqMan Fast Universal (Invitrogen) master mixes. Taqman probes for mature LET-7 miRNAs were obtained from Life Technologies (Cat. # 4427975, assay numbers 000377, 002406, 000382, and 002282). LET-7 levels were normalized to U6 snRNA (Cat. # 4427975, assay numbers 001973; Life Technologies), and mRNA levels were normalized to GAPDH or PPIA. Primer sequences are listed in Supplementary Table S2. Gene expression data are expressed as a fold change normalized to the mean values for controls. All experiments were conducted at least in three independent settings with technical replicates (duplicates) in each experiment. To analyze the mRNA expression levels of the LIN28B isoforms, we designed RT-PCR primer sets (Supplementary Fig. S1A). Primer set 1 can measure relative mRNA expression of LIN28B-long isoform; primer set 2 can measure relative mRNA expression of overall LIN28B. The relative mRNA expression of LIN28B-short isoform can be calculated by subtracting relative expression of LIN28B-long isoform from that of overall LIN28B. The primer sets were designed to match the efficiency (ref. 34; Supplementary Fig. S1B).

LIN28B shRNA knockdown and generation of LIN28B-long and -short isoform–expressing cells

LIN28B shRNA was cloned into the BII-mirT3G2B shRNA vector, which is a piggyBac (PB)-based vector that we generated for achieving inducible, stable shRNA expression. LIN28B shRNAs was inserted at unique BamHI and SalI sites in the BII-mirT3G2B vectors. Oligonucleotides for the LIN28B shRNA were obtained from Invitrogen (Supplementary Table S3), annealed as per the manufacturer's instructions, and then ligated 1:1 along with mirBXL adapters (Supplementary Table S3) into unique BamHI and SalI sites in the BII-mirT3G2B vector. The BII-mirT3GB vector is a tet-inducible vector containing the rtTA-M2 reverse tetracycline transactivator (35). Approximately 2.5 × 105 Caco-2 were seeded in 6-well plates and 16 to 24 hours later were transfected with 500 ng of the pCMV-hyPBase transposase (36) and 1,500 ng of the respective PB transposon vector using 6 μL of Lipofectamine 2000 (Life Technologies) in 1 mL of antibiotic-free DMEM containing 10% FBS. Fresh medium was exchanged after 16 to 24 hours, and 48 hours after transfection, and then cells were selected with 10 μg/mL blasticidin (B-800; Gold Biotechnology).

To generate LIN28B-short or -long isoform–expressing cells, LIN28B-short or -long isoform plasmids were cloned from the MSCV-PIG-LIN28B plasmid (21). To prevent the knockdown of transferred LIN28B isoforms by shLIN28B, we induced mutations in LIN28B-coding sequences (LIN28B-mutant) to be resistant to shLIN28B using the QuikChange Site-Directed Mutagenesis kit (#200518; Agilent), according to the manufacturer's instruction. The mutagenic oligonucleotides that are resistant to shLIN28B used are shown in Supplementary Table S3. The resulting mutant plasmids were verified by DNA sequencing.

To generate the LIN28B-short isoform plasmid, the MSCV-PIG-LIN28B–mutant plasmid was digested and inserted into the PB-EF1-MCS-IRES-NEO vector (PB533A-2; System Bioscience) 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 T4 DNA polymerase. The plasmid was then digested and inserted into 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 hours later were transfected. After 48 hours of transfection, Caco-2 or LoVo cells were selected with 0.6 or 1 μg/mL neomycin (G-418; Gold Biotechnology), respectively.

To generate the LIN28B-short and -long isoform coexpression cells, 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 the NheI and EcoRI sites. The LIN28B-long isoform–expressing Caco-2 cells or LoVo cells were transfected using the PiggyBac Transposon System. Caco-2 and LoVo cells were selected with 5 μg/mL or 0.5 μg/mL puromycin (P-600; Gold Biotechnology), respectively. In all experiments, Caco-2 cells with shLIN28B were treated with 250 ng/mL doxycycline (Sigma-Aldrich) for at least 48 hours to induce shLIN28B expression.

