Id1 mRNA Research Articles

Alk3, a BMP Type I Receptor Is Required for the Induction of Hepatic Hepcidin Gene Expression by Interleukin-6

Abstract 686 Introduction:Anemia of chronic disease (ACD), the second most prevalent form of anemia, is commonly associated with chronic inflammatory, infectious, or neoplastic conditions. ACD is characterized by high hepcidin levels that decrease serum iron levels by inducing degradation of the iron exporter ferroportin. In contrast, a relative deficiency of hepcidin leads to ferroportin overexpression and iron overload. Hepcidin is transcriptionally regulated by interleukin-6 (IL-6) and bone morphogenic protein (BMP) signaling. Binding of BMP ligands to type II and type I BMP receptors induces the type II receptor to phosphorylate and activate one of four type I receptors. We sought to identify the type I BMP receptor that participates in the ability of IL-6 to induce hepatic hepcidin gene expression. Methods:The four type I BMP receptors are Alk1, Alk2, Alk3, and Alk6. Alk1 is predominantly expressed in the endothelium. Alk6 is expressed at low levels in murine liver. In contrast, Alk2 and Alk3 are abundantly expressed in hepatocytes. Global deficiency of Alk2 or Alk3 is embryonic lethal. To selectively delete Alk2 or Alk3 in hepatocytes, we studied mice homozygous for Alk2 or Alk3 sequences flanked by loxP sites (Alk2fl/fl and Alk3fl/fl, respectively) that also carried a transgene specifying Cre recombinase under the control of the albumin gene promoter (Alb-Cre). Eight- to 12-week-old male mice (Alk2fl/fl, Alk2fl/fl; Alb-Cre, Alk3fl/fl, Alk3fl/fl; Alb-Cre) on a standard, iron-replete diet were injected via the tail vein with an adenovirus specifying IL-6 (Ad.IL-6) or an adenovirus specifying green fluorescent protein (GFP; Ad.GFP), as a control (1010 particles per ml for both). Seventy-two hours later, mice were euthanized, and blood was obtained for measurement of serum iron levels and transferrin saturations. Livers were harvested, and RNA was extracted. Hepatic levels of mRNAs encoding Alk2, Alk3, hepcidin, heme oxygenase-1 (HO-1, a transcriptional target of IL-6), and Id-1 (a BMP gene target) were measured by qRT-PCR. Hepatic STAT3 phosphorylation (a marker of IL-6 receptor activation) was measured using immunoblot techniques. Results:Liver-specific deletion of Alk2 or Alk3 caused mild and severe iron overload, respectively. Injection of Ad.IL-6, but not Ad.GFP, decreased serum iron levels and transferrin saturations in Alk2fl/fl, Alk2fl/fl; Alb-Cre, and Alk3fl/fl mice. In contrast, infection of Alk3fl/fl; Alb-Cre mice with Ad.IL-6 did not alter serum iron levels and only modestly reduced transferrin saturations. Infection with Ad.IL-6 induced of hepatic hepcidin gene expression in Alk2fl/fl, Alk2fl/fl; Alb-Cre, and Alk3fl/fl mice. Hepatic hepcidin mRNA levels were markedly reduced in Ad.GFP-infected Alk3fl/fl; Alb-Cre mice, and infection with Ad.IL-6 failed to increase hepcidin mRNA levels in this genotype. Ad.IL-6 infection induced hepatic Id-1 mRNA levels in Alk2fl/fl, Alk2fl/fl; Alb-Cre, and Alk3fl/fl mice, but not in Alk3fl/fl; Alb-Cre mice. Infection with Ad.IL-6 induced hepatic STAT-3 phosphorylation and HO-1 gene expression in all 4 genotypes of mice. Conclusions:Taken together, these results demonstrate that the response to IL-6, as reflected by STAT-3 phosphorylation and induction of HO-1 gene expression, does not require Alk2 or Alk3. In contrast, BMP signaling, predominantly via Alk3, is essential for the induction of hepcidin gene expression by IL-6. These results suggest that selective inhibition of Alk3 may represent a novel therapeutic approach to the treatment of ACD. Disclosures:Peterson:Massachusetts General Hospital: Patents & Royalties, The Massachusetts General Hospital has filed patents related to the use of small molecule inhibitors of BMP signaling to modulate iron metabolism, and PBY, RTP and KDB may be eligible to receive royalties. Yu:Massachusetts General Hospital: Patents & Royalties, The Massachusetts General Hospital has filed patents related to the use of small molecule inhibitors of BMP signaling to modulate iron metabolism, and PBY, RTP and KDB may be eligible to receive royalties. Bloch:Massachusetts General Hospital: Patents & Royalties, The Massachusetts General Hospital has filed patents related to the use of small molecule inhibitors of BMP signaling to modulate iron metabolism, and PBY, RTP and KDB may be eligible to receive royalties.

