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1.  Occurrence of HHIP gene CpG island methylation in gastric cancer 
Oncology Letters  2014;8(5):2340-2344.
The present study aimed to observe the methylation status of the CpG islands at the human hedgehog interacting protein (HHIP) gene in gastric cancer tissues, peritumoral tissues and the AGS cell line, to analyze the association between the methylation status of the CpG islands and the tumorigenesis of gastric cancer. The HHIP mRNA expression in 60 human gastric carcinnoma tissues, peritumoral tissues and the gastric carcinoma AGS cell line were detected by reverse transcription polymerase chain reaction (RT-PCR). The HHIP methylation status of the promoter region in the gastric carcinnoma tissues and peritumoral tissues was detected by methylation-specific PCR (MSP). Prior to and following treatment with methyl transferase inhibitor 5-aza-2′-deoxycitydine (5-aza-dc), the HHIP mRNA expression level, the methylation status of the promoter region and the methylation site loci on the CpG islands in the AGS cells were detected by RT-PCR, MSP and bisulfite sequencing PCR (BSP), respectively. The correlation between the methylation status of the CpG islands at the HHIP promoter region and the HHIP mRNA expression level were analyzed. It was found that the expression level of the HHIP mRNA in the gastric carcinoma tissues was significantly lower than that in the adjacent tissues (0.82±0.38 vs. 1.60±0.26, respectively; P<0.001). No significant correlations were observed between the expression of HHIP mRNA and age, gender, tumor-node-metastasis stage, differentiation degree and presence of lymph node metastasis (P>0.05). The degree of methylation of the HHIP gene promotor in the peritumoral tissues (17.7±3.59%) was significantly lower than that in the gastric cancer tissues (62.9±6.14%) and in the AGS cells (99.7±0.67%) (P<0.05). Compared with prior to 5-aza-dc intervention, the HHIP mRNA expression level in the AGS cells was significantly increased subsequent to intervention (0.21±0.12 vs. 4.68±0.22; P<0.01), while the degree of methylation in the AGS cells was significantly decreased (90.2±0.67 vs. 10.1±0.21%; P<0.01), and the methylation sites in CpG islands were significantly reduced. The degree of HHIP methylation showed a negative correlation with the level of mRNA expression (r=−0.693; P<0.01). It can be hypothesized that a high degree of methylation of the HHIP gene promoter CpG islands in gastric cancer tissues and cells causes a decrease in HHIP mRNA expression, which may be involved in the carcinogenesis of gastric cancer.
PMCID: PMC4186611  PMID: 25295121
HHIP gene; gastric cancer; methylation
2.  Gene expression analysis uncovers novel Hedgehog interacting protein (HHIP) effects in human bronchial epithelial cells 
Genomics  2013;101(5):263-272.
Hedgehog Interacting Protein (HHIP) was implicated in chronic obstructive pulmonary disease (COPD) by genome-wide association studies (GWAS). However, it remains unclear how HHIP contributes to COPD pathogenesis. To identify genes regulated by HHIP, we performed gene expression microarray analysis in a human bronchial epithelial cell line (Beas-2B) stably infected with HHIP shRNAs. HHIP silencing led to differential expression of 296 genes; enrichment for variants nominally associated with COPD was found. Eighteen of the differentially expressed genes were validated by real-time PCR in Beas-2B cells. Seven of 11 validated genes tested in human COPD and control lung tissues demonstrated significant gene expression differences. Functional annotation indicated enrichment for extracellular matrix and cell growth genes. Network modeling demonstrated that the extracellular matrix and cell proliferation genes influenced by HHIP tended to be interconnected. Thus, we identified potential HHIP targets in human bronchial epithelial cells that may contribute to COPD pathogenesis.
PMCID: PMC3659826  PMID: 23459001
Hedgehog interacting protein (HHIP); Gene expression profiling; COPD (Chronic obstructive pulmonary disease); extracellular matrix (ECM); network modeling
3.  Overactivation of Hedgehog Signaling Alters Development of the Ovarian Vasculature in Mice1  
Biology of Reproduction  2012;86(6):174.
The hedgehog (HH) signaling pathway is critical for ovarian function in Drosophila, but its role in the mammalian ovary has not been defined. Previously, expression of a dominant active allele of the HH signal transducer protein smoothened (SMO) in Amhr2cre/+SmoM2 mice caused anovulation in association with a lack of smooth muscle in the theca of developing follicles. The current study examined events during the first 2 wk of life in Amhr2cre/+SmoM2 mice to gain insight into the cause of anovulation. Expression of transcriptional targets of HH signaling, Gli1, Ptch1, and Hhip, which are used as measures of pathway activity, were elevated during the first several days of life in Amhr2cre/+SmoM2 mice compared to controls but were similar to controls in older mice. Microarray analysis showed that genes with increased expression in 2-day-old mutants compared to controls were enriched for the processes of vascular and tube development and steroidogenesis. The density of platelet endothelial cell adhesion molecule (PECAM)-labeled endothelial tubes was increased in the cortex of newborn ovaries of mutant mice. Costaining of preovulatory follicles for PECAM and smooth muscle actin showed that muscle-type vascular support cells are deficient in theca of mutant mice. Expression of genes for steroidogenic enzymes that are normally expressed in the fetal adrenal gland were elevated in newborn ovaries of mutant mice. In summary, overactivation of HH signaling during early life alters gene expression and vascular development and this is associated with the lifelong development of anovulatory follicles in which the thecal vasculature fails to mature appropriately.
Transgenic mice with conditional overactivation of hedgehog signaling in the ovary are anovulatory due to alterations in the development of the ovarian vasculature during the neonatal period.
PMCID: PMC3386146  PMID: 22402963
follicular development; ovary; ovulation
4.  Isoform-Specific Potentiation of Stem and Progenitor Cell Engraftment by AML1/RUNX1  
PLoS Medicine  2007;4(5):e172.
AML1/RUNX1 is the most frequently mutated gene in leukaemia and is central to the normal biology of hematopoietic stem and progenitor cells. However, the role of different AML1 isoforms within these primitive compartments is unclear. Here we investigate whether altering relative expression of AML1 isoforms impacts the balance between cell self-renewal and differentiation in vitro and in vivo.
Methods and Findings
The human AML1a isoform encodes a truncated molecule with DNA-binding but no transactivation capacity. We used a retrovirus-based approach to transduce AML1a into primitive haematopoietic cells isolated from the mouse. We observed that enforced AML1a expression increased the competitive engraftment potential of murine long-term reconstituting stem cells with the proportion of AML1a-expressing cells increasing over time in both primary and secondary recipients. Furthermore, AML1a expression dramatically increased primitive and committed progenitor activity in engrafted animals as assessed by long-term culture, cobblestone formation, and colony assays. In contrast, expression of the full-length isoform AML1b abrogated engraftment potential. In vitro, AML1b promoted differentiation while AML1a promoted proliferation of progenitors capable of short-term lymphomyeloid engraftment. Consistent with these findings, the relative abundance of AML1a was highest in the primitive stem/progenitor compartment of human cord blood, and forced expression of AML1a in these cells enhanced maintenance of primitive potential both in vitro and in vivo.
These data demonstrate that the “a” isoform of AML1 has the capacity to potentiate stem and progenitor cell engraftment, both of which are required for successful clinical transplantation. This activity is consistent with its expression pattern in both normal and leukaemic cells. Manipulating the balance of AML1 isoform expression may offer novel therapeutic strategies, exploitable in the contexts of leukaemia and also in cord blood transplantation in adults, in whom stem and progenitor cell numbers are often limiting.
The truncated "a" isoform of AML1 is shown to have the capacity to potentiate stem and progenitor cell engraftment, both of which are required for successful clinical transplantation.
