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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

Identification of Let-7 targets up-regulated specifically in transformed cells from intestinal adenocarcinomas.A) Schematic of experimental procedure where tumors were micro-dissected from the small intestine (S.I.) of Lin28bLo/Let7IEC-KO mice and cultured as epithelial tumoroid/enteroids (T/E), grown in ENR medium, and tumoroid cysts (TC), grown in medium lacking Noggin and Rspo1. B) Typical tumoroid grown in ENR medium. C) Tumoroid cysts grown in basal medium containing EGF. D) Let-7 miRNAs are repressed consistently in tumoroid/enteroids (TE) and tumoroid cysts (TC). E) Wnt (Tcf4/β-catenin) target genes Axin2, CD44 and cMyc mRNAs were up-regulated in tumors, T/E, and TC. F) Transcripts with highest expression in tumor or tumoroid, but tend to be down-regulated in tumoroid cysts. G) Transcripts that maintain high expression and/or are increased in tumoroid cysts. Note logarithmic scale where Hmga2 mRNA is induced approximately 200-fold in TC compared to wild-type S.I. Immunostaining in Lin28bLo/Let7IEC-KO adenomas (H, I) and adenocarcinomas (J, K) revealed that nuclear β-catenin (H, J) and Hmga2 (I, K) are often co-expressed at high levels. Expression analysis was performed by Q-RT-PCR, normalized to Hprt and Actb, with n = 3–4 for each tissue/organoid 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, relative to wild-type small intestine. One-way ANOVA standard weighted-means analysis was also performed in D-G, with p-values < 0.05 indicated above each gene. Tukey's HSD (honest significant difference) post-test was also performed in D-G, with samples p < 0.05 (red asterisk) and p < 0.01 (†) indicated, relative to mean of WT small intestine (jej.).
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pgen.1005408.g004: Identification of Let-7 targets up-regulated specifically in transformed cells from intestinal adenocarcinomas.A) Schematic of experimental procedure where tumors were micro-dissected from the small intestine (S.I.) of Lin28bLo/Let7IEC-KO mice and cultured as epithelial tumoroid/enteroids (T/E), grown in ENR medium, and tumoroid cysts (TC), grown in medium lacking Noggin and Rspo1. B) Typical tumoroid grown in ENR medium. C) Tumoroid cysts grown in basal medium containing EGF. D) Let-7 miRNAs are repressed consistently in tumoroid/enteroids (TE) and tumoroid cysts (TC). E) Wnt (Tcf4/β-catenin) target genes Axin2, CD44 and cMyc mRNAs were up-regulated in tumors, T/E, and TC. F) Transcripts with highest expression in tumor or tumoroid, but tend to be down-regulated in tumoroid cysts. G) Transcripts that maintain high expression and/or are increased in tumoroid cysts. Note logarithmic scale where Hmga2 mRNA is induced approximately 200-fold in TC compared to wild-type S.I. Immunostaining in Lin28bLo/Let7IEC-KO adenomas (H, I) and adenocarcinomas (J, K) revealed that nuclear β-catenin (H, J) and Hmga2 (I, K) are often co-expressed at high levels. Expression analysis was performed by Q-RT-PCR, normalized to Hprt and Actb, with n = 3–4 for each tissue/organoid 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, relative to wild-type small intestine. One-way ANOVA standard weighted-means analysis was also performed in D-G, with p-values < 0.05 indicated above each gene. Tukey's HSD (honest significant difference) post-test was also performed in D-G, with samples p < 0.05 (red asterisk) and p < 0.01 (†) indicated, relative to mean of WT small intestine (jej.).

