Blair B. Madison

Transforming Growth Factor β Receptor Type II Inactivation Induces the Malignant Transformation of Intestinal Neoplasms Initiated by Apc Mutation

The transforming growth factor-beta (TGF-beta) signaling pathway is a tumor-suppressor pathway that is commonly inactivated in colon cancer. TGF-beta is a secreted ligand that mediates its effects through a transmembrane heteromeric receptor complex, which consists of type I (TGFBR1) and type II subunits (TGFBR2). Approximately 30% of colon cancers carry TGFBR2 mutations, demonstrating that it is a common target for mutational inactivation in this cancer. To assess the functional role of TGFBR2 inactivation in the multistep progression sequence of colon cancer, we generated a mouse model that recapitulates two common genetic events observed in human colon cancer by mating Apc(1638N/wt) mice with mice that are null for Tgfbr2 in the intestinal epithelium, Villin-Cre;Tgfbr2(E2flx/E2flx) mice. In this model, we observed a dramatic increase in the number of intestinal adenocarcinomas in the Apc(1638N/wt);Villin-Cre;Tgfbr2(E2flx/E2flx) mice (called Apc(1638N/wt);Tgfbr2(IEKO)) compared with those mice with intact Tgfbr2 (Apc(1638N/wt);Tgfbr2(E2flx/E2flx)). Additionally, in vitro analyses of epithelial tumor cells derived from the Apc(1638N/wt);Tgfbr2(IEKO) mice showed enhanced expression and activity of matrix metalloproteinase MMP-2 and MMP-9, as well as increased TGF-beta1 secretion in the conditioned medium. Similarly, primary tumor tissues from the Apc(1638N/wt);Tgfbr2(IEKO) mice also showed elevated amounts of TGF-beta1 as well as higher MMP-2 activity in comparison with Apc(1638N/wt);Tgfbr2(E2flx/E2flx)-derived tumors. Thus, loss of TGFBR2 in intestinal epithelial cells promotes the invasion and malignant transformation of tumors initiated by Apc mutation, providing evidence that Wnt signaling deregulation and TGF-beta signaling inactivation cooperate to drive the initiation and progression, respectively, of intestinal cancers in vivo.

Paracrine Hedgehog Signaling in Stomach and Intestine: New Roles for Hedgehog in Gastrointestinal Patterning

BACKGROUND & AIMS Hedgehog signaling is critical in gastrointestinal patterning. Mice deficient in Hedgehog signaling exhibit abnormalities that mirror deformities seen in the human VACTERL (vertebral, anal, cardiac, tracheal, esophageal, renal, limb) association. However, the direction of Hedgehog signal flow is controversial and the cellular targets of Hedgehog signaling change with time during development. We profiled cellular Hedgehog response patterns from embryonic day 10.5 (E10.5) to adult in murine antrum, pyloric region, small intestine, and colon. METHODS Hedgehog signaling was profiled using Hedgehog pathway reporter mice and in situ hybridization. Cellular targets were identified by immunostaining. Ihh-overexpressing transgenic animals were generated and analyzed. RESULTS Hedgehog signaling is strictly paracrine from antrum to colon throughout embryonic and adult life. Novel findings include the following: mesothelial cells of the serosa transduce Hedgehog signals in fetal life; the hindgut epithelium expresses Ptch but not Gli1 at E10.5; the 2 layers of the muscularis externa respond differently to Hedgehog signals; organogenesis of the pyloric sphincter is associated with robust Hedgehog signaling; dramatically different Hedgehog responses characterize stomach and intestine at E16; and after birth, the muscularis mucosa and villus smooth muscle consist primarily of Hedgehog-responsive cells and Hh levels actively modulate villus core smooth muscle. CONCLUSIONS These studies reveal a previously unrecognized association of paracrine Hedgehog signaling with several gastrointestinal patterning events involving the serosa, pylorus, and villus smooth muscle. The results may have implications for several human anomalies and could potentially expand the spectrum of the human VACTERL association.