3D cultures

To grow Caco-2 cells in 3D conditions, 5 × 103 cells were embedded in 40 μL of Matrigel (#356234,BD Biosciences) and cultured in DMEM + 10% FBS + 1% P/S at 37°C in 5% CO2. The medium was changed every 2 days. In the MEK inhibitor treatment experiment, U0126 (#662005, Millipore) was added in the medium at a concentration of 10 μmol/L from day 2. In the control group, the same amount of dimethyl sulfoxide (DMSO) was added. To quantify the morphology of Caco-2 cells in 3D culture, cells were stained with Hoechst 33342 (#62249; Thermo Fisher Scientific). Hoechst 33342 was added in the medium at a concentration of 40 μmol/L and cultured for 30 minutes in the tissue culture incubator. After PBS washes (x3), cells were imaged using a spinning-disk confocal microscope (ECLIPSE Ti; Nikon) with an ORCA-ER camera (C4742-95-12ERG; Hamamatsu Photonics). 3D Caco-2 cells were imaged using a 20× objective (S Plan Fluor, NA 0.45; Nikon). Images were acquired and analyzed with MetaMorph software (Molecular Devices). 3D Caco-2 structures with a single epithelial cell layer were classified as empty cysts; 3D Caco-2 structures with multiple epithelial cell layers or with invading cells into lumen were classified as luminal accumulating cysts. The quantification of the morphology of 3D Caco-2 cells was performed in a blinded manner.

Cell viability assays

Cell 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 to obtain time 0 values. Absorbance was measured at 450 nm using a Microplate reader. Cells were incubated for 24 hours in 96-well plates. 5-Fluorouracil (5-FU, Sigma-Aldrich) or cisplatin (Sigma-Aldrich) was then added at the final concentration of 0, 5, 10, 20, 40, or 80 μg/mL. After 24-hour incubation, the WST-1 assay was performed. The cell viability was determined as percent viability compared with the vehicle control. All experiments were performed independently at least 3 times with 6 replicates.

LET-7 inhibition and transduction in Caco-2 cells

For 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 negative control 1 inhibitors (AM17010: Anti-miR miRNA Inhibitor Negative Control #1; Thermo Fisher Scientific) using Lipofectamine RNAiMAX (Thermo Fisher Scientific) according to the manufacture's instruction. Two days after transfection, LET-7 expression was checked by qPCR as described. For LET-7 mimetic transfection experiments, LIN28B-long Caco2-cells were transfected in 6-well plates with 90 pmol of mirVana miRNA mimic (#4464066, MC10050, hsa-let-7a-5p; Thermo Fisher Scientific) or mirVana miRNA Mimic, Negative Control #1 (#4464058; Thermo Fisher Scientific) using Lipofectamine RNAiMAX (Thermo Fisher Scientific) according to the manufacturer's instructions. Two days after transfection, LET-7a expression was checked by qPCR as described.

Electrophoretic mobility shift assay

The preparation of cell extracts was performed based upon a previous study (37). Briefly, cultured cells were washed twice with ice-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 μL of ice-cold lysis buffer containing 1% Triton x100, 25 mmol/L Tris-HCl (pH 7.4), and 40 mmol/L KCL per 107 cells was added and incubated for 20 minutes on ice. Samples were spun at full speed in a microfuge at 4°C, and supernatant was collected. Pre-let-7 (PM12304, PM10050; Thermo Fisher Scientific) was labeled with Texas-Red fluorescence using the 5′ EndTag Labeling DNA/RNA Kit and Texas Red Maleimide (Vector Laboratory), according to the manufacturer's instructions. Binding reactions were conducted with 1 ng of Texas-Red–labeled pre-let-7 in a total volume of 20 μL together with 50 μg of yeast tRNA and indicated amounts of protein extracts. The labeled RNA was incubated 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, 60 mmol/L KCL, 20 U of RNase inhibitor (Thermo Fisher Scientific; #10777019), and 1 mmol/L DTT. After 30 minutes of incubation at room temperature, 4 μL of loading buffer was added to the electrophoretic mobility shift assay (EMSA) sample and resolved on a DNA retardation gel (EC6365BOX; Thermo Fisher Scientific). RNA bands were visualized using ChemiDoc MP Imaging System (Bio-Rad).