ID’ing a Novel Inhibitor of β-Cell Function, Id1

It is well established that loss of β-cell mass and compromised β-cell function are telltale indicators of the development of type 2 diabetes (1). Even individuals at risk for developing diabetes show β-cell dysfunction such as reduction of first-phase insulin release (2). An early response to increased insulin demand, as brought about in states of insulin resistance such as obesity or pregnancy, is a compensatory upregulation of the pancreatic β-cell mass and function. However, when this compensation fails and an inadequate amount of insulin is released in response to a meal, diabetes develops. The inability of β-cells to compensate for insulin resistance is associated with several abnormalities in β-cell function such as mitochondrial stress and reactive oxygen species production, insufficient proinsulin-to-insulin processing, and increased expression of islet amyloid polypeptide. Many of these abnormalities are believed to be the result of a combination of genetic and environmental factors. In particular, the exposure of β-cells to high concentrations of free fatty acids in combination with hyperglycemia (glucolipotoxicity) have been shown to be detrimental to β-cells resulting in severe dysfunction, loss of differentiation markers, and apoptosis. The detailed cellular and molecular mechanism by which β-cell dysfunction develops leading to type 2 diabetes is yet to be fully understood. In a search for genes with altered expression in islets from diabetic mice, it was found that the mRNA level for the gene encoding the transcriptional regulator Id1 (inhibitor of DNA binding-1) was increased in islets from db/db mice (3), and furthermore, Id1 mRNA was increased in response to long-term exposure of β-cells to free fatty acids (4) as well as hyperglycemia (5). In order to investigate if Id1 plays …

USP1 Deubiquitinates ID Proteins to Preserve a Mesenchymal Stem Cell Program in Osteosarcoma

Inhibitors of DNA binding (IDs) antagonize basic-helix-loop-helix (bHLH) transcription factors to inhibit differentiation and maintain stem cell fate. ID ubiquitination and proteasomal degradation occur in differentiated tissues, but IDs in many neoplasms appear to escape degradation. We show that the deubiquitinating enzyme USP1 promotes ID protein stability and stem cell-like characteristics in osteosarcoma. USP1 bound, deubiquitinated, and thereby stabilized ID1, ID2, and ID3. A subset of primary human osteosarcomas coordinately overexpressed USP1 and ID proteins. USP1 knockdown in osteosarcoma cells precipitated ID protein destabilization, cell-cycle arrest, and osteogenic differentiation. Conversely, ectopic USP1 expression in mesenchymal stem cells stabilized ID proteins, inhibited osteoblastic differentiation, and enhanced proliferation. Consistent with USP1 functioning in normal mesenchymal stem cells, USP1-deficient mice were osteopenic. Our observations implicate USP1 in preservation of the stem cell state that characterizes osteosarcoma and identify USP1 as a target for differentiation therapy.

MAGE I Transcription Factors Regulate KAP1 and KRAB Domain Zinc Finger Transcription Factor Mediated Gene Repression

Class I MAGE proteins (MAGE I) are normally expressed only in developing germ cells but are aberrantly expressed in many cancers. They have been shown to promote tumor survival, aggressive growth, and chemoresistance but the underlying mechanisms and MAGE I functions have not been fully elucidated. KRAB domain zinc finger transcription factors (KZNFs) are the largest group of vertebrate transcription factors and regulate neoplastic transformation, tumor suppression, cellular proliferation, and apoptosis. KZNFs bind the KAP1 protein and direct KAP1 to specific DNA sequences where it suppresses gene expression by inducing localized heterochromatin characterized by histone 3 lysine 9 trimethylation (H3me3K9). Discovery that MAGE I proteins also bind to KAP1 prompted us to investigate whether MAGE I can affect KZNF and KAP1 mediated gene regulation. We found that expression of MAGE I proteins, MAGE-A3 or MAGE-C2, relieved repression of a reporter gene by ZNF382, a KZNF with tumor suppressor activity. ChIP of MAGE I (-) HEK293T cells showed KAP1 and H3me3K9 are normally bound to the ID1 gene, a target of ZNF382, but that binding is greatly reduced in the presence of MAGE I proteins. MAGE I expression relieved KAP1 mediated ID1 repression, causing increased expression of ID1 mRNA and ID1 chromatin relaxation characterized by loss of H3me3K9. MAGE I binding to KAP1 also induced ZNF382 poly-ubiquitination and degradation, consistent with loss of ZNF382 leading to decreased KAP1 binding to ID1. In contrast, MAGE I expression caused increased KAP1 binding to Ki67, another KAP1 target gene, with increased H3me3K9 and decreased Ki67 mRNA expression. Since KZNFs are required to direct KAP1 to specific genes, these results show that MAGE I proteins can differentially regulate members of the KZNF family and KAP1 mediated gene repression.