Editors' Summary
Blood contains red blood cells (which carry oxygen round the body), platelets (which help the blood to clot), and white blood cells (which fight off infections). All these cells, which are regularly replaced, are derived from hematopoietic stem cells, blood-forming cells present in the bone marrow. Like all stem cells, hematopoietic stem cells self-renew (reproduce themselves) and produce committed progenitor cells, which develop into mature blood cells in a process called hematopoiesis. Many proteins control hematopoiesis, some of which are called transcription factors; these factors bind to DNA through their DNA-binding domain and then control the expression of genes (that is, how DNA is turned into proteins) through particular parts of the protein (their transcription regulatory domains). An important hematopoietic transcription factor is AML1—a protein first identified because of its involvement in acute myelogenous leukemia (AML, a form of blood cancer). Mutations (changes) in the AML1 gene are now known to be present in other types of leukemia, which are often characterized by overproliferation of immature blood cells.
Why Was This Study Done?
Because of AML1′s crucial role in hematopoiesis, knowing more about which genes it regulates and how its activity is regulated could provide clues to treating leukemia and to improving hematopoietic cell transplantation. Many cancer treatments destroy hematopoietic stem cells, leaving patients vulnerable to infection. Transplants of bone marrow or cord blood (the cord that links mother and baby during pregnancy contains peripheral blood stem cells) can replace the missing cells, but cord blood in particular often contains insufficient stem cells for successful transplantation. It would be useful, therefore, to expand the stem cell content of these tissues before transplantation. In this study, the researchers investigated the effect of AML1 on self-renewal and differentiation of hematopoietic stem and progenitor cells in the laboratory (in vitro) and in animals (in vivo). In particular, they have asked how two isoforms (closely related versions) of AML1 affect the ability of these cells to grow and differentiate (engraft) in mice after transplantation.
What Did the Researchers Do and Find?
The researchers artificially expressed AML1a and AML1b (both isoforms contain a DNA binding domain, but only AML1b has transcription regulatory domains) in mouse hematopoietic stem and progenitor cells and then tested the cells' ability to engraft in mice. AML1a-expressing cells engrafted better than unaltered cells and outgrew unaltered cells when transplanted as a mixture. AML1b-expressing cells, however, did not engraft. In vitro, AML1a-expressing cells grew more than AML1b-expressing cells, whereas differentiation was promoted in AML1b-expressing cells. To investigate whether the isoforms have the same effects in human cells, the researchers measured the amount of AML1a and AML1b mRNA (the template for protein production) made by progenitor cells in human cord blood. Although AML1b (together with AML1c, an isoform with similar characteristics) mRNA predominated in all the progenitor cell types, the relative abundance of AML1a was greatest in the stem and progenitor cells. Furthermore, forced expression of AML1a in these cells improved their ability to divide in vitro and to engraft in mice.
What Do These Findings Mean?
These findings indicate that AML1a expression increases the self-renewal capacity of hematopoietic stem and progenitor cells and consequently improves their ability to engraft in mice, whereas AML1b expression encourages the differentiation of these cell types. These activities are consistent with the expression patterns of the two isoforms in normal hematopoietic cells and in leukemic cells—the mutated AML made by many leukemic cells resembles AML1a. Because the AML1 isoforms were expressed at higher than normal levels in these experiments, the physiological relevance of these findings needs to be confirmed by showing that normal levels of AML1a and AML1b produce similar results. Nevertheless, these results suggest that manipulating the balance of AML1 isoforms made by hematopoietic cells might be useful clinically. In leukemia, a shift toward AML1b expression might slow the proliferation of leukemic cells and encourage their differentiation. Conversely, in cord blood transplantation, a shift toward AML1a expression might improve patient outcomes by expanding the stem and progenitor cell populations.
Additional Information.
Please access these Web sites via the online version of this summary at
Wikipedia has pages on hematopoiesis and hematopoietic stem cells (note: Wikipedia is a free online encyclopedia that anyone can edit; available in several languages)
The US National Cancer Institute has a fact sheet on bone marrow and peripheral blood stem cell transplantation (in English and Spanish) and information for patients and professionals on leukemia (in English)
The American Society of Hematology provides patient information about blood diseases, including information on bone marrow and stem cell transplantation
PMCID: PMC1868041  PMID: 17503961
5.  Glioma stem cells enhance endothelial cell migration and proliferation via the Hedgehog pathway 
Oncology Letters  2013;6(5):1524-1530.
The aim of the present study was to determine the possible mechanism underlying the enhanced migration and proliferation of endothelial cells caused by glioma stem cells (GSCs). Tumor spheres enriched in GSCs derived from the mouse GL261 glioma cell line, and the brain microvessel endothelial cell line, b.END3, were used in this study. A Transwell co-culture system, RNAi experiments, quantitative polymerase chain reaction, western blotting and enzyme-linked immunosorbent, cell counting kit-8 (CCK-8) proliferation, Transwell migration and wound-healing assays were used in this study to determine the migration and proliferation ability, as well as the Hedgehog (HH) pathway-related gene expression in the b.END3 cells. Based on the results, it was demonstrated that the migration and proliferation of the endothelial cells were enhanced following co-culture with GSCs. The gene expression of the HH pathway-related genes, Sonic Hedgehog (Shh) and Hedgehog-interacting protein (Hhip) was altered in the endothelial cells when co-cultured with GSCs. Overexpression of glioma-associated oncogene homolog 1 indicated activation of the HH pathway. Following knockdown of smoothened (Smo) in the endothelial cells, the migration and proliferation abilities of the cells were inhibited. GSCs have little effect on enhancing these behaviors in endothelial cells following Smo-knockdown. Further investigation revealed that Shh levels in the supernatant of the co-culture system were elevated, indicating the importance of secreted Shh from the endothelial cells. In conclusion, GSCs enhanced the migration and proliferation of the endothelial cells in vitro, which was likely associated with the activation of the HH pathway in the endothelial cells, caused by the increased secretion of Shh.
PMCID: PMC3813800  PMID: 24179553
glioma stem cell; Hedgehog pathway; endothelial cell; migration; proliferation
6.  Identification of a chronic obstructive pulmonary disease genetic determinant that regulates HHIP 
Human Molecular Genetics  2011;21(6):1325-1335.
Multiple intergenic single-nucleotide polymorphisms (SNPs) near hedgehog interacting protein (HHIP) on chromosome 4q31 have been strongly associated with pulmonary function levels and moderate-to-severe chronic obstructive pulmonary disease (COPD). However, whether the effects of variants in this region are related to HHIP or another gene has not been proven. We confirmed genetic association of SNPs in the 4q31 COPD genome-wide association study (GWAS) region in a Polish cohort containing severe COPD cases and healthy smoking controls (P = 0.001 to 0.002). We found that HHIP expression at both mRNA and protein levels is reduced in COPD lung tissues. We identified a genomic region located ∼85 kb upstream of HHIP which contains a subset of associated SNPs, interacts with the HHIP promoter through a chromatin loop and functions as an HHIP enhancer. The COPD risk haplotype of two SNPs within this enhancer region (rs6537296A and rs1542725C) was associated with statistically significant reductions in HHIP promoter activity. Moreover, rs1542725 demonstrates differential binding to the transcription factor Sp3; the COPD-associated allele exhibits increased Sp3 binding, which is consistent with Sp3's usual function as a transcriptional repressor. Thus, increased Sp3 binding at a functional SNP within the chromosome 4q31 COPD GWAS locus leads to reduced HHIP expression and increased susceptibility to COPD through distal transcriptional regulation. Together, our findings reveal one mechanism through which SNPs upstream of the HHIP gene modulate the expression of HHIP and functionally implicate reduced HHIP gene expression in the pathogenesis of COPD.