Statistical analyses

Unless indicated otherwise in the text or figure legends, unpaired, two-tailed Student t tests were performed to determine statistical significance of comparisons between control and experimental cell lines with P < 0.05 as statistically significant. For all analyses, unless noted otherwise, data from a minimum of three independent experiments were presented as mean ± SEM.

Results

LIN28B-long isoform is predominantly expressed in human colorectal cancer tissues

LIN28B has two isoforms, namely the LIN28B-long and LIN28B-short isoforms. The LIN28B-long isoform consists of 250 amino acids and has two RNA-binding domains: the CSD in its N-terminus and two CCHC type ZFDs in its C-terminus. The LIN28B-short isoform lacks 70 amino acids in the N-terminus and does not have a complete CSD (ref. 33; Fig. 1A). To date, studies have not evaluated the functional and mechanistic differences between the isoforms.

Figure 1.
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-long isoform 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 by Western blotting (WB) analysis. C, WB analysis of LIN28B isoforms in 15 pairs of deidentified human colorectal cancers and adjacent normal mucosa.

To elucidate the role of the LIN28B-short isoform in the pathogenesis 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-long isoform. We could not detect any significant levels of the LIN28B isoforms in other colorectal cancer cell lines tested. We also evaluated LIN28B expression in HCC cell lines, HepG2 and Huh7.5. Both cell lines expressed high levels of the LIN28B-long isoform (Fig. 1B).

Next, we examined the distribution of LIN28B isoforms in 15 pairs of deidentified human colorectal cancers and matched normal colorectal tissues. Here, we used an anti-LIN28B antibody whose antigen surrounds 240 amino acid position of LIN28B, 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 cancer demonstrated that approximately 28% of these samples have high LIN28B expression (22), and our study is in concordance. We also found that the expression of LIN28B-short isoform was observed predominantly in the adjacent normal tissues (samples #1–6; Fig. 1C). LIN28A proteins are reported to have isoforms produced by alternative splicing in several animal species (1). Guo and colleagues identified the LIN28B isoforms from 5′-RACE analysis using a cDNA library of fetal liver (33). This suggests that alternative splicing may produce the LIN28B isoforms. Therefore, we designed RT-PCR primer sets to investigate the mRNA expression levels of the LIN28B isoforms (Supplementary Fig. S1). We evaluated the mRNA expression of each LIN28B isoform in human colorectal cancer tissues and matched adjacent normal mucosa in an independent cohort of 31 colorectal cancer patients (protein not available). Here, 7 samples showed more than a 2-fold increase of overall LIN28B expression in tumor tissue. In these samples, the LIN28B-long and -short isoforms are predominantly expressed in cancer and normal colon, respectively (Supplementary Fig. S2).

The LIN28B isoforms differentially regulate LET-7 expression

To investigate the functional roles of each LIN28B isoform in the pathogenesis of colon cancer, we generated specific LIN28B isoform–expressing colorectal cancer cells. For this purpose, we used Caco-2 cells and LoVo cells. Because Caco-2 cells have high levels of endogenous LIN28B-long and -short isoforms, we first knocked out the endogenous LIN28B by shRNA and generated LIN28B-null cells. Then, LIN28B-short or LIN28B-long isoforms were transduced stably into the LIN28B-null cells to generate LIN28B-short or -long–specific isoform-expressing Caco-2 cells. In LoVo cells, the specific LIN28B isoforms were transduced stably into wild-type LoVo cells because LoVo cells do not express endogenous LIN28B as shown in Fig. 1B (Fig. 2A).

Figure 2.
Figure 2.

