The LIN28B-IMP1 post-transcriptional regulon has opposing effects on oncogenic signaling in the intestine

Abstract

RNA-binding proteins (RBPs) are expressed broadly during both development and malignant transformation, yet their mechanistic roles in epithelial homeostasis or as drivers of tumor initiation and progression are incompletely understood. Here we describe a novel interplay between RBPs LIN28B and IMP1 in intestinal epithelial cells. Ribosome profiling and RNA sequencing identified IMP1 as a principle node for gene expression regulation downstream from LIN28B In vitro and in vivo data demonstrate that epithelial IMP1 loss increases expression of WNT target genes and enhances LIN28B-mediated intestinal tumorigenesis, which was reversed when we overexpressed IMP1 independently in vivo. Furthermore, IMP1 loss in wild-type or LIN28B-overexpressing mice enhances the regenerative response to irradiation. Together, our data provide new evidence for the opposing effects of the LIN28B-IMP1 axis on post-transcriptional regulation of canonical WNT signaling, with implications in intestinal homeostasis, regeneration and tumorigenesis.

Keywords: IMP1; LIN28B; intestinal tumorigenesis; post-transcriptional regulation; ribosome profiling.

Figure 1.

IMP1 is a significant translational regulator downstream from LIN28B. (A) Radar map demonstrating the top post-transcriptional regulators of differentially expressed genes with LIN28B overexpression in SW480 cells as compared with control cells. A single point on the radar map indicates −10 log P-value of enrichment of any post-transcriptional regulator. Differentially expressed genes at each level were identified using the DEGseq, and the list of post-transcriptional regulators was obtained from the AURA database (see the Materials and Methods). (B) Scatter plot of differential expression between LIN28B-overexpressing lines with and without IMP1 deletion.The log₂ fold changes between ribosome-bound RNAs (ribosome-protected fragments [RPFs]) and total mRNA are plotted. The plot indicates that IMP1 regulates both mRNA abundance and translation. (C) Scatter plot of genes with significant (in blue) differential translational efficiencies between LIN28B-overexpressing cells with and without IMP1 deletion. Translation efficiencies (TEs) of the transcripts were calculated as the ratio of reads of ribosome-protected fragments to the reads in total mRNA abundance. (D) Pathway analysis using DAVID software of the differentially expressed genes from C to see what signaling/effector pathways are enriched with IMP1 deletion (note the Wnt signaling pathway).

Figure 2.

IMP1 loss enhances LIN28B-mediated tumorigenesis in vivo. Imp1^fl/fl (wild type), Villin-Cre;Imp1^fl/fl, Villin-Lin28b, and Villin-Cre;Villin-Lin28b;Imp1^fl/fl mice were aged up to 1 yr and then sacrificed to evaluate tumor growth. (A) Igf2bp1/Imp1 expression in the epithelia isolated from the jejuna of 12-mo-old mice. n > 4 mice per genotype. (B) Representative immunofluorescence staining for EdU incorporation in mouse intestinal epithelium. Bars, 500 µm. (C) Quantification of EdU+ve cells using flow cytometry. n = 3 mice per genotype. (D) The number of intestinal tumors in mice with or without Imp1 in the context of Lin28b overexpression. n > 9 mice per genotype. (E) The percentage of tumors classified as adenocarcinomas by histological scoring. (F) Representative hematoxylin and eosin (H&E) staining of intestinal epithelia in aged mice. Mice lacking Lin28b overexpression exhibited normal intestinal morphology at 12 mo of age. Mice with Lin28b overexpression exhibited tumor development that worsened with Imp1 loss. Bars, 500 µm. (G) Relative expression of different let-7 targets in the intestinal epithelium via qPCR. n > 5 mice per genotype. (H) Relative expression of stem cell markers in the intestinal epithelium via qPCR expressed as fold change with respect to Imp1^fl/fl mice. n > 5 mice per genotype. (I) Relative expression of Wnt target genes in the intestinal epithelium via qPCR expressed as fold change with respect to Imp1^fl/fl mice. n > 5 mice per genotype. (J) Representative immunohistochemical staining for β-catenin in mouse intestinal epithelium (magnified in the inset). (K) Representative β-catenin protein levels in mouse intestinal crypts from the four genotypes. All data are expressed as mean ± SEM. (*) P < 0.05; (**) P < 0.01; (***) P < 0.001; (****) P<0.0001 by ordinary one-way ANOVA test followed by Tukey's multiple comparison test. The significance is shown compared with control/wild-type samples unless indicated otherwise.

Figure 3.