Roles of Id1 in Aβ-induced cell cycle re-entry in postmitotic neurons

Senile plaque containing aggregation of amyloid-beta peptide (Aß) is one of the major hallmarks of Alzheimer's disease (AD). Several lines of evidence suggest the correlation between Aß-induced neuronal death and cell cycle re-entry, but the underlying mechanisms remain unclear. Inhibitor of differentiation 1 (Id1) is involved in the regulation of cellular differentiation and cell cycle progression. Doublecortin (DCX) is a neuronal lineage marker that expresses at proliferative stage and early differentiating stage. Subsequent to exit from cell cycle, NeuN is expressed at early and late postmitotic maturation stage. Glial fibrillary acidic protein (GFAP) is a specific protein marker for astrocyte. Bromodeoxyuridine (BrdU) is incorporated during DNA synthesis in proliferating cells. Thus, colocalization of BrdU with DCX or NeuN in fully differentiated neurons may serve as a marker for cell cycle re-entry. We investigate whether Id1 induction by Aß causes cell cycle re-entry in postmitotic neurons leading to cell death. The expression profiles of Id1 and cyclins upon exposure to Aß25-35 were determined by real-time RT-PCR and Western blotting in primary cortical neurons. Immunocytochemistry was utilized to quantitatively determine the numbers of DCX+/BrdU+, NeuN+/BrdU+, GFAP+/BrdU+, and caspase 3+/BrdU+ cells after Aß25-35 treatment. Finally, siRNA was employed to suppress Id1 expression in cortical neurons. Aß25-35 induced Id1, cyclin D1, E1 and A2 mRNA in primary cortical neurons. Aß25-35 and Aß1-42 induced expression of Id1 protein. Aß25-35 increased the numbers of DCX+/BrdU+, NeuN+/BrdU+, GFAP+/BrdU+, and caspase 3+/BrdU+ cells, indicating Aß25-35-mediated interference of neuronal differentiation, increases in astrocytosis, and a heightened extent of apoptosis in cultured cortical neurons. Suppression of Id1 expression by siRNA in part recovered cell survival after Aß25-35 and Aß1-42 treatments in primary cortical neurons. Id1 induction by Aß may play a significant role in Aß-induced cell cycle re-entry and neuronal death.

Inhibitor of differentiation-1 as a novel prognostic factor in NSCLC patients with adenocarcinoma histology and its potential contribution to therapy resistance.

High inhibitor of differentiation-1 (Id1) levels have been found in some tumor types. We aimed to study Id1 levels and their prognostic impact in a large series of stages I to IV non-small cell lung cancer (NSCLC) patients. Experiments in cell lines and cells derived from malignant pleural effusions (MPE) were also carried out. A total of 346 NSCLC samples (three different cohorts), including 65 matched nonmalignant tissues, were evaluated for Id1 expression by using immunohistochemistry. Additional data from a fourth cohort including 111 patients were obtained for Id1 mRNA expression analysis by using publicly available microarrays. In vitro proliferation assays were conducted to characterize the impact of Id1 on growth and treatment sensitivity. Significantly higher Id1 protein levels were found in tumors compared with normal tissues (P < 0.001) and in squamous carcinomas compared with adenocarcinomas (P < 0.001). In radically treated stages I to III patients and stage IV patients treated with chemotherapy, higher Id1 levels were associated with a shorter disease-free survival and overall survival in adenocarcinoma patients in a log-rank test. A Cox model confirmed the independent prognostic value of Id1 levels for both stages I to III and stage IV patients. In silico analysis confirmed a correlation between higher Id1 mRNA levels and poor prognosis for adenocarcinoma subjects. In vitro Id1 silencing in radio/chemotherapy-resistant adenocarcinoma cells from MPEs restored sensitivity to both therapies. In our series, Id1 levels showed an independent prognostic value in patients with adenocarcinoma, regardless of the stage. Id1 silencing may sensitize adenocarcinoma cells to radiotherapy and chemotherapy.