LIN28B isoform differentially regulates LET-7. A, Western blot of WT, LIN28B-null, LIN28B-short, and LIN28B-long isoform–expressing Caco-2 cells and WT, empty vector, LIN28B-short isoform–, and LIN28B-long isoform–expressing LoVo cells. GAPDH was used as a loading control. Caco-2 cells were treated with 250 ng/mL of doxycycline (DOX) for at least 3 days. B, Measurement of individual LET-7 miRNAs by qPCR analysis. Results were normalized to U6 snRNA. Data are shown as mean ± SE. The experiment was performed independently 3 times each with duplicates. *, P < 0.05; **, P < 0.005; ***, P < 0.001. C, The expression ratio between the mRNAs of LIN28B-long and -short isoform negatively correlates with LET-7a, LET-7e, and LET-7g expression. Fourteen samples were used in this analysis.

LIN28B binds to LET-7 miRNA precursors and inhibits their processing, resulting in the decrease of mature LET-7 miRNAs (8, 9, 10, 11, 12). Because the LIN28B-short isoform lacks the CSD, we predicted that the maturation of LET-7 miRNA might be affected differentially between the LIN28B-short and -long isoform–expressing cells. Therefore, we first evaluated the expression levels of the mature LET-7 miRNAs using the cells that were generated. To that end, LET-7 expression was decreased in LIN28B-long isoform–expressing cells compared with LIN28B-null cells. By contrast, the expression levels of LET-7 miRNAs in LIN28B-short isoform–expressing cells were not changed compared with LIN28B-null cells and significantly higher than those of LIN28B-long isoform–expressing cells (Fig. 2B). We evaluated the relationship between LET-7 expression and the expression ratio of the LIN28B-long and -short isoform. We found that the LET-7 expression correlates negatively with the increase in the LIN28B-long isoform/LIN28B-short isoform ratio (Fig. 2C).

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

As there is a significant difference in the inhibitory effect on the levels of mature LET-7 miRNAs between the LIN28B isoforms, we next investigated the roles of each LIN28B isoform on the RAS signaling pathway, known to involve LET-7 (38). We found higher RAS levels in LIN28B-long isoform–expressing cells compared with LIN28B-null or LIN28B-short isoform–expressing cells. No significant difference was observed between LIN28B-null and LIN28B-short isoform–expressing cells (Fig. 3A). This expression pattern of RAS was compatible with the expression pattern of LET-7 miRNAs as shown in Fig. 2b.

Figure 3.
Figure 3.

LIN28B-long isoform upregulates RAS/ERK signaling through LET-7 suppression resulting in luminal cell accumulation in 3D Caco-2 cysts. A, The expression of RAS in Caco-2 or LoVo cells was analyzed by Western blot. The 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. B, Caco-2 cells were cultured in Matrigel and imaged at day 10. Representative images of bright field (BF) and nuclear staining by Hoechst 33342 are shown. Bar, 100 μm. The graph shows the quantification of the morphology of 3D Caco-2 structures. The mid-plane of >30 structures was imaged for each condition in three independent experiments and classified as having empty lumen (Empty) or luminal cell accumulation. Data are shown as mean ± SE. ***, P < 0.001. C, The expression of Phospho-ERK and ERK of Caco-2 cells was analyzed by Western blot. The graph shows the densitometry. Results were normalized to GAPDH. Data are expressed as the mean ± SE of five independent experiments. **, P < 0.005. D, 3D Caco-2 cells expressing LIN28B-long isoforms were treated with 10 μmol/L of U0126 or DMSO from day 2 and imaged at day 10. Left plots show the representative bright field and Hoechst 33342 staining images. Scale bar, 200 μm. The mid-plane of >30 structures was imaged for each condition in three independent experiments. Data represent mean ± SE. **, P < 0.005.

