Let-7 represses carcinogenesis and a stem cell phenotype in the intestine via regulation of Hmga2

Let-7 miRNAs comprise one of the largest and most highly expressed family of miRNAs among vertebrates, and is critical for promoting differentiation, regulating metabolism, inhibiting cellular proliferation, and repressing carcinogenesis in a variety of tissues. The large size of the Let-7 family of miRNAs has complicated the development of mutant animal models. Here we describe the comprehensive repression of all Let-7 miRNAs in the intestinal epithelium via low-level tissue-specific expression of the Lin28b RNA-binding protein and a conditional knockout of the MirLet7c-2/Mirlet7b locus. This ablation of Let-7 triggers the development of intestinal adenocarcinomas concomitant with reduced survival. Analysis of both mouse and human intestinal cancer specimens reveals that stem cell markers were significantly associated with loss of Let-7 miRNA expression, and that a number of Let-7 targets were elevated, including Hmga1 and Hmga2. Functional studies in 3-D enteroids revealed that Hmga2 is necessary and sufficient to mediate many characteristics of Let-7 depletion, namely accelerating cell cycle progression and enhancing a stem cell phenotype. In addition, inactivation of a single Hmga2 allele in the mouse intestine epithelium significantly represses tumorigenesis driven by Lin28b. In aggregate, we conclude that Let-7 depletion drives a stem cell phenotype and the development of intestinal cancer, primarily via Hmga2

Let-7 Represses Carcinogenesis and a Stem Cell Phenotype in the Intestine via Regulation of Hmga2

Let-7 miRNAs comprise one of the largest and most highly expressed family of miRNAs among vertebrates, and is critical for promoting differentiation, regulating metabolism, inhibiting cellular proliferation, and repressing carcinogenesis in a variety of tissues. The large size of the Let-7 family of miRNAs has complicated the development of mutant animal models. Here we describe the comprehensive repression of all Let-7 miRNAs in the intestinal epithelium via low-level tissue-specific expression of the Lin28b RNA-binding protein and a conditional knockout of the MirLet7c-2/Mirlet7b locus. This ablation of Let-7 triggers the development of intestinal adenocarcinomas concomitant with reduced survival. Analysis of both mouse and human intestinal cancer specimens reveals that stem cell markers were significantly associated with loss of Let-7 miRNA expression, and that a number of Let-7 targets were elevated, including Hmga1 and Hmga2. Functional studies in 3-D enteroids revealed that Hmga2 is necessary and sufficient to mediate many characteristics of Let-7 depletion, namely accelerating cell cycle progression and enhancing a stem cell phenotype. In addition, inactivation of a single Hmga2 allele in the mouse intestine epithelium significantly represses tumorigenesis driven by Lin28b. In aggregate, we conclude that Let-7 depletion drives a stem cell phenotype and the development of intestinal cancer, primarily via Hmga2

Let-7 Represses Carcinogenesis and a Stem Cell Phenotype in the Intestine via Regulation of Hmga2

Let-7 miRNAs comprise one of the largest and most highly expressed family of miRNAs among vertebrates, and is critical for promoting differentiation, regulating metabolism, inhibiting cellular proliferation, and repressing carcinogenesis in a variety of tissues. The large size of the Let-7 family of miRNAs has complicated the development of mutant animal models. Here we describe the comprehensive repression of all Let-7 miRNAs in the intestinal epithelium via low-level tissue-specific expression of the Lin28b RNA-binding protein and a conditional knockout of the MirLet7c-2/Mirlet7b locus. This ablation of Let-7 triggers the development of intestinal adenocarcinomas concomitant with reduced survival. Analysis of both mouse and human intestinal cancer specimens reveals that stem cell markers were significantly associated with loss of Let-7 miRNA expression, and that a number of Let-7 targets were elevated, including Hmga1 and Hmga2. Functional studies in 3-D enteroids revealed that Hmga2 is necessary and sufficient to mediate many characteristics of Let-7 depletion, namely accelerating cell cycle progression and enhancing a stem cell phenotype. In addition, inactivation of a single Hmga2 allele in the mouse intestine epithelium significantly represses tumorigenesis driven by Lin28b. In aggregate, we conclude that Let-7 depletion drives a stem cell phenotype and the development of intestinal cancer, primarily via Hmga2

Hmga1 and Hmga2 proteins are increased in invasive areas of adenocarcinomas.