Thrombospondin-1 Modulates the Angiogenic Phenotype of Human Cerebral Arteriovenous Malformation Endothelial Cells

The management of cerebral arteriovenous malformation (AVM) is challenging, and invasive therapies place vital intracranial structures at risk of injury. The development of noninvasive, pharmacologic approaches relies on identifying factors that mediate key angiogenic processes. Previous studies indicate that endothelial cells (ECs) derived from cerebral AVM (AVM-ECs) are distinct from control brain ECs with regard to important angiogenic characteristics. To determine whether thrombospondin-1 (TSP-1), a potent angiostatic factor, regulates critical angiogenic features of AVM-ECs and to identify factors that modulate TSP-1 production in AVM-ECs. EC proliferation, migration, and tubule formation were evaluated with bromodeoxyuridine incorporation, Boyden chamber, and Matrigel studies, respectively. TSP-1 and inhibitor of DNA binding/differentiation 1 (Id1) mRNA levels were quantified with microarray and quantitative real-time polymerase chain reaction analyses. TSP-1 protein expression was measured using Western blotting, immunohistochemical, and enzyme-linked immunosorbent assay techniques. The mechanistic link between Id1 and TSP-1 was established through small interfering RNA-mediated knockdown of Id1 in AVM-ECs followed by Western blot and enzyme-linked immunosorbent assay experiments assessing TSP-1 production. AVM-ECs proliferate faster, migrate more quickly, and form disorganized tubules compared with brain ECs. TSP-1 is significantly down-regulated in AVM-ECs. The addition of TSP-1 to AVM-EC cultures normalizes the rate of proliferation and migration and the efficiency of tubule formation, whereas brain ECs are unaffected. Id1 negatively regulates TSP-1 expression in AVM-ECs. These data highlight a novel role for TSP-1 in the pathobiology of AVM angiogenesis and provide a context for its use in the clinical management of brain AVMs.

Protein Kinase Cα Signaling Regulates Inhibitor of DNA Binding 1 in the Intestinal Epithelium

Increasing evidence supports a role for PKCα in growth arrest and tumor suppression in the intestinal epithelium. In contrast, the Id1 transcriptional repressor has pro-proliferative and tumorigenic properties in this tissue. Here, we identify Id1 as a novel target of PKCα signaling. Using a highly specific antibody and a combined morphological/biochemical approach, we establish that Id1 is a nuclear protein restricted to proliferating intestinal crypt cells. A relationship between PKCα and Id1 was supported by the demonstration that (a) down-regulation of Id1 at the crypt/villus junction coincides with PKCα activation, and (b) loss of PKCα in intestinal tumors is associated with increased levels of nuclear Id1. Manipulation of PKCα activity in IEC-18 nontransformed intestinal crypt cells determined that PKCα suppresses Id1 mRNA and protein via an Erk-dependent mechanism. PKCα, but not PKCδ, also inhibited Id1 expression in colon cancer cells. Id1 was found to regulate cyclin D1 levels in IEC-18 and colon cancer cells, pointing to a role for Id1 suppression in the antiproliferative/tumor suppressive activities of PKCα. Notably, Id1 expression was elevated in the intestinal epithelium of PKCα-knock-out mice, confirming that PKCα regulates Id1 in vivo. A wider role for PKCα in control of inhibitor of DNA binding factors is supported by its ability to down-regulate Id2 and Id3 in IEC-18 cells, although their suppression is more modest than that of Id1. This study provides the first demonstrated link between a specific PKC isozyme and inhibitor of DNA binding factors, and it points to a role for a PKCα → Erk ⊣ Id1 → cyclin D1 signaling axis in the maintenance of intestinal homeostasis.

Induction of Id-1 by FGF-2 involves activity of EGR-1 and sensitizes neuroblastoma cells to cell death

Inhibitor of differentiation-1 (Id-1) is a member of helix-loop-helix (HLH) family of proteins that regulate gene transcription through their inhibitory binding to basic-HLH transcription factors. Similarly to other members of this family, Id-1 is involved in the repression of cell differentiation and activation of cell growth. The dual function of Id-1, inhibition of differentiation, and stimulation of cell proliferation, might be interdependent, as cell differentiation is generally coupled with the exit from the cell cycle. Fibroblast growth factor-2 (FGF-2) has been reported to play multiple roles in different biological processes during development of the central nervous system (CNS). In addition, FGF-2 has been described to induce "neuronal-like" differentiation and trigger apoptosis in neuroblastoma SK-N-MC cells. Although regulation of Id-1 protein by several mitogenic factors is well-established, little is known about the role of FGF-2 in the regulation of Id-1. Using human neuroblastoma cell line, SK-N-MC, we found that treatment of these cells with FGF-2 resulted in early induction of both Id-1 mRNA and protein. The induction occurs within 1 h from FGF-2 treatment and is mediated by ERK1/2 pathway, which in turn stimulates expression of the early growth response-1 (Egr-1) transcription factor. We also demonstrate direct interaction of Egr-1 with Id-1 promoter in vitro and in cell culture. Finally, inhibition of Id-1 expression results in G(2) /M accumulation of FGF-2-treated cells and delayed cell death.