Caco-2 cells are known to form clear cystic structures when they are cultured under 3D conditions (39). The activation of RAS/ERK signaling is reported to induce cell accumulation in the lumens of the 3D cysts (40). Since the LIN28B-long isoform increases RAS expression, we examined the morphology of Caco-2 cells expressing each LIN28B isoform in 3D. LIN28B-null and LIN28B-short isoform–expressing Caco-2 cells formed clear cystic structures. By contrast, LIN28B-long isoform–expressing Caco-2 cells showed a significant increase of 3D cysts accumulating cells in the lumen (Fig. 3B). The phosphorylation of ERK was activated in LIN28B-long isoform–expressing Caco-2 cells in 3D (Fig. 3C). The luminal cell accumulation in LIN28B-long isoform–expressing cells was significantly inhibited by the MEK inhibitor, U0126 (Fig. 3D). Taken together, these results indicate that the LIN28B-long isoform upregulates RAS/ERK signaling through the inhibition of LET-7 miRNAs, thereby resulting in the accumulation of cells in the lumens of Caco-2 3D cysts.

The LIN28B-long isoform induces chemoresistance through the upregulation 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 chemotherapy resistance in cancers (26–29). To test this, we evaluated the viability of the cell lines expressing the LIN28B isoforms treated with either cisplatin or 5-FU, two standard chemotherapeutic agents. LIN28B-long isoform–expressing cells demonstrated a significant increase in drug resistance compared with LIN28B-null cells. Notably, LIN28B-short isoform–expressing cells did not confer any drug resistance either (Fig. 4A). Previous studies have reported that excision repair cross-complementing group 1 (ERCC1) expression is associated with drug resistance in colon cancer (41, 42). We found that ERCC1 expression was upregulated only in LIN28B-long isoform–expressing cells (Fig. 4B). As chemoresistance and ERCC1 expression showed a negative correlation pattern with LET-7 miRNAs (Figs. 2B, 4A, and 4B), we examined whether ERCC1 expression is negatively regulated by LET-7. We transfected a LET-7 inhibitor or negative control into LIN28B-null Caco-2 cells and compared ERCC1 expression (Fig. 4C). ERCC1 expression was higher in LET-7–inhibited cells (Fig. 4D). We also transduced a LET-7 mimetic into LIN28B-long isoform–expressing Caco-2 cells and found that ERCC1 expression was significantly decreased in LET-7–transduced cells compared with control cells (Fig. 4E and F). These results suggest that the LIN28B-long isoform may upregulate ERCC1 in a LET-7–dependent manner.

Figure 4.
Figure 4.

LIN28B-long isoform contributes to the acquisition of drug resistance. A, Cell viability was analyzed by the WST-1 assay. 1 × 104 Caco-2 or LoVo cells were seeded in triplicate. 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 and LIN28B-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-2 or 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-7a miRNAs in LIN28B-null Caco-2 cells transfected with LET-7a inhibitor or negative control precursor miRNA by qPCR 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 plot shows the representative WB images of ERCC1 and GAPDH in LIN28B-null Caco-2 cells transfected with LET-7a inhibitor or negative control precursor miRNA. Right graphs 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-7a miRNAs in LIN28B-long Caco-2 cells transfected with LET-7a mimetic or negative control miRNA by qPCR analysis. Results were normalized to U6 snRNA. Data are shown as mean ± SE. The experiment was performed independently 3 times. ***, P < 0.0001. F, Left plots show the representative WB images of ERCC1 and GAPDH in LIN28B-long Caco-2 cells transfected with LET-7a mimetic or negative control miRNA. Right graphs show the densitometry. Results were normalized to GAPDH. Data are expressed as the mean ± SE of three independent experiments. *, P < 0.05.

