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

Madison BB, Jeganathan AN, Mizuno R, Winslow MM, Castells A, Cuatrecasas M, Rustgi AK - PLoS Genet. (2015)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Division of Gastroenterology, Washington University School of Medicine, Saint Louis, Missouri, United States of America; Department of Medicine, Washington University School of Medicine, Saint Louis, Missouri, United States of America.

ABSTRACT
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.

No MeSH data available.


Related in: MedlinePlus

Let-7 and HMGA2 are associated with a stem cell signature in intestinal adenocarcinomas.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. HMGA2 (B), Let-7a vs. PLAGL2 (C), Let-7a vs. HMGA2 (D), and Let-7a vs. IGF2BP2 (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 EPHB2, ASCL2, and LGR5 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 EPHB2 and LGR5, suggesting that Let-7 may repress a stem cell signature. J) Expression of stem cell markers is dramatically up-regulated in Lin28bLo/Let7IEC-KO tumors, relative to WT jejunum, with a trend for up-regulation in Lin28bLo/Let7IEC-KO jejunum, relative to WT. K) Comparison of stem cell marker expression and Let-7 target mRNA expression levels in WT jejunum, Lin28bLo/Let7IEC-KO jejunum, and Lin28bLo/Let7IEC-KO tumors by linear regression yielded Pearson correlation coefficients, with Arid3a, Hmga1, and Hmga2 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 Hprt and Actb, with n = 3–4 for each mouse genotype with error bars representing +/–the S.E.M. Human QPCR was normalized to PPIA and B2M, 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.
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pgen.1005408.g005: Let-7 and HMGA2 are associated with a stem cell signature in intestinal adenocarcinomas.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. HMGA2 (B), Let-7a vs. PLAGL2 (C), Let-7a vs. HMGA2 (D), and Let-7a vs. IGF2BP2 (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 EPHB2, ASCL2, and LGR5 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 EPHB2 and LGR5, suggesting that Let-7 may repress a stem cell signature. J) Expression of stem cell markers is dramatically up-regulated in Lin28bLo/Let7IEC-KO tumors, relative to WT jejunum, with a trend for up-regulation in Lin28bLo/Let7IEC-KO jejunum, relative to WT. K) Comparison of stem cell marker expression and Let-7 target mRNA expression levels in WT jejunum, Lin28bLo/Let7IEC-KO jejunum, and Lin28bLo/Let7IEC-KO tumors by linear regression yielded Pearson correlation coefficients, with Arid3a, Hmga1, and Hmga2 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 Hprt and Actb, with n = 3–4 for each mouse genotype with error bars representing +/–the S.E.M. Human QPCR was normalized to PPIA and B2M, 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.

Mentions: To extrapolate relevance to human CRC from these mouse models, we examined expression data from human samples from The Cancer Genome Atlas (TCGA) [35] by querying for expression of Let-7 target mRNAs, with a focus on targets that exhibited significant up-regulation in either Vil-Lin28bMed or Lin28bLo/Let7IEC-KO mouse models (namely, ARID3A, PLAGL2, HMGA1, HMGA2, MYCN, IGF2BP1, IGF2BP2, and E2F5). We examined a cohort of 416 CRC patients from a TCGA dataset and found that all transcripts except HIF3A mRNA were significantly elevated in cancer tissue compared to expression levels in normal tissues (Fig 5A). IGF2BP1 expression in primary tumors was also associated with an increased likelihood of having nodal metastases (Fig 5A). Levels of HMGA1, HMGA2, PLAGL2, IGF2BP2, E2F5, and ARID3A transcripts were also inversely proportional to levels of Let-7 miRNA by examination of a cohort of 199 CRC patients from the TCGA Pan-Cancer analysis project [20] (Fig 5B–5E and S1D–S1I Fig). Since Let-7a and Let-7b appear to be the most highly expressed Let-7 miRNAs in normal colonic epithelium, and are significantly depleted in CRC specimens [20,30] (S1A, S1B and S1C Fig), we examined these miRNAs in a subset of colon cancer specimens. We also compared their expression with the crypt-base-columnar (CBC) stem cell markers EPHB2, ASCL2, and LGR5, which are markers of stem cells in human intestine/colon and CRC, and are associated with aggressive CRC [36]. We found that Let-7a and Let-7b were significantly down-regulated in CRC specimens, while stem cell markers were significantly up-regulated (Fig 5F and 5G). Let-7a and Let-7b levels were also correlated tightly, suggesting co-regulation (Fig 5H), and were also inversely proportional to the expression of the stem cell markers EPHB2 and LGR5 (Fig 5I). This suggests provocatively that Let-7a and Let-7b depletion may contribute to a stem cell phenotype in the intestine, and perhaps CRC.