Abstract 2219: Inhibitor of differentiation-1 is a novel prognostic factor among NSCLC patients with adenocarcinoma histology and contributes to therapy resistance

Abstract High Id1 levels have been found in some tumor types, particularly in advance stages. We aimed to study Id1 levels and their prognostic value in a large series of different histologies of stage I-IV NSCLC patients and to test Id1 function in cell lines and cells derived from malignant pleural effusions (MPE) from patients (pts). Id1 expression was analyzed in NSCLC samples from 311 pts and 65 normal lung tissues by immunohistochemistry. Id1 mRNA expression was also studied in 111 pts using publicly available microarrays. Significantly higher Id1 protein expression levels were found in tumors compared to normal tissue (p&amp;lt;0.001) and in adenocarcinomas (AC) compared so squamous histology (p&amp;lt;0.001). Among the localized NSCLC pts undergoing surgery, higher Id1 expression levels were associated with a shorter overall survival (OS) for AC histology (p=0.017). In the surgery+adjuvant chemo-radiotherapy pts, the same correlation was found between Id1 levels and OS among AC samples (p=0.038). Consistently, in stage IV pts receiving palliative chemotherapy, Id1 maintained its prognostic value (shorter OS in pts with higher levels) for AC (p=0.003). Id1 was also correlated with time-to-progression (TTP) after chemotherapy (lower levels, prolonged TTP) in AC pts (p=0.008). The in silico analysis confirmed this association. High expression of Id1 was also found by western blots in squamous cell lines (H520, HCC15, H157 and H58) and in lesser extent in AC cell lines (SKLU-1, LXF-289, H322, H23) and MPE-derived AC cells. Id1 knockdown resulted in a significant reduction of the clonogenic activity of squamous and AC cells. Id1 silencing on radiotherapy and chemotherapy-resistant MPE-derived cells restored sensitivity to both therapies. We conclude that Id1 protein and mRNA expression is an independent prognostic factor among pts with AC but no other histologies, regardless of the stage or treatment received. In cell lines derived from MPE pts, Id1 downregulation decreases NSCLC cell proliferation and sensitizes cells to radiotherapy and chemotherapy, in vitro. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2219. doi:10.1158/1538-7445.AM2011-2219

Abstract LB-141: Expression and localization of Id3 in human prostate cancer

Abstract Purpose: The inhibitor of differentiation (Id) proteins are dominant negative basic helix-loop-helix family of transcription factors that lack the DNA-binding basic domain but have intact HLH domain. The four members of the Id protein family (Id1-Id4) are known to play a critical role in the coordinated regulation of gene expression during cell growth, differentiation and tumorigenesis. Although in vitro studies have demonstrated the role of Id1 in regulating prostate cancer (PCa) cell proliferation, apoptosis and androgen independence, its clinical significance in PCa remains controversial. On the other hand there is lack of evidence on the expression of Id2 and Id3 in PCa progression. Studies have demonstrated that silencing of Id3 is significantly more effective in blocking proliferation of PCa cells as compared to Id1 whereas Id2 silencing has been primarily shown to be involved in apoptotic pathway. Hence in this study we re-evaluated the role of Id1 in PCa and extended our study to show that Id3 protein expression has better clinical relevance compared to Id1 and Id2 in PCa. Experimental design: The expression of Id-1, -2 and -3 was determined by real time and Western blotting along with their sub-cellular localization by ICC in PCa cell lines LNCaP, DU145 and PC3. To assess their significance in prostate cancer progression, the protein expression was investigated in clinical prostate tumor samples on high density tissue microarray by IHC. Results: The expression of Id1 and Id3 mRNA was low in LNCaP and DU145 as compared to PC3 whereas the level of Id2 expression was similar between all the PCa cell lines. The protein expression data was consistent with the mRNA levels in the PCa cell lines. All three Id's were primarily cytoplasmic with some nuclear presence. The IHC data showed a significant increase in the expression of Id1 and Id3 in clinical prostate tumor tissue samples compared to normal prostate tissue whereas Id2 showed elevated levels but was not significant as compared to Id1 and Id3. The expression of Id3 protein correlated with the Gleason grading and stage of PCa. Conclusions: This is the first study demonstrating a correlation between increased expression of Id3 and prostate cancer progression. It also reconfirms and establishes the association of Id1with PCa. The results warrant further investigation into isoform specific effects of Id1and Id3 on prostate tumorigenesis. (Research Support: NIH/NCI RO1 CA128914 and NIH/NCRR/RCMI G12RR03062. The first two authors contributed equally to this work. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-141. doi:10.1158/1538-7445.AM2011-LB-141

Abstract 3987: Src regulates inhibitor of differentiation 1 (Id1) via c-Myc induced repression of microRNA-29b: Implications for invasion of lung cancer cells and their resistance against Src inhibitors