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

We next examined whether the differential regulation of mature LET-7 by the LIN28B isoforms is caused by the loss of the binding ability to LET-7 precursor due to the incomplete CSD in the LIN28B-short isoform. For this purpose, we performed EMSA to check the binding ability of each isoform to the LET-7 precursor, pre-let-7. Protein extract from LIN28B-null cells did not form protein-pre-let-7 complex (Fig. 5A). Protein extracts from either LIN28B-long isoform–expressing cells or LIN28B-short isoform–expressing cells formed the LIN28B protein-pre-let-7 complex in a dose-dependent manner (Fig. 5B–E). These data suggested that the LIN28B-short isoform does not have the inhibitory effects on the maturation of LET-7 miRNAs while preserving the binding ability to pre-let-7. These data prompted us to hypothesize that the LIN28B-short isoform could antagonize the LIN28B-long isoform–mediated binding to pre-let-7. To test this hypothesis, we performed EMSA using pre-let-7 and protein extracts from LIN28B-long isoform–expressing cells at the concentration that free pre-let-7 completely diminishes with increasing concentration of protein extract from LIN28B-null or LIN28B-short isoform–expressing cells. Protein extracts from LIN28B-null cells did not prevent the formation of the LIN28B-long isoform-pre-let-7 complex (Fig. 5F). By contrast, as the concentration of protein extract of LIN28B-short isoform–expressing cells increased, the LIN28B-long isoform-pre-let-7 complex decreased and the LIN28B-short isoform-pre-let-7 complex increased (Fig. 5G). This indicates that LIN28B-short isoform competes with the LIN28B-long isoform for binding to pre-let-7 and may function as an antagonist to the LIN28B-long isoform.

Figure 5.
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, mixed with increasing concentration of whole-cell protein extracts from LIN28B-null LoVo cells. A total of 0, 3, 6, 12, or 24 μg of protein extracts were used. B, EMSA with pre-let-7a 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 μg 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 μg of protein extracts 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 increasing concentration 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 μg of protein extracts were used. Right graph shows the bound fraction of RNA against the total amount. E, EMSA with pre-let-7e as a probe, mixed with increasing concentration 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. F, EMSA using the mixture of Texas-Red–labeled pre-let-7a or pre-let-7e and 12 μg 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 μg). G, EMSA using the mixture of Texas-Red–labeled pre-let-7a or pre-let-7e and 12 μg of protein extract from LIN28B-long isoform–expressing LoVo cells with increasing concentration of protein from LIN28B-short isoform–expressing LoVo cells (Lin28B-short cell extract: 0, 3, 6, 12, 24 μg).

The LIN28B-short isoform antagonizes the phenotypes induced by the LIN28B-long isoform

We next examined whether the LIN28B-short isoform antagonizes the phenotypes induced by the LIN28B-long isoform in a LET-7-dependent manner. For this purpose, we generated LIN28B-short isoform and -long isoform–coexpressing Caco-2 or LoVo cells (Fig. 6A and B). Coexpression of LIN28B-short isoform significantly increased the expression of mature LET-7 miRNAs compared with the cells expressing only the LIN28B-long isoform (Fig. 6C). We also expressed the LIN28B-short isoform in Huh7.5 cells, known to have high endogenous LIN28B-long isoform expression (Fig. 1B; Supplementary Fig. S3A and S3B). The expression of mature LET-7a was significantly increased in LIN28B-short isoform–expressing Huh7.5 cells compared with wild-type Huh7.5 cells (Supplementary Fig. S3C). Coexpression of the LIN28B-short isoform decreased the RAS expression induced by LIN28B-long isoform in Caco-2 or LoVo cells (Fig. 6D). Coexpression of LIN28B-short isoform inhibited the luminal cell accumulation in 3D-cultured Caco-2 cells and increased the formation of 3D cysts with clear lumens (Fig. 6E). The LIN28B-short isoform also decreased ERCC1 expression and increased the sensitivity to chemotherapy (Fig. 6F and G).