Mentions: We next examined Let-7 targets that might mediate programs of tumorigenesis in Lin28bLo/Let7IEC-KO mice in the context of tumors and cellular transformation. To model intestinal epithelial carcinogenesis we developed a 3-D culture model to examine only the epithelium and to select transformed tumor cells (Fig 4A). Enteroids derived from CRC tumors appear to faithfully recapitulate the major expression signatures of un-manipulated whole tumors [33]. To pursue this, we micro-dissected and dissociated adenocarcinomas from Lin28bLo/Let7IEC-KO mice and cultured “tumoroids” from these lesions in medium supplemented with EGF, Noggin, and Rspo1, as described previously for enteroid culture [34]. These tumoroid/enteroids (T/E) resembled normal small intestine enteroids (Fig 4B) and are likely a mixture of different cell types, but upon withdrawal of Noggin and Rspo1, a small population of growth-factor independent cells expanded into tumoroid cysts (TC) (Fig 4C), which likely possess cell-autonomous activation of Wnt signaling and Noggin-independent resistance to BMP signaling. Quantification by Taqman RT-PCR confirmed that Let-7 miRNAs are severely repressed in tumoroid/enteroids and transformed tumoroid cysts (Fig 4D). Tumors and tumoroids, but not normal tissue from Lin28bLo/Let7IEC-KO mice, also exhibited up-regulation of Wnt target genes Axin2, CD44, and cMyc (Fig 4E), suggesting spontaneous and constitutive activation of Wnt signaling. Analysis of Let-7 target mRNAs revealed two basic patterns of expression, with one group displaying expression highest in intact tumors or tumoroids/enteroids (Fig 4F). The other group displayed increasing or plateauing expression, with higher levels in tumoroid/enteroids or tumoroid cysts (Fig 4G). In this latter group we find known and suspected oncogenes, such as Hmga1, Hmga2, Igf2bp1, Igf2bp2, and Mycn. As Hmga2 appeared to exhibit pronounced up-regulation (>200-fold) in the tumoroid/enteroid and tumoroid cyst populations, and increased staining in invasive areas of adenocarcinomas (Fig 3H), we evaluated Hmga2 co-localization with nuclear β-catenin in mouse tumors, to assay potential coincident activation of canonical Wnt signaling with nuclear Hmga2. Immunostaining in both adenomas and adenocarcinomas from Lin28bLo/Let7IEC-KO mice revealed frequent and intense co-staining of Hmga2 with nuclear β-catenin (Fig 4H–4K). This pattern of co-staining was not observed for Hmga1, Arid3a, or Hif3a.


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)