LIN28B fosters colon cancer migration, invasion and transformation through let-7-dependent and -independent mechanisms.

Lin28b is an RNA-binding protein that inhibits biogenesis of let-7 microRNAs. LIN28B is overexpressed in diverse cancers, yet a specific role in the molecular pathogenesis of colon cancer has to be elucidated. We have determined that human colon tumors exhibit decreased levels of mature let-7 isoforms and increased expression of LIN28B. To determine LIN28Bs mechanistic role in colon cancer, we expressed LIN28B in immortalized colonic epithelial cells and human colon cancer cell lines. We found that LIN28B promotes cell migration, invasion and transforms immortalized colonic epithelial cells. In addition, constitutive LIN28B expression increases expression of intestinal stem cell markers LGR5 and PROM1 in the presence of let-7 restoration. This may occur as a result of Lin28b protein binding LGR5 and PROM1 mRNA, suggesting that a subset of LIN28B functions is independent of its ability to repress let-7. Our findings establish a new role for LIN28B in human colon cancer pathogenesis, and suggest LIN28B post-transcriptionally regulates LGR5 and PROM1 through a let-7-independent mechanism.

LIN28B promotes growth and tumorigenesis of the intestinal epithelium via Let-7.

The RNA-binding proteins LIN28A and LIN28B have diverse functions in embryonic stem cells, cellular reprogramming, growth, and oncogenesis. Many of these effects occur via direct inhibition of Let-7 microRNAs (miRNAs), although Let-7-independent effects have been surmised. We report that intestine targeted expression of LIN28B causes intestinal hypertrophy, crypt expansion, and Paneth cell loss. Furthermore, LIN28B fosters intestinal polyp and adenocarcinoma formation. To examine potential Let-7-independent functions of LIN28B, we pursued ribonucleoprotein cross-linking, immunoprecipitation, and high-throughput sequencing (CLIP-seq) to identify direct RNA targets. This revealed that LIN28B bound a substantial number of mRNAs and modestly augmented protein levels of these target mRNAs in vivo. Conversely, Let-7 had a profound effect; modulation of Let-7 levels via deletion of the mirLet7c2/mirLet7b genes recapitulated effects of Lin28b overexpression. Furthermore, intestine-specific Let-7 expression could reverse hypertrophy and Paneth cell depletion caused by Lin28b. This was independent of effects on insulin-PI3K-mTOR signaling. Our study reveals that Let-7 miRNAs are critical for repressing intestinal tissue growth and promoting Paneth cell differentiation. Let-7-dependent effects of LIN28B may supersede Let-7-independent effects on intestinal tissue growth. In summary, LIN28B can definitively act as an oncogene in the absence of canonical genetic alterations.

Hedgehog Is an Anti-Inflammatory Epithelial Signal for the Intestinal Lamina Propria

BACKGROUND & AIMS Epithelial Hedgehog (Hh) ligands regulate several aspects of fetal intestinal organogenesis, and emerging data implicate the Hh pathway in inflammatory signaling in the adult colon. Here, we investigated the effects of chronic Hh inhibition in vivo and profiled molecular pathways acutely modulated by Hh signaling in the intestinal mesenchyme. METHODS The progression of inflammatory disease was characterized in a bi-transgenic mouse model of chronic Hh inhibition (VFHhip). In parallel, microarray and bioinformatic analyses (Gene Ontology terms overrepresentation analysis, hierarchical clustering, and MeSH term filtration) were performed on isolated cultured intestinal mesenchyme acutely exposed to Hh ligand. RESULTS Six- to 10-month-old VFHhip animals exhibited villus smooth muscle loss and subsequent villus atrophy. Areas of villus loss became complicated by spontaneous inflammation and VFHhip animals succumbed to wasting and death. Phenotypic similarities were noted between the VFHhip phenotype and human inflammatory disorders, especially human celiac disease. Microarray analysis revealed that inflammatory pathways were acutely activated in intestinal mesenchyme cultured in the absence of epithelium, and the addition of Hh ligand alone was sufficient to largely reverse this inflammatory response within 24 hours. CONCLUSIONS Hh ligand is a previously unrecognized anti-inflammatory epithelial modulator of the mesenchymal inflammatory milieu. Acute modulation of Hh signals results in changes in inflammatory pathways in intestinal mesenchyme, while chronic inhibition of Hh signaling in adult animals leads to spontaneous intestinal inflammation and death. Regulation of epithelial Hh signaling may be an important mechanism to modulate tolerogenic versus proinflammatory signaling in the small intestine.