<p>Immunohistochemical staining for Hmga2 (A-C, G, H) and Hmga2 (D-F, I, J), in sections from WT small intestine (S.I.) (A, D), <i>Lin28b</i>^<i>Lo</i><i>/Let7</i>^{<i>IEC-KO</i>} S.I. (B, E), <i>Lin28b</i>^<i>Lo</i><i>/Let7</i>^{<i>IEC-KO</i>} adenoma (C, F), and <i>Lin28b</i>^<i>Lo</i><i>/Let7</i>^{<i>IEC-KO</i>} adenocarcinoma (AdenoCA) (G-J). An enlargement of a region containing invasive HMGA1-positive tumor cells from G (dotted yellow box) is pictured in H, while a region containing invasive HMGA2-positive tumor cells from I is likewise displayed in J. Pictures in A-F, H, and J are at same magnification (200x), with scale bar = 100 μm, while pictures in G and I are both at 40x, with scale bar = 250 μm.</p

Quantification of Let-7 target mRNA levels in intestinal epithelium crypts.

<p>A) Expression of Let-7 target mRNA levels in small intestine crypts isolated from <i>wild-type</i> (WT) and <i>Vil-Lin28b</i>^<i>Med</i> mice. B) Expression of Let-7 target mRNA levels in small intestine (jejunum) crypts isolated from <i>wild-type</i> (WT), <i>Vil-Lin28b</i>^<i>Lo</i>, <i>Let7</i>^{<i>IEC-KO</i>}, <i>Lin28b</i>^<i>Lo</i><i>/Let7</i>^<i>+/-</i>, and <i>Lin28b</i>^<i>Lo</i><i>/Let7</i>^{<i>IEC-KO</i>} mice. C) Comparison of Let-7 target mRNA changes in small intestine crypts from <i>Vil-Lin28b</i>^<i>Med</i> mice vs. <i>Lin28b</i>^<i>Lo</i><i>/Let7</i>^{<i>IEC-KO</i>} mice reveals similar expression changes in each model of Let-7 depletion, with significant correlation (Pearson correlation shown). Expression analysis was performed by Q-RT-PCR, normalized to <i>Hprt</i> and <i>Actb</i>, with n = 3 mice for each genotype at 12 weeks of age with error bars representing +/–the S.E.M. D) Identification of conserved Let-7 target genes in ten of eleven Let-7 target genes based upon TargetScan.org prediction. Student’s two-tailed T-tests were performed to determine significance with * p-value < 0.05, ** p-value < 0.01, and *** p-value < 0.001, relative to WT small intestine. One-way ANOVA standard weighted-means analysis was also performed in B, with p-values < 0.05 indicated above each gene. Tukey's HSD (honest significant difference) post-test was also performed in B, with samples p < 0.05 (red asterisk) and p < 0.01 (†) indicated, relative to mean of WT small intestine.</p

Hmga2 mediates Lin28b effects on stem cell colony formation and enteroid proliferation.

<p>Colony formation assay of small intestine enteroids from wild-type (WT) mice (A) and <i>Vil-Lin28b</i>^<i>Med</i> mice (B). Two wells of a 24-well plate are pictured. C) Quantification of colony forming assay of WT mice and <i>Vil-Lin28b</i>^<i>Med</i> mice. D) Schematic of lentiviral vector-mediated transduction of enteroids with a “tet-on” doxycycline (dox)-inducible vector. Inducible expression of a turbo RFP reporter in the unmodified pTRIPz vector is readily observed in enteroids (H) vs. untreated cells (G). Phase contrast images of untreated and doxycycline treated enteroids are pictured in (E) and (F), respectively. Immunostaining for Hmga2 in sectioned enteroids from pTRIPz-transduced (I), pTRIPz-Hmga2-transduced (J), and pTRIPz-Hmga2-transduced plus doxycycline (K). L) Comparison of colony forming potential of cells from dissociated enteroids transduced with pTRIPz (empty), Arid3a, Hif3a, or Hmga2 vectors. Phase contrast images of colony formation assays from enteroids transduced with pTRIPz (empty) (M) or Hmga2 (N) vectors. O) Colony forming assays of non-transgenic mice (NTG) or <i>Vil-Lin28b</i>^<i>Med</i> mice with and without inactivation of one conditional Hmga2 allele using <i>Vil-Cre</i>. EdU incorporation of enteroids, as assayed by flow cytometry, transduced with pTRIPz-Hmga2 (P) or pTRIPz-Hif3a (Q) vectors. Cultures were treated with 100 ng/ml doxycycline in all experiments except in (F) and (H), which were treated with 500 ng/ml doxycycline. All experiments were performed at least in triplicate. Student’s two-tailed T-tests were performed to determine significance with * p-value < 0.05, ** p-value < 0.01, and *** p-value < 0.001, relative to pTRIPz vector controls, unless noted otherwise.</p

Let-7 and HMGA2 are associated with a stem cell signature in intestinal adenocarcinomas.