Identification of Let-7 targets up-regulated specifically in transformed cells from intestinal adenocarcinomas.A) Schematic of experimental procedure where tumors were micro-dissected from the small intestine (S.I.) of Lin28bLo/Let7IEC-KO mice and cultured as epithelial tumoroid/enteroids (T/E), grown in ENR medium, and tumoroid cysts (TC), grown in medium lacking Noggin and Rspo1. B) Typical tumoroid grown in ENR medium. C) Tumoroid cysts grown in basal medium containing EGF. D) Let-7 miRNAs are repressed consistently in tumoroid/enteroids (TE) and tumoroid cysts (TC). E) Wnt (Tcf4/β-catenin) target genes Axin2, CD44 and cMyc mRNAs were up-regulated in tumors, T/E, and TC. F) Transcripts with highest expression in tumor or tumoroid, but tend to be down-regulated in tumoroid cysts. G) Transcripts that maintain high expression and/or are increased in tumoroid cysts. Note logarithmic scale where Hmga2 mRNA is induced approximately 200-fold in TC compared to wild-type S.I. Immunostaining in Lin28bLo/Let7IEC-KO adenomas (H, I) and adenocarcinomas (J, K) revealed that nuclear β-catenin (H, J) and Hmga2 (I, K) are often co-expressed at high levels. Expression analysis was performed by Q-RT-PCR, normalized to Hprt and Actb, with n = 3–4 for each tissue/organoid 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, relative to wild-type small intestine. One-way ANOVA standard weighted-means analysis was also performed in D-G, with p-values < 0.05 indicated above each gene. Tukey's HSD (honest significant difference) post-test was also performed in D-G, with samples p < 0.05 (red asterisk) and p < 0.01 (†) indicated, relative to mean of WT small intestine (jej.).
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pgen.1005408.g004: Identification of Let-7 targets up-regulated specifically in transformed cells from intestinal adenocarcinomas.A) Schematic of experimental procedure where tumors were micro-dissected from the small intestine (S.I.) of Lin28bLo/Let7IEC-KO mice and cultured as epithelial tumoroid/enteroids (T/E), grown in ENR medium, and tumoroid cysts (TC), grown in medium lacking Noggin and Rspo1. B) Typical tumoroid grown in ENR medium. C) Tumoroid cysts grown in basal medium containing EGF. D) Let-7 miRNAs are repressed consistently in tumoroid/enteroids (TE) and tumoroid cysts (TC). E) Wnt (Tcf4/β-catenin) target genes Axin2, CD44 and cMyc mRNAs were up-regulated in tumors, T/E, and TC. F) Transcripts with highest expression in tumor or tumoroid, but tend to be down-regulated in tumoroid cysts. G) Transcripts that maintain high expression and/or are increased in tumoroid cysts. Note logarithmic scale where Hmga2 mRNA is induced approximately 200-fold in TC compared to wild-type S.I. Immunostaining in Lin28bLo/Let7IEC-KO adenomas (H, I) and adenocarcinomas (J, K) revealed that nuclear β-catenin (H, J) and Hmga2 (I, K) are often co-expressed at high levels. Expression analysis was performed by Q-RT-PCR, normalized to Hprt and Actb, with n = 3–4 for each tissue/organoid 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, relative to wild-type small intestine. One-way ANOVA standard weighted-means analysis was also performed in D-G, with p-values < 0.05 indicated above each gene. Tukey's HSD (honest significant difference) post-test was also performed in D-G, with samples p < 0.05 (red asterisk) and p < 0.01 (†) indicated, relative to mean of WT small intestine (jej.).
Mentions: We next examined Let-7 targets that might mediate programs of tumorigenesis in Lin28bLo/Let7IEC-KO mice in the context of tumors and cellular transformation. To model intestinal epithelial carcinogenesis we developed a 3-D culture model to examine only the epithelium and to select transformed tumor cells (Fig 4A). Enteroids derived from CRC tumors appear to faithfully recapitulate the major expression signatures of un-manipulated whole tumors [33]. To pursue this, we micro-dissected and dissociated adenocarcinomas from Lin28bLo/Let7IEC-KO mice and cultured “tumoroids” from these lesions in medium supplemented with EGF, Noggin, and Rspo1, as described previously for enteroid culture [34]. These tumoroid/enteroids (T/E) resembled normal small intestine enteroids (Fig 4B) and are likely a mixture of different cell types, but upon withdrawal of Noggin and Rspo1, a small population of growth-factor independent cells expanded into tumoroid cysts (TC) (Fig 4C), which likely possess cell-autonomous activation of Wnt signaling and Noggin-independent resistance to BMP signaling. Quantification by Taqman RT-PCR confirmed that Let-7 miRNAs are severely repressed in tumoroid/enteroids and transformed tumoroid cysts (Fig 4D). Tumors and tumoroids, but not normal tissue from Lin28bLo/Let7IEC-KO mice, also exhibited up-regulation of Wnt target genes Axin2, CD44, and cMyc (Fig 4E), suggesting spontaneous and constitutive activation of Wnt signaling. Analysis of Let-7 target mRNAs revealed two basic patterns of expression, with one group displaying expression highest in intact tumors or tumoroids/enteroids (Fig 4F). The other group displayed increasing or plateauing expression, with higher levels in tumoroid/enteroids or tumoroid cysts (Fig 4G). In this latter group we find known and suspected oncogenes, such as Hmga1, Hmga2, Igf2bp1, Igf2bp2, and Mycn. As Hmga2 appeared to exhibit pronounced up-regulation (>200-fold) in the tumoroid/enteroid and tumoroid cyst populations, and increased staining in invasive areas of adenocarcinomas (Fig 3H), we evaluated Hmga2 co-localization with nuclear β-catenin in mouse tumors, to assay potential coincident activation of canonical Wnt signaling with nuclear Hmga2. Immunostaining in both adenomas and adenocarcinomas from Lin28bLo/Let7IEC-KO mice revealed frequent and intense co-staining of Hmga2 with nuclear β-catenin (Fig 4H–4K). This pattern of co-staining was not observed for Hmga1, Arid3a, or Hif3a.

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