PMCID: PMC3284120  PMID: 22140090
7.  Overexpression of Smoothened activates the Sonic Hedgehog signaling pathway in pancreatic cancer associated fibroblasts 
Accumulating evidence suggests that cancer associated stromal fibroblasts contribute to tumor growth by actively communicating with cancer cells. Our aim is to identify signaling pathways involved in tumor-stromal cell interactions in human pancreatic cancer.
Experimental Design
We established primary fibroblast cultures from human pancreatic adenocarcinomas and non-neoplastic pancreas tissues. To identify differentially expressed genes in CAFs, we performed gene expression profiling of human pancreatic CAFs and non-neoplastic pancreatic fibroblasts.
The Hedgehog receptor Smoothened (SMO) was upregulated in cancer associated fibroblasts relative to control fibroblasts. CAFs expressing SMO could transduce the Shh signal to activate Gli1 expression, and siRNA knockdown of SMO blocked the induction of Gli1 in these cells. Stromal fibroblasts of human primary pancreatic adenocarcinomas overexpressed Smo compared to normal pancreatic fibroblasts.
These findings implicate overexpression of Smo as a mechanism for the activation of Hedgehog signaling in human pancreatic CAFs and suggest that stromal cells may be a therapeutic target for Smo antagonists in pancreatic cancer.
PMCID: PMC2846609  PMID: 20215540
8.  Reduction of Human Embryonal Rhabdomyosarcoma Tumor Growth by Inhibition of the Hedgehog Signaling Pathway 
Genes & Cancer  2010;1(9):941-951.
Rhabdomyosarcoma (RMS) is the most frequent soft-tissue sarcoma in children. Embryonal rhabdomyosarcoma (E-RMS) represents the most common RMS subtype, but the molecular events driving this tumor are still largely unknown. The hedgehog (HH) pathway, a major signal transduction cascade, is linked with many cancers, including RMS. As we previously have detected loss of heterozygosity of PTCH1 in E-RMS, we now examined 8 E-RMS tumor samples and 5 E-RMS cell lines for the presence of PTCH1 mutations, but none was detected. However, in the E-RMS cell lines, a variable pattern of up-regulated expression of certain HH signaling target genes, including HHIP, PTCH1, SFRP1, and GLI1, was observed. Moreover, treatment with the small molecule HH signaling inhibitors cyclopamine and GANT61 inhibited cell proliferation in all E-RMS cell lines analyzed. Interestingly, GANT61 was more effective, and this was accompanied by increased apoptosis, while cyclopamine promoted necrotic events. Specific knockdown of SMO had no effect on the proliferation of E-RMS cells, indicating the presence of an SMO-independent HH signaling pathway in the E-RMS cell lines. Furthermore, in an in vivo xenograft model, tumor growth was significantly reduced by GANT61 treatment of E-RMS cells. Additionally, siRNA experiments provided evidence that inhibition of GLI1 or GLI3 but not GLI2 was sufficient to reduce proliferation of these cell lines. As GANT61 is known to block GLI1/GLI2 transcriptional activity, the inhibition of E-RMS growth by GANT61 is likely to be mediated through GLI1. In conclusion, our findings implicate that GLI1 could constitute an effective therapeutic target in pediatric E-RMS.
PMCID: PMC3092259  PMID: 21779473
hedgehog signaling; embryonal rhabdomyosarcoma; GLI; GANT
9.  A Broadly Conserved G-Protein-Coupled Receptor Kinase Phosphorylation Mechanism Controls Drosophila Smoothened Activity 
PLoS Genetics  2014;10(7):e1004399.
Hedgehog (Hh) signaling is essential for normal growth, patterning, and homeostasis of many tissues in diverse organisms, and is misregulated in a variety of diseases including cancer. Cytoplasmic Hedgehog signaling is activated by multisite phosphorylation of the seven-pass transmembrane protein Smoothened (Smo) in its cytoplasmic C-terminus. Aside from a short membrane-proximal stretch, the sequence of the C-terminus is highly divergent in different phyla, and the evidence suggests that the precise mechanism of Smo activation and transduction of the signal to downstream effectors also differs. To clarify the conserved role of G-protein-coupled receptor kinases (GRKs) in Smo regulation, we mapped four clusters of phosphorylation sites in the membrane-proximal C-terminus of Drosophila Smo that are phosphorylated by Gprk2, one of the two fly GRKs. Phosphorylation at these sites enhances Smo dimerization and increases but is not essential for Smo activity. Three of these clusters overlap with regulatory phosphorylation sites in mouse Smo and are highly conserved throughout the bilaterian lineages, suggesting that they serve a common function. Consistent with this, we find that a C-terminally truncated form of Drosophila Smo consisting of just the highly conserved core, including Gprk2 regulatory sites, can recruit the downstream effector Costal-2 and activate target gene expression, in a Gprk2-dependent manner. These results indicate that GRK phosphorylation in the membrane proximal C-terminus is an evolutionarily ancient mechanism of Smo regulation, and point to a higher degree of similarity in the regulation and signaling mechanisms of bilaterian Smo proteins than has previously been recognized.
Author Summary
Hedgehog proteins are critical regulators of embryonic tissue growth and organization in species ranging from flies to humans. Binding of the secreted Hh protein to its receptor at the surface of cells triggers an intracellular signal that is initiated by Smoothened (Smo). Upon exposure of cells to Hh, Smo becomes active and signals through a series of downstream proteins to regulate gene expression. Although Smo proteins in flies and mammals are similar, the critical regions involved in activation and signal initiation differ between the two, implying that different mechanisms have evolved in different organisms. Using the fruit fly as a model organism, we identified regions in Smo that are phosphorylated by a protein kinase called Gprk2 to enhance Smo activity. These phosphorylation sites overlap with previously identified sites in mouse Smo and are conserved in Smo proteins in many animals. Phosphorylation at these sites regulates the recruitment of Costal2 to Smo, a critical step in signal initiation, through a region of the protein that is also highly conserved. Our results indicate that Gprk2 phosphorylation represents an evolutionarily ancient and conserved mechanism for regulating Smo activity, and suggest that Smo regulation and signaling are more similar between different species than previously thought.
PMCID: PMC4091690  PMID: 25009998
10.  HSulf-1 suppresses cell growth and down-regulates Hedgehog signaling in human gastric cancer cells 
Oncology Letters  2011;2(6):1291-1295.
Gastric cancer is the second most lethal cancer worldwide. Despite the current surgical and adjuvant therapies, 5-year survival remains less than 20–25% in the US, Europe and China. Therefore, there is an urgent need to identify new therapeutic targets for treating this malignant disease. Accumulating evidence has supported that aberrant activation of the Hedgehog signaling pathway plays a crucial role in tumorigenesis and progression of gastric cancer. Human sulfatase-1 (HSulf-1) is a recently identified enzyme that desulfates cell surface heparan sulfate proteoglycans (HSPGs), which is critical for Hedgehog signal transduction under a highly sulfated state. HSulf-1 has recently emerged as a tumor suppressor gene in certain types of cancer, including ovarian, breast, myeloma and hepatocellular carcinoma; however, its role in gastric cancer remains to be elucidated. Therefore, we established HSulf-1-expressing monoclonal MKN28 gastric cancer cells to investigate its function in gastric cancer. Expression of HSulf-1 significantly suppressed cellular proliferation and growth in MKN28 gastric cancer cells. Notably, HSulf-1 inhibits Gli-mediated transcription and down-regulates the expression of Hedgehog target genes, including GLI1, PTCH1/2, HHIP, CCND1, C-MYC and BCL-2. Collectively, the study provides evidence that HSulf-1 may function as a tumor suppressor in gastric cancer. It suppresses gastric cancer cell proliferation, possibly through abrogating the Hedgehog signaling pathway. The study provides new mechanistic insight into HSulf-1- mediated tumor suppression, and supports the use of HSulf-1 as a potential new therapeutic target in treating gastric cancer.