Abstract Background: Src and Id1 mediate cancer cell invasion and are frequently co-expressed in non-small cell lung cancer (NSCLC). We previously demonstrated that Src regulates Id1 in human lung cancer cells. MicroRNAs (miRNAs) are noncoding RNAs that regulate gene expression by inducing translational inhibition or cleavage of their target mRNAs. The current study aimed at identifying miRNAs controlled by Src that target Id1, and to test if modulation of these miRNAs and Id1 in NSCLC cells leads to resistance against Src inhibitors. Methods: The study included the following human lung cancer cell lines: A549, H460, H1299, H820, HCC827. Cells were incubated with the Src inhibitors Saracatinib and Dasatinib and miRNA was extracted. To assess miRNA expression profiles, we used TaqMan® Array MicroRNA Cards (Applied Biosystems). Western Blotting was performed using antibodies for Src, phospho-Src, Id1, c-Myc, active beta-Catenin and Actin. Id1 and miR-29b transcript levels were measured using real-time quantitative RT-PCR. Lentiviral vectors expressing pre-miR-29b, anti-sense miR-29b, Id1 and c-Myc were used. Migration was tested with wound healing assays. For invasion matrigel coated membranes for transwell assays were used. Formalin-fixed and paraffin-embedded adenocarcinoma samples and matched lung tissue from 23 patients were used for miRNA expression analysis. Results: Upon incubation of A549 cells with Saracatinib, miR-29b was the most highly upregulated miRNA with a predicted binding site in the Id1 3’UTR. Indeed, Id1 3’UTR luciferase reporter assays revealed direct binding of miR-29b to the Id1 3’UTR, but not to a mutated Id1 3’UTR. Anti-miR-29b enhanced Id1 mRNA and protein levels and significantly increased cell migration and invasion (p=0.0006). Anti-miR-29b or Id1 overexpression abolished the effects of Src inhibitors on invasion and migration. In contrast, transfection with pre-miR-29b suppressed Id1 and significantly reduced cell migration and invasion (p&amp;lt;0.00001). Overexpression of c-Myc repressed the level of miR-29b and induced the expression of Id1. Saracatinib and Dasatinib decreased active beta-Catenin, c-Myc and Id1 levels, and increased miR-29b levels in a dose-dependent manner. In 19 of 23 human lung adenocarcinoma biopsies, miR-29b was significantly downregulated compared with matched normal lung tissue (p=0.031). miR-29b was a strong prognostic factor for event-free (p=0.003) and overall survival (p=0.04) Conclusions: These results demonstrate that Id1 is a direct target of miR-29b, which in turn is regulated by Src, active beta-Catenin and c-Myc. Therefore, miR-29b is involved in lung cancer cell migration and invasion. Furthermore, miR-29b is downregulated in human lung adenocarcinomas, and may be involved in the resistance of lung cancer cells against Src kinase inhibitors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3987. doi:10.1158/1538-7445.AM2011-3987

Prenatal Androgen Exposure Leads to Alterations in Gene and Protein Expression in the Ovine Fetal Ovary

Exposure of a female fetus to increased androgens in utero results in an adult phenotype reminiscent of polycystic ovary syndrome. We investigated whether prenatal androgens could directly alter the structure and function of the fetal ovary. We examined fetal ovarian cell proliferation, germ cell volume, and the expression of steroid receptors and steroidogenic enzymes. In addition, we studied the inhibitors of differentiation (Ids) and the SLIT/Roundabout developmental pathways. Female fetuses were collected from ewes treated with 100 mg testosterone propionate (TP) or vehicle control (C), twice weekly from d 60 to 70 (C = 3, TP = 6) or d 90 (C = 6, TP = 8). Female fetuses were also collected at d 70 after a single injection of TP (20 mg) or vehicle C into the fetal flank at d 60 (C = 4, TP = 8). Prenatal androgenization had no effect on fetal ovarian morphology, cell proliferation, or germ cell volume. However, there was a reduction in the expression of StAR, CYP11A, CYP17, and LHR at d 90 of gestation. There was also an increase in Id1 immunostaining at d 90 and an increase in Id3 immunostaining at d 70. Direct injection of TP into the fetus down-regulated ovarian CYP11A, estrogen receptor α and β mRNA, and ROBO1 and up-regulated CYP19, androgen receptor immunostaining, and Id3 mRNA and protein. Although at d 90 prenatal androgenization does not result in structural changes of the fetal ovary, there are functional changes that may impact on ovarian development. TP has direct actions on the fetal ovary, and these may contribute to the adult ovarian phenotype in the ovine model of polycystic ovary syndrome.