Figure 6.
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-short vectors were transfected intoLIN28B-long isoform–expressing cells. Control cells (L+Emp) and coexpressing cells (L+S) are generated. B, The expression of LIN28B-long or -short isoforms was confirmed by WB. The graphs show the densitometry. Results were normalized to GAPDH. Data are obtained from three independent 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 RAS were analyzed by WB. The graphs indicate the densitometry of WB. Results were normalized to GAPDH. Data are obtained from three independent experiments. *, P < 0.05 and **, P < 0.005. E, Caco-2 cells expressing LIN28B-long (L+Emp) or LIN28B-long and short isoforms (L+S) are cultured in 3D condition. Representative images of bright field and Hoechst 33342 staining are shown. Scale bar, 200 μm. The mid-plane of >40 structures was imaged for each condition 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 by Western blot. The graphs indicate the densitometry. Results were normalized to GAPDH. Data were obtained 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 treated with 10 μg/mL 5-FU or 40 μg/mL of Cisplatin for 24 hours. LoVo cells were treated with 20 μg/mL 5-FU or 40 μg/mL of Cisplatin for 24 hours. Data are obtained from three independent experiments each with 5 replicates. All the graph data are the mean ± SE. *, P < 0.05.

Discussion

LIN28B is a critical regulator of genes in development, stem cell biology, tissue regeneration, and tumorigenesis. LIN28B consists of 250 amino acids with two RNA-binding domains, CSD and ZFDs. During normal development, LIN28B is highly expressed in stem cells or progenitor cells and regulates genes related to selfrenewal or proliferation. As progenitor cells differentiate, LIN28B expression is decreased (17). LIN28B is also known to be upregulated in cancers, such as chronic myelogenous leukemia, HCC, neuroblastoma, lung, breast, ovary, cervix, and colorectal (18, 19, 20, 21, 23). In human colorectal cancer, about 30% of the tumors overexpress LIN28B (22). LIN28B functions as an oncogene because transgenic mice expressing LIN28B develop intestinal polyps and adenocarcinomas tumors (24, 25). Furthermore, LIN28B cooperates with WNT signaling to drive invasive intestinal and colorectal adenocarcinomas (22).

LIN28B has two isoforms, LIN28B-long and LIN28B-short isoforms. The LIN28B-short isoform lacks 70 amino acids in the N-terminus with an incomplete CSD but intact ZFDs. The LIN28B isoforms were identified originally by 5′-RACE analysis using a cDNA library of fetal liver, which suggests that mRNAs corresponding to each LIN28B isoform could be generated by alternative splicing (33). In fact, LIN28A proteins were reported to have isoforms produced by alternatively spliced first exons in several animal species (1). In silico prediction of alternative splicing sites using Human Splicing Finder (HSF; ref. 43) suggests possible alternative splicing sites for the LIN28B-short isoform. The LIN28B-long isoform mRNA consists of 4 exons. The initiation of LIN28B-short isoform translation likely starts from the first ATG codon in exon 3 (33).The HSF predicts the existence of several alternative donor splicing sites in the region upstream of the start codon of LIN28B-long isoform in exon 1. Several possible acceptor 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 splicing might occur. Further investigation into the mechanisms through which LIN28B isoforms are produced would be very interesting.

Here, we demonstrated that LET-7 expression was downregulated only by the LIN28B-long isoform but not by the LIN28B-short isoform. Consequently, RAS/ERK signaling, which is negatively regulated by LET-7 (38), was activated only in the LIN28B-long isoform–expressing cells. This activation of RAS/ERK signaling induced luminal cell accumulation of cells in the lumen of 3D Caco-2 cysts in the LIN28B-long isoform–expressing cells. The upregulation of LIN28B or downregulation of LET-7 has been reported to contribute to the acquisition of chemoresistance in various types of cancer (26, 27, 28, 29). LIN28A/B is reported to confer 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 resistance in Caco-2 or LoVo cells. By contrast, the LIN28B-short isoform did not change drug response compared with LIN28B-null cells. ERCC1, which is associated with DNA repair, has been linked with poor responses to chemotherapy in several types of cancers (44, 45, 46, 47, 48). In colorectal cancer, the upregulation of ERCC1 also contributes to the acquisition of drug resistance (41, 42). We demonstrated that ERCC1 expression was negatively regulated by LET-7 using LET-7 inhibition and transduction experiments. Although we checked whether the 3′UTR in Ercc1 contains the conserved LET-7 site using TargetScan.org prediction, 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 signaling by LET-7 (49, 50, 51).