PMCID: PMC3406501  PMID: 22848304
HSulf-1; Hedgehog signaling; gastric cancer; proliferation
11.  Analysis of Germline GLI1 Variation Implicates Hedgehog Signalling in the Regulation of Intestinal Inflammatory Pathways 
PLoS Medicine  2008;5(12):e239.
Ulcerative colitis (UC) and Crohn's disease (CD) are polygenic chronic inflammatory bowel diseases (IBD) of high prevalence that are associated with considerable morbidity. The hedgehog (HH) signalling pathway, which includes the transcription factor glioma-associated oncogene homolog 1 (GLI1), plays vital roles in gastrointestinal tract development, homeostasis, and malignancy. We identified a germline variation in GLI1 (within the IBD2 linkage region, 12q13) in patients with IBD. Since this IBD-associated variant encodes a GLI1 protein with reduced function and our expression studies demonstrated down-regulation of the HH response in IBD, we tested whether mice with reduced Gli1 activity demonstrate increased susceptibility to chemically induced colitis.
Methods and Findings
Using a gene-wide haplotype-tagging approach, germline GLI1 variation was examined in three independent populations of IBD patients and healthy controls from Northern Europe (Scotland, England, and Sweden) totalling over 5,000 individuals. On log-likelihood analysis, GLI1 was associated with IBD, predominantly UC, in Scotland and England (p < 0.0001). A nonsynonymous SNP (rs2228226C→G), in exon 12 of GLI1 (Q1100E) was strongly implicated, with pooled odds ratio of 1.194 (confidence interval = 1.09–1.31, p = 0.0002). GLI1 variants were tested in vitro for transcriptional activity in luciferase assays. Q1100E falls within a conserved motif near the C terminus of GLI1; the variant GLI protein exhibited reduced transactivation function in vitro. In complementary expression studies, we noted the colonic HH response, including GLI1, patched (PTCH), and hedgehog-interacting protein (HHIP), to be down-regulated in patients with UC. Finally, Gli1+/lacZ mice were tested for susceptibility to dextran sodium sulphate (DSS)-induced colitis. Clinical response, histology, and expression of inflammatory cytokines and chemokines were recorded. Gli1+/lacZ mice rapidly developed severe intestinal inflammation, with considerable morbidity and mortality compared with wild type. Local myeloid cells were shown to be direct targets of HH signals and cytokine expression studies revealed robust up-regulation of IL-12, IL-17, and IL-23 in this model.
HH signalling through GLI1 is required for appropriate modulation of the intestinal response to acute inflammatory challenge. Reduced GLI1 function predisposes to a heightened myeloid response to inflammatory stimuli, potentially leading to IBD.
Charlie Lees and colleagues identify a reduced-function variant of the hedgehog signaling pathway protein GLI1 that associates with inflammatory bowel disease, and investigate its role in a mouse model of colitis.
Editors' Summary
Inflammatory bowel diseases (IBDs) are common disorders in which parts of the digestive tract become repeatedly or continuously inflamed. The immune system normally protects the body from entities it identifies as foreign, but in IBD it mistakenly recognizes gut tissue, and immune system cells accumulate in the lining of the bowel, which causes inflammation. There are two main types of IBD—Crohn's disease (CD), which mainly affects the small bowel, and ulcerative colitis (UC), which affects only the large bowel (colon). Both types tend to run in families and usually develop between the ages of 15 and 35 years. Symptoms—including diarrhea, abdominal cramps, and unexplained weight loss—can be mild or severe and the disease can develop slowly or suddenly. There is no cure for IBD except surgical removal of the affected part of the digestive tract. However, drugs that modulate the immune system (for example, corticosteroids) or that specifically inhibit “proinflammatory cytokines” (proteins made by the immune system that stimulate inflammation) are often helpful in reducing symptoms.
Why Was This Study Done?
Why the immune system becomes unbalanced in people with IBD is not clear but it is known that IBD is “polygenic,” that is, a disease caused by the combined actions of two or more inherited gene variants. Although UC and CD are clinically different diseases, they share several “susceptibility loci” (regions of the genome that harbor disease-associated gene variants), including the IBD2 locus. The identification of the actual gene within the IBD2 locus that is altered in people who are susceptible to IBD might provide new insights into what causes the immune imbalance in IBD and into how to treat the disease. In this study, the researchers test the hypothesis that a variant of a gene called GLI1, which lies in the IBD2 locus, is associated with IBD susceptibility. GLI1 encodes a transcription factor (a protein that regulates the production of proteins) that is a central component in the signaling pathway named for a protein called “hedgehog.” This pathway is involved in the development of many organs, including the digestive tract.
What Did the Researchers Do and Find?
The researchers used a technique called gene-wide haplotype tagging to look for inherited GLl1 variants in patients with IBD and in healthy people living in Scotland, England, and Sweden. A specific variant of the GLI1 gene, resulting in alteration of a single amino acid component of the GLI1 protein, was associated with IBD (particularly with UC) in both Scotland and England; the same variant was weakly associated with IBD in the Swedish population. The variant GLI1 protein was only half as active as the normal protein in a laboratory assay, and, consistent with this result, the expression of several components of the hedgehog signaling pathway was lower in colon samples taken from patients with UC than in samples taken from healthy individuals. Finally, Gli1+/lacZ mice (which express half the normal amount of Gli1 protein) developed severe intestinal inflammation more rapidly than wild-type mice when they were treated with dextran sodium sulfate (DSS), a chemical that induces acute (sudden) colitis. Cellular analysis revealed that myeloid cells (cells that sense and modify the inflammatory response) are direct targets of the hedgehog signaling pathway. Furthermore, the expression of several pro-inflammatory cytokines (in particular, one called IL-23) increased more markedly in the Gli1+/lacZ mice than in the wild-type mice after DSS treatment.
What Do These Findings Mean?
These findings suggest that the normal response of the mammalian gut to challenge with inflammatory substances involves hedgehog signaling through GLI1 and that reduced GLI1 function might be one trigger for IBD. More specifically, the human genetic studies identify a GLI1 variant that is associated with IBD (at least in certain north European populations), the laboratory experiments indicate that this GLI1 variant encodes a protein with reduced activity, and the animal studies show that a similar reduction in Gli1 activity is sufficient to heighten intestinal inflammatory responses. Although this last result needs to be confirmed in animal models of chronic colitis that more closely resemble human IBD, these findings suggest that drugs that modulate hedgehog signaling might be useful in the treatment of IBD.
Additional Information.
Please access these Web sites via the online version of this summary at
The MedlinePlus Encyclopedia has pages on Crohn's disease and on ulcerative colitis (in English and Spanish)
MedlinePlus provides links to other information Crohn's disease and ulcerative colitis (in English and Spanish)
The US National Institute of Diabetes and Digestive and Kidney Diseases provides information on Crohn's disease and ulcerative colitis
The UK National Health Service Direct Encyclopedia also provides information on Crohn's disease and on ulcerative colitis
Wikipedia has a page on the hedgehog signaling pathway (note: Wikipedia is a free online encyclopedia that anyone can edit; available in several languages)
PMCID: PMC2596854  PMID: 19071955
12.  ELMO1 Is Upregulated in AML CD34+ Stem/Progenitor Cells, Mediates Chemotaxis and Predicts Poor Prognosis in Normal Karyotype AML 
PLoS ONE  2014;9(10):e111568.