First randomized trial comparing balloon kyphoplasty (BKP) to non-surgical management among cancer patients with vertebral compression fractures

but overexpression of type II receptor did not rescue Hfg inhibition of TGF-β-responsive promoter activity. Further, Hfg increased mRNA expression of the inhibitory Smad, Smad7 but knockdown of Smad7 did not rescue Hfg inhibition of TGF-β responsive genes. These data suggested that the effect of Hfg was not completely TGF-βdependent. We next studied the effect of Hfg on BMP signaling. Hfg reduced BMP–Smad-reporter (BRE)4 in MDA-MB-231 or PC-3 cells treated with BMP7, as well as significantly reduced phosphoSmad2/ 3 and phospho-Smad1/5/8 proteins. Further Hfg reduced ID1 mRNA, a BMP-regulated gene. These data suggest that Hfg is an effective therapy for bone metastases through both TGF-β-dependent and independent effects.

Altered hepatic BMP signaling pathway in human HFE hemochromatosis

Human hemochromatosis (HC) has been associated with the common C282Y polymorphism of HFE or rare pathogenic mutations of TfR2, HJV, FPN and HAMP. All forms of human HC seem to be caused by low or inadequate levels of hepcidin, the iron hormone. We and others have recently shown that Hfe −/− mice exhibit an impairment in the bone morphogenetic protein (BMP) signaling pathway controlling hepcidin. However, all data indicating the central role of BMPs in hepcidin regulation and an impaired BMP/SMAD signaling in HC have been collected in mice. In this study we investigated whether also in humans the expression of BMP signaling targets, SMAD7 and Id1, are associated with liver iron concentration (LIC) and whether such regulation is disrupted in HFE-HC. We correlated the mRNA expression, assessed by RT-PCR, of HAMP, SMAD7 and Id1 with LIC in liver biopsies from patients with normal iron status, HFE-HC or non-HC hepatic iron overload. We found that in human liver, not only HAMP, but also SMAD7 and Id1 mRNA significantly correlate with the extent of hepatic iron burden. However, this correlation is lost in patients with HFE-HC, but maintained in subjects with non-hemochromatotic iron overload. These data indicate that in human HFE-HC a disrupted BMP/SMAD signaling in the liver is key in the pathogenesis of the disease.

Overexpressed Id-1 is associated with patient prognosis and HBx expression in hepatitis B virus-related hepatocellular carcinoma

Id-1 is a member of the helix-loop-helix protein family and is involved in multiple biological processes, including development, proliferation, angiogenesis and carcinogenesis. However, the role of Id-1 in hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC) is still unclear. In this study, Id-1 expression in 96 tumor specimens of HBV-related HCC was detected by immunohistochemical (IHC) staining. The relationship between the Id-1 expression grade and patient clinicopathological features was studied. In addition, the relationship between Id-1 expression and disease-free and overall survival times was analyzed. Colocalization of Id-1 and HBx was investigated by confocal laser scanning microscopy. The effect of HBx on Id-1 expression was studied in vitro using the HepG2 HCC cell line. Overexpression of Id-1 was found in 64.6% (62/96) of tumor specimens and was correlated with the histological grade, portal vein invasion, lymph node metastasis, HBsAg and Child-Pugh classification. Patients with Id-1 overexpression had both shorter disease-free and overall survival times. Besides, colocalization of Id-1 and HBx was found by paired IHC and confocal study. The expression of Id-1 was positively correlated to that of HBx. In vitro ectopic expression of HBx in HepG2 cells significantly increased Id-1 mRNA and protein expression. To our knowledge, this is the first report suggesting that Id-1 expression is at least partially regulated by HBx and may serve as a potential prognostic marker for HBV-related HCC.

Dynamic regulation of pituitary mRNAs for bone morphogenetic protein (BMP) 4, BMP receptors, and activin/inhibin subunits in the ewe during the estrous cycle and in cultured pituitary cells

Recently, bone morphogenetic protein (BMP) 4 has been shown to inhibit FSH secretion in ewe. The detection of BMP4 mRNA and BMP receptors in the pituitary suggests that BMP4 can exert paracrine actions on FSH production. This work aimed at determining whether BMP4 and/or BMP receptor mRNA as well as activin/inhibin subunit mRNA fluctuates during the estrous cycle when FSHβ mRNA and FSH release changed. The estrous cycles of ewes were synchronized with progestagen sponges. Ewes were killed in late follicular stage (n=5), before the secondary FSH surge (n=4), and in luteal phase (n=4). Using quantitative reverse transcription-PCR, we showed that the levels of mRNA for BMP4, BMP receptor, the inhibitor of differentiation 2 (Id2), a target gene of BMP4, and noggin did not change significantly across the estrous cycle. In contrast, the level of activin βB mRNA and the percentage of immunoreactive cells for activin βB-subunit were higher before the secondary surge of FSH compared to other groups. In ewe pituitary cell cultures, activin, GnRH, or estradiol-17β (E(2)) did not significantly affect the levels of BMP4, BMP receptor, and Id2 mRNA. E(2), but not GnRH, increased the level of activin βB mRNA. Moreover, the in vitro FSH release was not modified by noggin, a BMP antagonist. In contrast, SB431542, an inhibitor of activin pathway, inhibited FSH release. Collectively, our data showed that pituitary BMP4 would not play a crucial role in the regulation of FSH production during the estrous cycle, whereas local activin B would be a major stimulus of FSH synthesis necessary for the secondary FSH surge.