Although the LIN28B-short isoform loses the ability to inhibit the maturation of LET-7 miRNAs, EMSA demonstrated that the LIN28B-short isoform preserves the ability to bind the LET-7 precursor, 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 be similar between protein extracts from LIN28B-long and -short isoform–expressing cells. It is difficult to measure the precise binding affinity of each LIN28B isoform to pre-let-7 because we did not use purified proteins but rather protein extracts from cells in our experimental conditions. However, the binding affinity of each isoform to pre-let-7 can be assumed to be similar or at least not significantly different because they have the similar relative expression levels to the housekeeping gene, GAPDH, which might suggest that the concentration of each isoform protein in the cell extracts may be similar. A previous study revealed that the binding affinity between the ZFDs of LIN28 and pre-let-7g was similar to the binding affinity between the whole LIN28 and pre-let-7g, whereas the binding affinity between CSD and pre-let-7g was significantly lower (52). Taken together, the LIN28B-short isoform, which has ZFDs, could have similar or at least not significantly lower affinity to ple-let-7 compared with the LIN28B-long isoform. Furthermore, it has been noted that both the CSD and ZFDs in LIN28A are necessary for the inhibition of the maturation of LET-7 (9). The addition of the LIN28B-short isoform to the mixture of the LIN28B-long isoform and pre-let-7 decreased the formation of LIN28B-long isoform-pre-let-7 complex in a dose-dependent manner. This suggests that the LIN28B-short isoform could potentially antagonize the LIN28B-long isoform–mediated binding to the pre-let-7 in our experimental conditions (Fig. 7C). In fact, the coexpression of the LIN28B-short isoform in colorectal cancer cell lines rescued the phenotypes induced by LIN28B-long isoform in a LET-7–dependent manner. Coexpression of the LIN28B-short isoform increased LET-7 expression, decreased RAS expression with a decrease in luminal cell accumulation in 3D Caco-2, and decreased ERCC1 expression with increased chemotherapy sensitivity.

Figure 7.
Figure 7.

A model of the antagonistic function of the LIN28B-short isoform against the LIN28B-long isoform though LET-7 regulation. A, The LIN28B-long isoform binds to pre-let-7 and inhibits its maturation affecting the downstream signaling of LET-7. B, The LIN28B-short isoform preserves binding ability to pre-let-7 without inhibiting its maturation. C, The LIN28B-short isoform antagonizes the LIN28B-long isoform by competing for binding to pre-let-7.

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

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Authors' Contributions

Conception and design: R. Mizuno, A. Jeganathan, A.K. Rustgi

Development of methodology: R. Mizuno, P. Chatterji, A. Jeganathan, B. Madison, A.K. Rustgi

Acquisition 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. Rustgi

Writing, review, and/or revision of the manuscript: R. Mizuno, P. Chatterji, S. Andres, K. Hamilton, B.D. Gregory, B. Madison, A.K. Rustgi

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): A. Jeganathan, A.K. Rustgi

Study supervision: B.D. Gregory, A.K. Rustgi

Acknowledgments

This work was supported by the NIH/NIDDK P30DK050306, R01DK056645, K01DK093885, NIH K01DK100485 (KEH), Hansen Foundation, Lustgarten Family colon cancer grants, and the Penn Colon Cancer Translational Center of Excellence.

The authors thank the Molecular Pathology and Imaging Core (J. Katz, A. Bedenbaugh, and D. Budo), the Human Microbial and Analytic Repository Core (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 article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Footnotes

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

  • Received September 18, 2017.
  • Revision received November 7, 2017.
  • Accepted November 29, 2017.

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Differential Regulation of LET-7 by LIN28B Isoform–Specific Functions
Rei Mizuno, Priya Chatterji, Sarah Andres, Kathryn Hamilton, Lauren Simon, Shawn W. Foley, Arjun Jeganathan, Brian D. Gregory, Blair Madison and Anil K. Rustgi
Mol Cancer Res March 1 2018 (16) (3) 403-416; DOI: 10.1158/1541-7786.MCR-17-0514
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