Both normal as well leukemic hematopoietic stem cells critically depend on their microenvironment in the bone marrow for processes such as self-renewal, survival and differentiation, although the exact pathways that are involved remain poorly understood. We performed transcriptome analysis on primitive CD34+ acute myeloid leukemia (AML) cells (n = 46), their more differentiated CD34− leukemic progeny, and normal CD34+ bone marrow cells (n = 31) and focused on differentially expressed genes involved in adhesion and migration. Thus, Engulfment and Motility protein 1 (ELMO1) was identified amongst the top 50 most differentially expressed genes. ELMO1 is a crucial link in the signaling cascade that leads to activation of RAC GTPases and cytoskeleton rearrangements. We confirmed increased ELMO1 expression at the mRNA and protein level in a panel of AML samples and showed that high ELMO1 expression is an independent negative prognostic factor in normal karyotype (NK) AML in three large independent patient cohorts. Downmodulation of ELMO1 in human CB CD34+ cells did not significantly alter expansion, progenitor frequency or differentiation in stromal co-cultures, but did result in a decreased frequency of stem cells in LTC-IC assays. In BCR-ABL-transduced human CB CD34+ cells depletion of ELMO1 resulted in a mild decrease in proliferation, but replating capacity of progenitors was severely impaired. Downregulation of ELMO1 in a panel of primary CD34+ AML cells also resulted in reduced long-term growth in stromal co-cultures in two out of three cases. Pharmacological inhibition of the ELMO1 downstream target RAC resulted in a severely impaired proliferation and survival of leukemic cells. Finally, ELMO1 depletion caused a marked decrease in SDF1-induced chemotaxis of leukemic cells. Taken together, these data show that inhibiting the ELMO1-RAC axis might be an alternative way to target leukemic cells.
PMCID: PMC4216115  PMID: 25360637
13.  RepSox Slows Decay of CD34+ Acute Myeloid Leukemia Cells and Decreases T Cell Immunoglobulin Mucin-3 Expression 
To facilitate development of therapies that target leukemic stem/progenitor cells (LPCs), in vitro ways to enhance the survival and immunogenicity of a patient's CD34+ acute myeloid leukemia (AML) cells were explored. RepSox was identified as a candidate cell-engineering tool because it slows in vitro decay of CD34+ AML cells (which often contain LPCs) and accelerates loss of the immune checkpoint receptor T cell immunoglobulin mucin-3 (Tim-3).
Despite initial response to therapy, most acute myeloid leukemia (AML) patients relapse. To eliminate relapse-causing leukemic stem/progenitor cells (LPCs), patient-specific immune therapies may be required. In vitro cellular engineering may require increasing the “stemness” or immunogenicity of tumor cells and activating or restoring cancer-impaired immune-effector and antigen-presenting cells. Leukapheresis samples provide the cells needed to engineer therapies: LPCs to be targeted, normal hematopoietic stem cells to be spared, and cancer-impaired immune cells to be repaired and activated. This study sought to advance development of LPC-targeted therapies by exploring nongenetic ways to slow the decay and to increase the immunogenicity of primary CD34+ AML cells. CD34+ AML cells generally displayed more colony-forming and aldehyde dehydrogenase activity than CD34− AML cells. Along with exposure to bone marrow stromal cells and low (1%–5%) oxygen, culture with RepSox (a reprogramming tool and inhibitor of transforming growth factor-β receptor 1) consistently slowed decline of CD34+ AML and myelodysplastic syndrome (MDS) cells. RepSox-treated AML cells displayed higher CD34, CXCL12, and MYC mRNA levels than dimethyl sulfoxide-treated controls. RepSox also accelerated loss of T cell immunoglobulin mucin-3 (Tim-3), an immune checkpoint receptor that impairs antitumor immunity, from the surface of AML and MDS cells. Our results suggest RepSox may reduce Tim-3 expression by inhibiting transforming growth factor-β signaling and slow decay of CD34+ AML cells by increasing CXCL12 and MYC, two factors that inhibit AML cell differentiation. By prolonging survival of CD34+ AML cells and reducing Tim-3, RepSox may promote in vitro immune cell activation and advance development of LPC-targeted therapies.
PMCID: PMC4073822  PMID: 24855276
Acute myeloid leukemia; Cancer stem cells; Immunotherapy; Immunogenicity; Tim-3; CD34+
14.  Altered differentiation and paracrine stimulation of mammary epithelial cell proliferation by conditionally activated Smoothened 
Developmental biology  2011;352(1):116-127.
The Hedgehog (Hh) signaling network is critical for patterning and organogenesis in mammals, and has been implicated in a variety of cancers. Smoothened (Smo), the gene encoding the principal signal transducer, is overexpressed frequently in breast cancer, and constitutive activation in MMTV-SmoM2 transgenic mice caused alterations in mammary gland morphology, increased proliferation, and changes in stem/progenitor cell number. Both in transgenic mice and in clinical specimens, proliferative cells did not usually express detectable Smo, suggesting the hypothesis that Smo functioned in a non-cell autonomous manner to stimulate proliferation. Here, we employed a genetically tagged mouse model carrying a Cre-recombinase-dependent conditional allele of constitutively active Smo (SmoM2) to test this hypothesis. MMTV-Cre- or adenoviral-Cre-mediated SmoM2 expression in the luminal epithelium, but not in the myoepithelium, was required for the hyper-proliferative phenotypes. High levels of proliferation were observed in cells adjacent or in close-proximity to Smo expressing cells demonstrating that SmoM2 expressing cells were stimulating proliferation via a paracrine or juxtacrine mechanism. In contrast, Smo expression altered luminal cell differentiation in a cell-autonomous manner. SmoM2 expressing cells, purified by fluorescence activated cell sorting (FACS) via the genetic fluorescent tag, expressed high levels of Ptch2, Gli1, Gli2, Jag2 and Dll-1, and lower levels of Notch4 and Hes6, in comparison to wildtype cells. These studies provide insight into the mechanism of Smo activation in the mammary gland and its possible roles in breast tumorigenesis. In addition, these results also have potential implications for the interpretation of proliferative phenotypes commonly observed in other organs as a consequence of hedgehog signaling activation.
PMCID: PMC3057274  PMID: 21276786
SmoM2; ductal hyperplasia; paracrine tissue interactions; hedgehog signaling; microenvironment; notch signaling
15.  Hedgehog-Regulated Ubiquitination Controls Smoothened Trafficking and Cell Surface Expression in Drosophila 
PLoS Biology  2012;10(1):e1001239.
Hedgehog transduces signal by promoting cell surface expression of the seven-transmembrane protein Smoothened (Smo) in Drosophila, but the underlying mechanism remains unknown. Here we demonstrate that Smo is downregulated by ubiquitin-mediated endocytosis and degradation, and that Hh increases Smo cell surface expression by inhibiting its ubiquitination. We find that Smo is ubiquitinated at multiple Lysine residues including those in its autoinhibitory domain (SAID), leading to endocytosis and degradation of Smo by both lysosome- and proteasome-dependent mechanisms. Hh inhibits Smo ubiquitination via PKA/CK1-mediated phosphorylation of SAID, leading to Smo cell surface accumulation. Inactivation of the ubiquitin activating enzyme Uba1 or perturbation of multiple components of the endocytic machinery leads to Smo accumulation and Hh pathway activation. In addition, we find that the non-visual β-arrestin Kurtz (Krz) interacts with Smo and acts in parallel with ubiquitination to downregulate Smo. Finally, we show that Smo ubiquitination is counteracted by the deubiquitinating enzyme UBPY/USP8. Gain and loss of UBPY lead to reciprocal changes in Smo cell surface expression. Taken together, our results suggest that ubiquitination plays a key role in the downregulation of Smo to keep Hh pathway activity off in the absence of the ligand, and that Hh-induced phosphorylation promotes Smo cell surface accumulation by inhibiting its ubiquitination, which contributes to Hh pathway activation.