Hedgehog signaling controls homeostasis of adult intestinal smooth muscle

The Hedgehog (Hh) pathway plays multiple patterning roles during development of the mammalian gastrointestinal tract, but its role in adult gut function has not been extensively examined. Here we show that chronic reduction in the combined epithelial Indian (Ihh) and Sonic (Shh) hedgehog signal leads to mislocalization of intestinal subepithelial myofibroblasts, loss of smooth muscle in villus cores and muscularis mucosa as well as crypt hyperplasia. In contrast, chronic over-expression of Ihh in the intestinal epithelium leads to progressive expansion of villus smooth muscle, but does not result in reduced epithelial proliferation. Together, these mouse models show that smooth muscle populations in the adult intestinal lamina propria are highly sensitive to the level of Hh ligand. We demonstrate further that Hh ligand drives smooth muscle differentiation in primary intestinal mesenchyme cultures and that cell-autonomous Hh signal transduction in C3H10T1/2 cells activates the smooth muscle master regulator Myocardin (Myocd) and induces smooth muscle differentiation. The rapid kinetics of Myocd activation by Hh ligands as well as the presence of an unusual concentration of Gli sties in this gene suggest that regulation of Myocd by Hh might be direct. Thus, these data indicate that Hh is a critical regulator of adult intestinal smooth muscle homeostasis and suggest an important link between Hh signaling and Myocd activation. Moreover, the data support the idea that lowered Hh signals promote crypt expansion and increased epithelial cell proliferation, but indicate that chronically increased Hh ligand levels do not dampen crypt proliferation as previously proposed.

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.

A LIN28B-RAN-AURKA Signaling Network Promotes Neuroblastoma Tumorigenesis.

A more complete understanding of aberrant oncogenic signaling in neuroblastoma, a malignancy of the developing sympathetic nervous system, is paramount to improving patient outcomes. Recently, we identified LIN28B as an oncogenic driver in high-risk neuroblastoma. Here, we identify the oncogene RAN as a LIN28B target and show regional gain of chromosome 12q24 as an additional somatic alteration resulting in increased RAN expression. We show that LIN28B influences RAN expression by promoting RAN Binding Protein 2 expression and by directly binding RAN mRNA. Further, we demonstrate a convergence of LIN28B and RAN signaling on Aurora kinase A activity. Collectively, these findings demonstrate that LIN28B-RAN-AURKA signaling drives neuroblastoma oncogenesis, suggesting that this pathway may be amenable to therapeutic targeting.

Mutagenesis in rodents using the L1 retrotransposon.

LINE1 (L1) retrotransposons are genetic elements that are present in all mammalian genomes. L1s are active in both humans and mice, and are capable of copying themselves and inserting the copy into a new genomic location. These de novo insertions occasionally result in disease. Endogenous L1 retrotransposons can be modified to increase their activity and mutagenic power in a variety of ways. Here we outline the advantages of using modified L1 retrotransposons for performing random mutagenesis in rodents and discuss several potential applications.

Srebp2: A master regulator of sterol and fatty acid synthesis

Journal of Lipid Research Volume 57, 2016 333 Copyright