IMP1 regulates intestinal epithelial regeneration following irradiation. Mice were evaluated for Imp1 expression in nonirradiated conditions and 4 d after 12 Gy of whole-body irradiation. (A) qRT–PCR and representative Western blot for Imp1 in isolated crypts from these mice. (*) P < 0.05 versus nonirradiated. n = 3–4 mice per genotype. (B) Representative immunohistochemical staining showing Imp1 increase in wild-type (Imp1^fl/fl) mice following radiation. VillinCre;Imp1^fl/fl mice were used as controls. Bars, 500 µm. (C) Villin-Lin28b;Villin-Cre;Imp1^fl/fl mice lost significantly less weight at sacrifice following irradiation than Villin-Lin28b mice (22.23% ± 0.9905% mean weight loss in Villin-Cre;Villin-Lin28b;Imp1^fl/fl mice [n = 3] vs. 26.68% ± 0.4076% in Villin-Lin28b [n = 5]). The weight loss in Villin-Lin28b mice was significantly higher than in controls (22.24% ± 0.2556% mean weight loss). (D) Analysis of Ki67⁺ cells revealed a significant increase in Ki67⁺ regenerative crypt foci in VillinCre Villin-Lin28b;Imp1^fl/fl mice at 4 d following irradiation. n = 3–4 mice per genotype; n = 20–30 high-power fields (HPFs) per mouse. Representative immunohistochemical staining for Ki67⁺ foci in the mouse intestinal epithelium is shown. Bars, 500 µm. (E) VillinCre;Imp1^fl/fl mice lost significantly less weight at sacrifice following irradiation than controls (18.83% ± 0.98% in VillinCre;Imp1^fl/fl mice vs. 23.34% ± 0.56% mean weight loss in controls). n = 14 Imp1^fl/fl mice; n = 12 VillinCre;Imp1^fl/fl mice. (F) Analysis of Ki67⁺ cells revealed a robust increase in Ki67⁺ regenerative crypt foci in VillinCre;Imp1^fl/fl mice at 4 d following irradiation. n = 4 mice per genotype; n = 20–30 HPFs per mouse. (G) Representative immunohistochemical staining for Ki67⁺ foci in the mouse intestinal epithelium. Bars, 500 µm. (H) Representative pictures of the enterosphere and enteroid with buds. Enhanced growth of post-irradiation enteroids from VillinCre;Imp1^fl/fl mice. Regenerative crypt units were plated in enteroid culture on the day of sacrifice to evaluate ex vivo survival and growth. (I) Evaluation of enterosphere growth 1 d following plating and enteroid growth 4 d after plating revealed a significant increase in VillinCre;Imp1^fl/fl compared with Imp1^fl/fl. All data are expressed as mean ± SEM. (*) P < 0.05; (**) P < 0.01; (***) P < 0.001; (****) P < 0.0001 by ordinary one-way ANOVA test followed by Tukey's multiple comparison test. The significance is shown compared with control/wild-type samples unless indicated otherwise.

Figure 4.

IMP1 overexpression does not initiate tumors in vivo. (A) The VillinCre;Imp1^OE mice did not exhibit tumor formation between 10 and 12 mo of age. (B) Representative H&E and Ki67 staining of intestinal epithelia in aged (10–12 mo) mice. Mice overexpressing IMP1 exhibited normal intestinal morphology and did not show a tumor phenotype. Bars, 500 µm. (C) Relative expression of Wnt target genes in the intestinal epithelium via qPCR expressed as fold change with respect to wild-type control mice at 10–12 mo of age. n = >4 mice per genotype. All data are expressed as mean ± SEM. (*) P < 0.05; (**) P < 0.01; (***) P < 0.001 by ordinary one-way ANOVA test followed by Tukey's multiple comparison test.

Figure 5.

IMP1 plays a functional role in reserve intestinal stem cells. (A) qRT–PCR for stem cell markers and Wnt target genes in isolated crypts from VillinCre;Imp1fl/fl mice compared with Impfl/fl mice. n = 3–4 mice per genotype. (B) qPCR in Sox9-EGFP reporter mice, where epithelial cell populations are subclassified viafluorescent-activated cell sorting (FACS) into Sox9-EGFP0-negative, Sox9-EGFP0 sublow, Sox9-EGFP0 low, and Sox9-EGFP0 high cells. Imp1 is expressed at low levels in all represented cell types except Sox9-EGFP-negative cells (white bars). Imp1 expression is significantly higher in Sox9-EGFP low and Sox9-EGFP high cells, which encompass intestinal stem cells. n = 5 animals per group. (C) HopxCreERT2;Imp1^fl/fl mice lost significantly less weight at sacrifice following irradiation than controls (18.8% ± 1.951% mean weight loss in HopxCreERT2;Imp1^fl/fl mice vs. 23.92% ± 1.015% in controls). n = 8 HopxCreERT2;Imp1^fl/fl; n = 12 control mice. (D) Analysis of Ki67⁺ cells revealed there was a robust increase in Ki67⁺ regenerative crypt foci at 4 d following irradiation in HopxCreERT2;Imp1^fl/fl mice compared with control mice. n = 4 mice per genotype; n = 20–30 HPFs per animal. Bars, 500 µm. (E) Representative immunohistochemical staining for Ki67⁺ foci in the mouse intestinal epithelium quantified in D. (F) qPCR for Imp1 expression in Lgr5⁺ and Paneth (CD24/cKit/SSC high) cells sorted from Lgr5-eGFP-IRES-CreERT2 mouse crypts. All data are expressed as mean ± SEM. (*) P < 0.05; (**) P < 0.01; (***) P < 0.001; (****) P < 0.0001 by unpaired t-test.

Figure 6.

IMP1 is the principal node for post-transcriptional regulation downstream from LIN28B. We propose a model in which IMP1 plays an important regulatory role downstream from LIN28B. Both LIN28B overexpression and whole-body irradiation enhance IMP1 expression in the intestinal epithelium. Deletion of IMP1 causes a significant increase in LIN28B-mediated tumorigenesis, likely due in part to observed increases in Wnt signaling and, potentially, stem cell signature. Furthermore, IMP1 loss (specifically in Hopx⁺ stem cells) causes increased regeneration following radiation injury. Taken together, these data suggest IMP1 as a regulator of intestinal epithelial homeostasis downstream from both LIN28B and radiation.