Author Summary
The Hedgehog (Hh) family of secreted proteins governs cell growth and patterning in diverse species ranging from Drosophila to human. Hh signals across the cell surface membrane by regulating the subcellular location and conformation of a membrane protein called Smoothened (Smo). In Drosophila, Smo accumulates on the cell surface in response to Hh, whereas in the absence of Hh it is internalized and degraded. The molecular mechanisms that control this intracellular trafficking and degradation of Smo were unknown, but here we show that Smo is modified by attachment of several molecules of a small protein called ubiquitin, which tags it for internalization and degradation within the cell. Hh inhibits this ubiquitination of Smo by inducing another modification, phosphorylation, of its intracellular tail by two types of protein kinase enzymes. This loss of ubiquitination and gain of phosphorylation causes the accumulation of Smo at the cell surface. What's more, we find that another protein called Kurtz interacts with Smo and acts in parallel with the ubiquitination process to promote internalization of Smo, and that the deubiquitinating enzyme UBPY/USP8 counteracts ubiquitination of Smo to promote its cell surface accumulation. Our study demonstrates that reversible ubiquitination plays a key role in regulating Smo trafficking to and from the cell surface and thus it provides novel insights into the mechanism of Hh signaling from the outside to the inside of the cell.
PMCID: PMC3254653  PMID: 22253574
16.  Synergism between Hedgehog-GLI and EGFR Signaling in Hedgehog-Responsive Human Medulloblastoma Cells Induces Downregulation of Canonical Hedgehog-Target Genes and Stabilized Expression of GLI1 
PLoS ONE  2013;8(6):e65403.
Aberrant activation of Hedgehog (HH) signaling has been identified as a key etiologic factor in many human malignancies. Signal strength, target gene specificity, and oncogenic activity of HH signaling depend profoundly on interactions with other pathways, such as epidermal growth factor receptor-mediated signaling, which has been shown to cooperate with HH/GLI in basal cell carcinoma and pancreatic cancer. Our experimental data demonstrated that the Daoy human medulloblastoma cell line possesses a fully inducible endogenous HH pathway. Treatment of Daoy cells with Sonic HH or Smoothened agonist induced expression of GLI1 protein and simultaneously prevented the processing of GLI3 to its repressor form. To study interactions between HH- and EGF-induced signaling in greater detail, time-resolved measurements were carried out and analyzed at the transcriptomic and proteomic levels. The Daoy cells responded to the HH/EGF co-treatment by downregulating GLI1, PTCH, and HHIP at the transcript level; this was also observed when Amphiregulin (AREG) was used instead of EGF. We identified a novel crosstalk mechanism whereby EGFR signaling silences proteins acting as negative regulators of HH signaling, as AKT- and ERK-signaling independent process. EGFR/HH signaling maintained high GLI1 protein levels which contrasted the GLI1 downregulation on the transcript level. Conversely, a high-level synergism was also observed, due to a strong and significant upregulation of numerous canonical EGF-targets with putative tumor-promoting properties such as MMP7, VEGFA, and IL-8. In conclusion, synergistic effects between EGFR and HH signaling can selectively induce a switch from a canonical HH/GLI profile to a modulated specific target gene profile. This suggests that there are more wide-spread, yet context-dependent interactions, between HH/GLI and growth factor receptor signaling in human malignancies.
PMCID: PMC3677915  PMID: 23762360
17.  USP8 Promotes Smoothened Signaling by Preventing Its Ubiquitination and Changing Its Subcellular Localization 
PLoS Biology  2012;10(1):e1001238.
Hedgehog regulates the activity of its signal transducer Smoothened by enhancing its interaction with the deubiquitinase USP8, thereby promoting Smoothened translocation to the cell surface and so enhancing Hh signaling.
The seven transmembrane protein Smoothened (Smo) is a critical component of the Hedgehog (Hh) signaling pathway and is regulated by phosphorylation, dimerization, and cell-surface accumulation upon Hh stimulation. However, it is not clear how Hh regulates Smo accumulation on the cell surface or how Hh regulates the intracellular trafficking of Smo. In addition, little is known about whether ubiquitination is involved in Smo regulation. In this study, we demonstrate that Smo is multi-monoubiquitinated and that Smo ubiquitination is inhibited by Hh and by phosphorylation. Using an in vivo RNAi screen, we identified ubiquitin-specific protease 8 (USP8) as a deubiquitinase that down-regulates Smo ubiquitination. Inactivation of USP8 increases Smo ubiquitination and attenuates Hh-induced Smo accumulation, leading to decreased Hh signaling activity. Moreover, overexpression of USP8 prevents Smo ubiquitination and elevates Smo accumulation, leading to increased Hh signaling activity. Mechanistically, we show that Hh promotes the interaction of USP8 with Smo aa625–753, which covers the three PKA and CK1 phosphorylation clusters. Finally, USP8 promotes the accumulation of Smo at the cell surface and prevents localization to the early endosomes, presumably by deubiquitinating Smo. Our studies identify USP8 as a positive regulator in Hh signaling by down-regulating Smo ubiquitination and thereby mediating Smo intracellular trafficking.
Author Summary
The Hedgehog (Hh) signaling pathway is well known for its role in directing processes such as cell growth, proliferation, and differentiation during embryogenesis. The signal initiated by Hh binding to its receptor, Patched, is transduced by another protein called Smoothened (Smo), which moves from membranes inside the cell to accumulate on the cell surface when Hh binds. This accumulation of Smo on the cell surface is thought to play a central role in maintaining Hh signaling. In this study, we investigated how Hh controls the stability and movement of Smo inside the cell. We found that Smo is modified by addition of a small protein called ubiquitin (Ub), and that Hh regulates the ubiquitination of Smo. We identified an enzyme called USP8 that can remove the ubiquitin modification from Smo, thereby enhancing its signaling activity. Furthermore we show that Hh can enhance the interaction between Smo and USP8. Finally, we discovered that USP8 promotes the movement of Smo from inside the cell to the cell surface. We conclude that Hh promotes the deubiquitination of Smo by USP8, resulting in the relocation of Smo to the cell surface where it enhances Hh signaling.
PMCID: PMC3254663  PMID: 22253573
18.  Aberrant Hedgehog Ligands Induce Progressive Pancreatic Fibrosis by Paracrine Activation of Myofibroblasts and Ductular Cells in Transgenic Zebrafish 
PLoS ONE  2011;6(12):e27941.
Hedgehog (Hh) signaling is frequently up-regulated in fibrogenic pancreatic diseases including chronic pancreatitis and pancreatic cancer. Although recent series suggest exclusive paracrine activation of stromal cells by Hh ligands from epithelial components, debates still exist on how Hh signaling works in pathologic conditions. To explore how Hh signaling affects the pancreas, we investigated transgenic phenotypes in zebrafish that over-express either Indian Hh or Sonic Hh along with green fluorescence protein (GFP) to enable real-time observation, or GFP alone as control, at the ptf1a domain. Transgenic embryos and zebrafish were serially followed for transgenic phenotypes, and investigated using quantitative reverse transcription-polymerase chain reaction (qRT-PCR), in situ hybridization, and immunohistochemistry. Over-expression of Ihh or Shh reveals virtually identical phenotypes. Hh induces morphologic changes in a developing pancreas without derangement in acinar differentiation. In older zebrafish, Hh induces progressive pancreatic fibrosis intermingled with proliferating ductular structures, which is accompanied by the destruction of the acinar structures. Both myofibroblasts and ductular are activated and proliferated by paracrine Hh signaling, showing restricted expression of Hh downstream components including Patched1 (Ptc1), Smoothened (Smo), and Gli1/2 in those Hh-responsive cells. Hh ligands induce matrix metalloproteinases (MMPs), especially MMP9 in all Hh-responsive cells, and transform growth factor-ß1 (TGFß1) only in ductular cells. Aberrant Hh over-expression, however, does not induce pancreatic tumors. On treatment with inhibitors, embryonic phenotypes are reversed by either cyclopamine or Hedgehog Primary Inhibitor-4 (HPI-4). Pancreatic fibrosis is only prevented by HPI-4. Our study provides strong evidence of Hh signaling which induces pancreatic fibrosis through paracrine activation of Hh-responsive cells in vivo. Induction of MMPs and TGFß1 by Hh signaling expands on the current understanding of how Hh signaling affects fibrosis and tumorigenesis. These transgenic models will be a valuable platform in exploring the mechanism of fibrogenic pancreatic diseases which are induced by Hh signaling activation.