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

Madison BB, Jeganathan AN, Mizuno R, Winslow MM, Castells A, Cuatrecasas M, Rustgi AK - PLoS Genet. (2015)

Let-7 and HMGA2 are associated with a stem cell signature in intestinal adenocarcinomas.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. HMGA2 (B), Let-7a vs. PLAGL2 (C), Let-7a vs. HMGA2 (D), and Let-7a vs. IGF2BP2 (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 EPHB2, ASCL2, and LGR5 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 EPHB2 and LGR5, suggesting that Let-7 may repress a stem cell signature. J) Expression of stem cell markers is dramatically up-regulated in Lin28bLo/Let7IEC-KO tumors, relative to WT jejunum, with a trend for up-regulation in Lin28bLo/Let7IEC-KO jejunum, relative to WT. K) Comparison of stem cell marker expression and Let-7 target mRNA expression levels in WT jejunum, Lin28bLo/Let7IEC-KO jejunum, and Lin28bLo/Let7IEC-KO tumors by linear regression yielded Pearson correlation coefficients, with Arid3a, Hmga1, and Hmga2 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 Hprt and Actb, with n = 3–4 for each mouse genotype with error bars representing +/–the S.E.M. Human QPCR was normalized to PPIA and B2M, 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.
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pgen.1005408.g005: Let-7 and HMGA2 are associated with a stem cell signature in intestinal adenocarcinomas.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. HMGA2 (B), Let-7a vs. PLAGL2 (C), Let-7a vs. HMGA2 (D), and Let-7a vs. IGF2BP2 (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 EPHB2, ASCL2, and LGR5 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 EPHB2 and LGR5, suggesting that Let-7 may repress a stem cell signature. J) Expression of stem cell markers is dramatically up-regulated in Lin28bLo/Let7IEC-KO tumors, relative to WT jejunum, with a trend for up-regulation in Lin28bLo/Let7IEC-KO jejunum, relative to WT. K) Comparison of stem cell marker expression and Let-7 target mRNA expression levels in WT jejunum, Lin28bLo/Let7IEC-KO jejunum, and Lin28bLo/Let7IEC-KO tumors by linear regression yielded Pearson correlation coefficients, with Arid3a, Hmga1, and Hmga2 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 Hprt and Actb, with n = 3–4 for each mouse genotype with error bars representing +/–the S.E.M. Human QPCR was normalized to PPIA and B2M, 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.
Mentions: To extrapolate relevance to human CRC from these mouse models, we examined expression data from human samples from The Cancer Genome Atlas (TCGA) [35] by querying for expression of Let-7 target mRNAs, with a focus on targets that exhibited significant up-regulation in either Vil-Lin28bMed or Lin28bLo/Let7IEC-KO mouse models (namely, ARID3A, PLAGL2, HMGA1, HMGA2, MYCN, IGF2BP1, IGF2BP2, and E2F5). We examined a cohort of 416 CRC patients from a TCGA dataset and found that all transcripts except HIF3A mRNA were significantly elevated in cancer tissue compared to expression levels in normal tissues (Fig 5A). IGF2BP1 expression in primary tumors was also associated with an increased likelihood of having nodal metastases (Fig 5A). Levels of HMGA1, HMGA2, PLAGL2, IGF2BP2, E2F5, and ARID3A transcripts were also inversely proportional to levels of Let-7 miRNA by examination of a cohort of 199 CRC patients from the TCGA Pan-Cancer analysis project [20] (Fig 5B–5E and S1D–S1I Fig). Since Let-7a and Let-7b appear to be the most highly expressed Let-7 miRNAs in normal colonic epithelium, and are significantly depleted in CRC specimens [20,30] (S1A, S1B and S1C Fig), we examined these miRNAs in a subset of colon cancer specimens. We also compared their expression with the crypt-base-columnar (CBC) stem cell markers EPHB2, ASCL2, and LGR5, which are markers of stem cells in human intestine/colon and CRC, and are associated with aggressive CRC [36]. We found that Let-7a and Let-7b were significantly down-regulated in CRC specimens, while stem cell markers were significantly up-regulated (Fig 5F and 5G). Let-7a and Let-7b levels were also correlated tightly, suggesting co-regulation (Fig 5H), and were also inversely proportional to the expression of the stem cell markers EPHB2 and LGR5 (Fig 5I). This suggests provocatively that Let-7a and Let-7b depletion may contribute to a stem cell phenotype in the intestine, and perhaps CRC.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Division of Gastroenterology, Washington University School of Medicine, Saint Louis, Missouri, United States of America; Department of Medicine, Washington University School of Medicine, Saint Louis, Missouri, United States of America.

ABSTRACT
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.

No MeSH data available.


Related in: MedlinePlus