Down-regulation of Bmp/Smad signaling by Tmprss6 is required for maintenance of systemic iron homeostasis

AbstractIron-refractory, iron-deficiency anemia (IRIDA) is a familial disorder characterized by iron deficiency anemia unresponsive to oral iron treatment but partially responsive to intravenous iron therapy. Previously, we showed that IRIDA patients harbor loss-of-function mutations in TMPRSS6, a type II transmembrane serine protease primarily expressed by the liver. Both humans and mice with TMPRSS6 mutations show inappropriately elevated levels of the iron-regulatory hormone hepcidin, suggesting that TMPRSS6 acts to negatively regulate hepcidin expression. Here we investigate the relationship between Tmprss6 and the bone morphogenetic protein (BMP)–Smad signaling pathway, a key pathway promoting hepcidin transcription in hepatocytes. We show that livers from mice deficient for Tmprss6 have decreased iron stores and decreased Bmp6 mRNA, but markedly increased mRNA for Id1, a target gene of Bmp6 signaling. In contrast, mice deficient for both Tmprss6 and hemojuvelin (Hjv), a BMP coreceptor that augments hepcidin expression in hepatocytes, showed markedly decreased hepatic levels of hepcidin and Id1 mRNA, markedly increased hepatic Bmp6 mRNA levels, and systemic iron overload similar to mice deficient for Hjv alone. These findings suggest that down-regulation of Bmp/Smad signaling by Tmprss6 is required for regulation of hepcidin expression and maintenance of systemic iron homeostasis.

Loss of Id2 Potentiates the Tumorigenic Effect of Rb Inactivation in a Mouse Model of Retinoblastoma

Purpose: In some cancers, the oncogenic consequences of inactivating the retinoblastoma protein (Rb) appear to be mediated by unrestrained activity of the inhibitor of DNA binding protein Id2. The role of Id2 has not yet been investigated in the prototype cancer Rb-defective cancer, retinoblastoma itself. This study investigated whether loss of Id2 modified the effects of Rb inactivation in a mouse model of retinoblastoma.Methods: Id2 was analyzed in cultured cells using qPCR, Western blot, and colony formation assays. LHβ-Tag transgenic mice were crossed with Id2 heterozygotes to obtain mice with all three Id2 genotypes. Intraocular tumors were assessed for size, degree of differentiation, mitotic index, and tumor vascular density at 15 weeks of age.Results: Retinoblastoma cell lines expressed low levels of Id2 mRNA and protein. Depletion of Id2 in Rb-inactivated cells increased clonogenic activity. Id2-deficient tumors in vivo were significantly larger, less differentiated, and more vascularized than Id2-wild-type tumors (P = 0.02, P = 0.01, P = 0.0001, respectively). There was a dosage effect for loss of each Id2 allele with respect to differentiation and vascular density.Conclusions: Id2 suppresses rather than promotes tumor progression in this mouse model of retinoblastoma. Id2 can act as either an oncogene or a tumor suppressor depending on context.

Characterization of Functional Domains Necessary for Mutant p53 Gain of Function

Tumor cells, including SW480 carcinoma cells that carry a mutant p53, are addicted to the mutant for their survival and resistance to growth suppression by chemotherapeutic agents. Here, we investigated whether various classes of p53 mutants share a common property and functional domains necessary for mutant p53 gain of function. To test this, we generated SW480 cell lines in which endogenous mutant R273H/P309S can be inducibly or stably knocked down, whereas a small interfering RNA-resistant mutant p53 along with a mutated functional domain can be inducibly or stably expressed. We found that both contact-site (R248W and R273H) and conformation (G245S and R249S) mutants are able to maintain the transformed phenotypes of SW480 cells conferred by endogenous mutant p53. We also found that activation domains 1-2 and the proline-rich domain are required for mutant p53 gain of function. Interestingly, we showed that the C-terminal basic domain, which is required for wild-type p53 activity, is an inhibitory domain for mutant p53. Furthermore, we showed that deletion of the basic domain enhances, whereas a mutation in activation domains 1-2 and deletion of the proline-rich domain abolish mutant p53 to regulate Gro1 and Id2, both of which are regulated by and mediate endogenous mutant p53 gain of function. These results indicate that both conformation and contact-site mutants share a property for cell transformation, and the domains critical for wild-type p53 tumor suppression are also required for mutant p53 tumor promotion. Thus, the inhibitory basic domain and the common property for p53 mutants can be explored for targeting tumors with mutant p53.