PMCID: PMC3229500  PMID: 22164219
19.  Hedgehog signaling regulates liver sinusoidal endothelial cell capillarisation 
Gut  2012;62(2):299-309.
Vascular remodeling during liver damage involves loss of healthy liver sinusoidal endothelial cell (LSEC) phenotype via capillarisation. Hedgehog (Hh) signaling regulates vascular development and increases during liver injury. Therefore, we examined its role in capillarisation.
Primary LSEC were cultured for 5 days to induce capillarisation. Pharmacologic, antibody-mediated, and genetic approaches were used to manipulate Hh signaling. Effects on mRNA and protein expression of Hh-regulated genes and capillarisation markers were evaluated by qRT-PCR and immunoblot. Changes in LSEC function were assessed by migration and tube forming assay, and gain/loss of fenestrae was examined by electron microscopy. Mice with acute or chronic liver injury were treated with Hh inhibitors; effects on capillarisation were assessed by immunohistochemistry.
Freshly isolated LSEC expressed Hh ligands, Hh receptors, and Hh ligand antagonist Hhip. Capillarisation was accompanied by repression of Hhip and increased expression of Hh-regulated genes. Treatment with Hh agonist further induced expression of Hh ligands and Hh-regulated genes, and up-regulated capillarisation-associated genes; whereas Hh signaling antagonist or Hh ligand neutralizing antibody each repressed expression of Hh target genes and capillarisation markers. LSEC isolated from SmoloxP/loxP transgenic mice that had been infected with adenovirus expressing Cre-recombinase to delete Smoothened showed over 75% knockdown of Smoothened. During culture, Smoothened-deficient LSEC had inhibited Hh signaling, less induction of capillarisation-associated genes, and retention of fenestrae. In mice with injured livers, inhibiting Hh signaling prevented capillarisation.
LSEC produce and respond to Hh ligands, and use Hh signaling to regulate complex phenotypic changes that occur during capillarisation.
PMCID: PMC3595101  PMID: 22362915
20.  Constitutive Activation of Smoothened Leads to Female Infertility and Altered Uterine Differentiation in the Mouse1 
Biology of Reproduction  2010;82(5):991-999.
Previous work has identified Indian hedgehog (Ihh) as a major mediator of progesterone signaling during embryo implantation. Ihh acts through its downstream effector smoothened (Smo) to activate the GLI family of transcription factors. In order to gain a better understanding of Ihh action during embryo implantation, we expressed a Cre-recombinase-dependent constitutively activated SMO in the murine uterus using the Pgrtm2(cre)Lyd (PRcre) mouse model [Pgrtm2(cre)Lyd+Gt(ROSA)26Sortm1(Smo/EYFP)Amc+ (PRcre/+SmoM2+)]. Female PRcre/+SmoM2+ mice were infertile. They exhibited normal serum progesterone levels and normal ovulation, but their ova failed to be fertilized in vivo and their uterus failed to undergo the artificially induced decidual response. Examination of the PRcre/+SmoM2+ uteri revealed numerous features such as uterine hypertrophy, the presence of a stratified luminal epithelial cell layer, a reduced number of uterine glands, and an endometrial stroma that had lost its normal morphologic characteristics. Microarray analysis of 3-mo-old PRcre/+SmoM2+ uteri demonstrated a chondrocytic signature and confirmed that constitutive activation of PRcre/+SmoM2+ increased extracellular matrix production. Thus, constitutive activation of Smo in the mouse uterus alters postnatal uterine differentiation which interferes with early pregnancy. These results provide new insight into the role of Hedgehog signaling during embryo implantation.
Activation of smoothened in the mouse uterus reveals a requirement for the precise regulation of hedgehog signaling during early pregnancy and postnatal uterine development.
PMCID: PMC2857637  PMID: 20130264
differentiation; implantation; mouse; smoothened; uterus
21.  Dual functions of the AML1/Evi-1 chimeric protein in the mechanism of leukemogenesis in t(3;21) leukemias. 
Molecular and Cellular Biology  1995;15(5):2383-2392.
The chromosomal translocation t(3;21)(q26;q22), which is found in blastic crisis in chronic myelogenous leukemias and myelodysplastic syndrome-derived leukemias, produces AML1/Evi-1 chimeric transcription factor and is thought to play important roles in acute leukemic transformation of hemopoietic stem cells. We report here the functional analyses of AML1/Evi-1. It was revealed that AML1/Evi-1 itself does not alter the transactivation level through mouse polyomavirus enhancer-binding protein 2 (PEBP2; PEA2) sites (binding site of AML1) but dominantly suppresses the transactivation by intact AML1, which is assumed to be a stimulator of myeloid cell differentiation. DNA-binding competition is a putative mechanism of such dominant negative effects of AML1/Evi-1 because it binds to PEBP2 sites with higher affinity than AML1 does. Furthermore, AML1/Evi-1 stimulated c-fos promoter transactivation and increased AP-1 activity, as Evi-1 (which is not normally expressed in hemopoietic cells) did. Experiments using deletion mutants of AML1/Evi-1 showed that these two functions are mutually independent because the dominant negative effects on intact AML1 and the stimulation of AP-1 activity are dependent on the runt domain (DNA-binding domain of AML1) and the zinc finger domain near the C terminus, respectively. Furthermore, we showed that AML1/Evi-1 blocks granulocytic differentiation, otherwise induced by granulocyte colony-stimulating factor, of 32Dcl3 myeloid cells. It was also suggested that both AML1-derived and Evi-1-derived portions of the fusion protein play crucial roles in this differentiation block. We conclude that the leukemic cell transformation in t(3;21) leukemias is probably caused by these dual functions of AML1/Evi-1 chimeric protein.
PMCID: PMC230467  PMID: 7739522
22.  Response to inhibition of smoothened in diverse epithelial cancer cells that lack smoothened or patched 1 mutations 
International Journal of Oncology  2012;41(5):1751-1761.