<p>A) Heat map of TCGA mRNA-seq colon and rectal adenocarcinoma dataset from UCSC Cancer Genome Browser (genome-cancer.ucsc.edu) comparing expression of Let-7 target mRNAs in normal tissue (N.T.) vs. cancer. Significant up-regulation (red) or down-regulation (green) is indicated below heatmap in plots of the-log(p-value) of Benjamini-Hochberg-corrected T-tests on the y-axis. T-test results are shown for expression in tumors vs. N.T. and in tumors associated with at least one lymph node metastases vs. tumors with no associated lymph node metastases. Inverse relationships for Let-7 and target mRNAs could be discerned by plotting miRNA-seq data against mRNA-seq data for Let-7c vs. <i>HMGA2</i> (B), Let-7a vs. <i>PLAGL2</i> (C), Let-7a vs. <i>HMGA2</i> (D), and Let-7a vs. <i>IGF2BP2</i> (E). F) Taqman QPCR for mature Let-7a and Let-7b miRNAs in a cohort of colon adenocarcinomas (N = 20) indicates that Let-7a and Let-7b are down-regulated. G) Intestinal epithelial stem cell markers <i>EPHB2</i>, <i>ASCL2</i>, and <i>LGR5</i> are significantly up-regulated in colon cancer vs. normal adjacent tissues. H) Mature Let-7a and Let-7b levels are tightly correlated in these tissue specimens. I) Let-7a and Let-7b levels are inversely proportional to mRNA levels of stem cell markers <i>EPHB2</i> and <i>LGR5</i>, suggesting that Let-7 may repress a stem cell signature. J) Expression of stem cell markers is dramatically up-regulated in <i>Lin28b</i>^<i>Lo</i><i>/Let7</i>^{<i>IEC-KO</i>} tumors, relative to WT jejunum, with a trend for up-regulation in <i>Lin28b</i>^<i>Lo</i><i>/Let7</i>^{<i>IEC-KO</i>} jejunum, relative to WT. K) Comparison of stem cell marker expression and Let-7 target mRNA expression levels in WT jejunum, <i>Lin28b</i>^<i>Lo</i><i>/Let7</i>^{<i>IEC-KO</i>} jejunum, and <i>Lin28b</i>^<i>Lo</i><i>/Let7</i>^{<i>IEC-KO</i>} tumors by linear regression yielded Pearson correlation coefficients, with <i>Arid3a</i>, <i>Hmga1</i>, and <i>Hmga2</i> correlating very highly with expression of stem cell markers. L) HMGA2 and LGR5 expression from the TCGA mRNA-seq colon and rectal adenocarcinoma dataset exhibit significant positive correlation. Expression analysis (F-K) was performed by QPCR, normalized to <i>Hprt</i> and <i>Actb</i>, with n = 3–4 for each mouse genotype with error bars representing +/–the S.E.M. Human QPCR was normalized to <i>PPIA</i> and <i>B2M</i>, with error bars representing +/–the S.E.M. Student’s two-tailed T-tests were performed to determine significance with * p-value < 0.05, ** p-value < 0.01, and *** p-value < 0.001.</p

Comprehensive depletion of all Let-7 miRNAs leads to the development of intestinal adenocarcinomas.

<p>A) Schematic of the intestine-specific deletion of the <i>Mirlet7c-2/Mirlet7b</i> floxed locus via <i>Villin-Cre</i> and expression of Lin28b with a <i>Villin-Lin28b-ires-tdTomato</i> transgene, which repress all 8 of the Let-7 clusters. Let-7 miRNA genes are shown as black hairpins while non-let-7 miRNA genes are depicted as gray hairpins. B) Kaplan-Meier plot showing survival over 10 months. C) Representative small intestine from a <i>Lin28b</i>^<i>Lo</i><i>/Let7</i>^{<i>IEC-KO</i>} mouse containing two tumors, T1 and T2 (box outline with yellow dotted lines). D) Large tumor from (C) dissected with luminal side facing outward. E) H&E stained paraffin section of adenocarcinoma from a <i>Lin28b</i>^<i>Lo</i><i>/Let7</i>^{<i>IEC-KO</i>} mouse. F) Representative section of adenocarcinoma from a <i>Lin28b</i>^<i>Lo</i><i>/Let7</i>^{<i>IEC-KO</i>} mouse immunostained for β-catenin, showing a nuclear pattern of localization. Scale bars in (E) and (F) = 100 μm.</p