Hedgehog (HH) pathway Smoothened (Smo) inhibitors are active against Gorlin syndrome-associated basal cell carcinoma (BCC) and medulloblastoma where Patched (Ptch) mutations occur. We interrogated 705 epithelial cancer cell lines for growth response to the Smo inhibitor cyclopamine and for expressed HH pathway-regulated species in a linked genetic database. Ptch and Smo mutations that respectively conferred Smo inhibitor response or resistance were undetected. Previous studies revealed HH pathway activation in lung cancers. Therefore, findings were validated using lung cancer cell lines, transgenic and transplantable murine lung cancer models, and human normal-malignant lung tissue arrays in addition to testing other Smo inhibitors. Cyclopamine sensitivity most significantly correlated with high cyclin E (P=0.000009) and low insulin-like growth factor binding protein 6 (IGFBP6) (P=0.000004) levels. Gli family members were associated with response. Cyclopamine resistance occurred with high GILZ (P=0.002) expression. Newer Smo inhibitors exhibited a pattern of sensitivity similar to cyclopamine. Gain of cyclin E or loss of IGFBP6 in lung cancer cells significantly increased Smo inhibitor response. Cyclin E-driven transgenic lung cancers expressed a gene profile implicating HH pathway activation. Cyclopamine treatment significantly reduced proliferation of murine and human lung cancers. Smo inhibition reduced lung cancer formation in a syngeneic mouse model. In human normal-malignant lung tissue arrays cyclin E, IGFBP6, Gli1 and GILZ were each differentially expressed. Together, these findings indicate that Smo inhibitors should be considered in cancers beyond those with activating HH pathway mutations. This includes tumors that express genes indicating basal HH pathway activation.
PMCID: PMC3583816  PMID: 22923130
hedgehog; smoothened; patched; lung cancer
23.  The tumor suppressive role of miRNA-370 by targeting FoxM1 in acute myeloid leukemia 
Molecular Cancer  2012;11:56.
Recent evidence has accumulated that MicroRNA (miRNA) dysregulation occurs in the majority of human malignancies including acute myeloid leukemia (AML) and may contribute to onco-/leukemo-genesis.
The expression levels of miR-370 and FoxM1 were assessed in 48 newly diagnosed AML patients, 40 AML patients in 1st complete remission (CR) and 21 healthy controls. Quantitative real-time PCR, western blots, colony formation assay, and β-Galactosidase ( SA-β-Gal) staining were used to characterize the changes induced by overexpression or inhibition of miR-370 or FoxM1.
We found that the down-regulation of miR-370 expression was a frequent event in both leukemia cell lines and primary leukemic cells from patients with de novo AML. Lower levels of miR-370 expression were found in 37 of 48 leukemic samples from AML patients compared to those in bone marrow cells derived from healthy adult individuals. Ectopic expression of miR-370 in HL60 and K562 cells led to cell growth arrest and senescence. In contrast, depletion of miR-370 expression using RNA interference enhanced the proliferation of those leukemic cells. Mechanistically, miR-370 targets the transcription factor FoxM1, a well established oncogenic factor promoting cell cycle progression. Moreover, when HL60 and K562 cells were treated with 5-aza-2′-deoxycytidine, a DNA methylation inhibitor, miR-370 expression was up-regulated, which indicates epigenetic silencing of miR-370 in leukemic cells.
Taken together, miR-370 may function as a tumor suppressor by targeting FoxM1, and the epigenetic silence of miR-370 thus leads to derepression of FoxM1 expression and consequently contributes to AML development and progression.
PMCID: PMC3533721  PMID: 22900969
miR-370; FoxM1; AML; Cellular senescence
24.  Lentivirus-mediated SMO RNA interference inhibits SMO expression and cell proliferation, and affects the cell cycle in LNCaP and PC3 cancer cell lines 
Asian Journal of Andrology  2009;12(2):196-202.
Smoothened (SMO) is an important member of the Hedgehog signaling pathway. We constructed a specific recombinant lentiviral vector for RNA interference, targeting the SMO gene (NM_005631) to observe its effect on SMO expression, cell proliferation and the cell cycle in the human androgen-sensitive prostate cancer cell line, LNCaP, and in the androgen-independent prostate cancer cell line, PC3. Four siRNA sequences were designed and inserted into a lentiviral vector pGCSIL-GFP to construct four recombinant vectors. The vector with the highest interfering efficiency was co-transfected with packaging vectors (pHelper1.0 and pHelper2.0) in 293T cells to assemble lentivirus particles by liposome for infecting LNCaP and PC3 cell lines, respectively. The expression level of SMO mRNA, tumor cell proliferation and cell cycle were measured by quantitative realtime polymerase chain reaction (qRT-PCR), 3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di-phenytetrazoliumromide (MTT) assay and flow cytometry, respectively. Sequence results showed that recombinant lentiviral vectors were constructed successfully. pGCSIL-GFP-723 had the highest interfering efficiency, named Lv-SIL-SMO723 after co-transfection, with which LNCaP and PC3 cell lines were infected. Compared with the control groups, results showed significantly decreased (P < 0.05) SMO mRNA expressions of LNCaP and PC3, lower mean percentage of S-phase cells and higher mean percentage of G2/M phase cells, as well as obviously slow proliferation (P < 0.01) of LNCaP in the infected group. Yet, the proliferation of PC3 was not altered (P > 0.05). In conclusion, the recombinant lentivirus particles were able to suppress SMO expression, regulate the cell cycle in the LNCaP and PC3 cell lines and markedly inhibit proliferation of LNCaP cells but not PC3 cells.
PMCID: PMC3739093  PMID: 20023691
lentivirus; prostate neoplasm; RNAi; smoothened
25.  Activation of Smurf E3 Ligase Promoted by Smoothened Regulates Hedgehog Signaling through Targeting Patched Turnover 
PLoS Biology  2013;11(11):e1001721.
Protein turnover of Patched, the Hedgehog receptor and key negative regulator of Hedgehog signaling, is controlled by the ubiquitin E3 ligase, Smurf, in a manner that depends on activation of signal transducer, Smoothened.
Hedgehog signaling plays conserved roles in controlling embryonic development; its dysregulation has been implicated in many human diseases including cancers. Hedgehog signaling has an unusual reception system consisting of two transmembrane proteins, Patched receptor and Smoothened signal transducer. Although activation of Smoothened and its downstream signal transduction have been intensively studied, less is known about how Patched receptor is regulated, and particularly how this regulation contributes to appropriate Hedgehog signal transduction. Here we identified a novel role of Smurf E3 ligase in regulating Hedgehog signaling by controlling Patched ubiquitination and turnover. Moreover, we showed that Smurf-mediated Patched ubiquitination depends on Smo activity in wing discs. Mechanistically, we found that Smo interacts with Smurf and promotes it to mediate Patched ubiquitination by targeting the K1261 site in Ptc. The further mathematic modeling analysis reveals that a bidirectional control of activation of Smo involving Smurf and Patched is important for signal-receiving cells to precisely interpret external signals, thereby maintaining Hedgehog signaling reliability. Finally, our data revealed an evolutionarily conserved role of Smurf proteins in controlling Hh signaling by targeting Ptc during development.
Author Summary
Hedgehog (Hh) signaling is a pathway renowned for its roles in controlling embryonic development and tumorigenesis. Signaling via this pathway proceeds when Hh ligands bind to the receptor Patched (Ptc), thereby preventing Ptc from inhibiting the signal transducer, Smoothened (Smo), and thus allowing Smo to accumulate on the cell surface where it becomes activated and promotes downstream signal transduction. In the absence of Hh ligands, Ptc inhibits Smo and is a key negative regulator of Hh signaling. In this study, we investigate how protein turnover of Ptc is controlled to ensure tight regulation of Hh signaling. Using Drosophila as a model system, we provide biochemical and genetic evidence to show that the E3 ligase, Smurf, directly controls Ptc protein turnover in developing wing discs. Moreover, we found that Smurf mediates Ptc degradation in a manner that depends on Smo signaling activity: activated Smo forms a complex with Smurf to preferentially promote degradation of the ligand-unbound Ptc receptor. Using mathematic modeling we reveal that the control of Smo activation by the opposing activities of Smurf and Ptc, is important for cells receiving the Hh signal to precisely interpret and relay external signals. We show that this control mechanism is also active in vertebrates with evidence that zebrafish Smurf proteins target Ptc1 protein for degradation to control late somitogenesis during zebrafish embryogenesis.
PMCID: PMC3841102  PMID: 24302888

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