Function Of Id1 Research Articles

Identification of ID1 and miR-150 interaction and effects on proliferation and apoptosis in ovine granulosa cells

Granulosa cells (GCs) proliferation and apoptosis play a significantly role in follicular development and atresia. ID1 and miR-150 are involved in cell apoptosis and follicular atresia, but the interaction and function of ID1 and miR-150 in GCs are unclear. This study focuses on ID1 and miR-150 in terms of the interaction and effects on proliferation and apoptosis in ovine granulosa cells. Our findings revealed that ID1 decreased the promoter activity and expression level of oar-miR-150. However, the expression of ID1 was downregulated by miR-150, and ID1 was identified as a target gene of oar-miR-150. miR-150 mimic inhibited proliferation and upregulated apoptosis rate in ovine GCs, while the results of miR-150 inhibitor were opposite. Overexpression of ID1 significantly inhibited ovine GCs proliferation and cell cycle-related genes (CDK1, CDK2, CDK4, CCND2, CDC20, and PCNA) expression, whereas knockdown of ID1 promoted cell proliferation and those genes expression. Overexpression of ID1 significantly downregulated mitochondrial membrane potential and Bcl-2 expression in ovine GCs, and upregulated the expression of pro-apoptosis genes Bax, Caspase-3, and Caspase-9, whereas the results of ID1 knockdown were reversed. Collectively, these findings indicate the interaction and the vital role of ID1 and miR-150 on proliferation and apoptosis in ovine granulosa cells, which may suggest a novel target for ovine follicular development and atresia.

The non-cell-autonomous function of ID1 promotes AML progression via ANGPTL7 from the microenvironment

AbstractThe bone marrow microenvironment (BMM) can regulate leukemia stem cells (LSCs) via secreted factors. Increasing evidence suggests that dissecting the mechanisms by which the BMM maintains LSCs may lead to the development of effective therapies for the eradication of leukemia. Inhibitor of DNA binding 1 (ID1), a key transcriptional regulator in LSCs, previously identified by us, controls cytokine production in the BMM, but the role of ID1 in acute myeloid leukemia (AML) BMM remains obscure. Here, we report that ID1 is highly expressed in the BMM of patients with AML, especially in BM mesenchymal stem cells, and that the high expression of ID1 in the AML BMM is induced by BMP6, secreted from AML cells. Knocking out ID1 in mesenchymal cells significantly suppresses the proliferation of cocultured AML cells. Loss of Id1 in the BMM results in impaired AML progression in AML mouse models. Mechanistically, we found that Id1 deficiency significantly reduces SP1 protein levels in mesenchymal cells cocultured with AML cells. Using ID1-interactome analysis, we found that ID1 interacts with RNF4, an E3 ubiquitin ligase, and causes a decrease in SP1 ubiquitination. Disrupting the ID1-RNF4 interaction via truncation in mesenchymal cells significantly reduces SP1 protein levels and delays AML cell proliferation. We identify that the target of Sp1, Angptl7, is the primary differentially expression protein factor in Id1-deficient BM supernatant fluid to regulate AML progression in mice. Our study highlights the critical role of ID1 in the AML BMM and aids the development of therapeutic strategies for AML.

Neuron-Radial Glial Cell Communication via BMP/Id1 Signaling Is Key to Long-Term Maintenance of the Regenerative Capacity of the Adult Zebrafish Telencephalon.

The central nervous system of adult zebrafish displays an extraordinary neurogenic and regenerative capacity. In the zebrafish adult brain, this regenerative capacity relies on neural stem cells (NSCs) and the careful management of the NSC pool. However, the mechanisms controlling NSC pool maintenance are not yet fully understood. Recently, Bone Morphogenetic Proteins (BMPs) and their downstream effector Id1 (Inhibitor of differentiation 1) were suggested to act as key players in NSC maintenance under constitutive and regenerative conditions. Here, we further investigated the role of BMP/Id1 signaling in these processes, using different genetic and pharmacological approaches. Our data show that BMPs are mainly expressed by neurons in the adult telencephalon, while id1 is expressed in NSCs, suggesting a neuron-NSC communication via the BMP/Id1 signaling axis. Furthermore, manipulation of BMP signaling by conditionally inducing or repressing BMP signaling via heat-shock, lead to an increase or a decrease of id1 expression in the NSCs, respectively. Induction of id1 was followed by an increase in the number of quiescent NSCs, while knocking down id1 expression caused an increase in NSC proliferation. In agreement, genetic ablation of id1 function lead to increased proliferation of NSCs, followed by depletion of the stem cell pool with concomitant failure to heal injuries in repeatedly injured mutant telencephala. Moreover, pharmacological inhibition of BMP and Notch signaling suggests that the two signaling systems cooperate and converge onto the transcriptional regulator her4.1. Interestingly, brain injury lead to a depletion of NSCs in animals lacking BMP/Id1 signaling despite an intact Notch pathway. Taken together, our data demonstrate how neurons feedback on NSC proliferation and that BMP1/Id1 signaling acts as a safeguard of the NSC pool under regenerative conditions.

ID1 and ID3 are Negative Regulators of TGFβ2-Induced Ocular Hypertension and Compromised Aqueous Humor Outflow Facility in Mice.

PurposeIn POAG, elevated IOP remains the major risk factor in irreversible vision loss. Increased TGFβ2 expression in POAG aqueous humor and in the trabecular meshwork (TM) amplifies extracellular matrix (ECM) deposition and reduces ECM turnover in the TM, leading to a decreased aqueous humor (AH) outflow facility and increased IOP. Inhibitor of DNA binding proteins (ID1 and ID3) inhibit TGFβ2-induced fibronectin and PAI-1 production in TM cells. We examined the effects of ID1 and ID3 gene expression on TGFβ2-induced ocular hypertension and decreased AH outflow facility in living mouse eyes.MethodsIOP and AH outflow facility changes were determined using a mouse model of Ad5-hTGFβ2C226S/C288S-induced ocular hypertension. The physiological function of ID1 and ID3 genes were evaluated using Ad5 viral vectors to enhance or knockdown ID1/ID3 gene expression in the TM of BALB/cJ mice. IOP was measured in conscious mice using a Tonolab impact tonometer. AH outflow facilities were determined by constant flow infusion in live mice.ResultsOver-expressing ID1 and ID3 significantly blocked TGFβ2-induced ocular hypertension (P < 0.0001). Although AH outflow facility was significantly decreased in TGFβ2-transduced eyes (P < 0.04), normal outflow facility was preserved in eyes injected concurrently with ID1 or ID3 along with TGFβ2. Knockdown of ID1 or ID3 expression exacerbated TGFβ2-induced ocular hypertension.ConclusionsIncreased expression of ID1 and ID3 suppressed both TGFβ2-elevated IOP and decreased AH outflow facility. ID1 and/or ID3 proteins thus may show promise as future candidates as IOP-lowering targets in POAG.

Id Proteins Promote a Cancer Stem Cell Phenotype in Mouse Models of Triple Negative Breast Cancer via Negative Regulation of Robo1.

Breast cancers display phenotypic and functional heterogeneity and several lines of evidence support the existence of cancer stem cells (CSCs) in certain breast cancers, a minor population of cells capable of tumor initiation and metastatic dissemination. Identifying factors that regulate the CSC phenotype is therefore important for developing strategies to treat metastatic disease. The Inhibitor of Differentiation Protein 1 (Id1) and its closely related family member Inhibitor of Differentiation 3 (Id3) (collectively termed Id) are expressed by a diversity of stem cells and are required for metastatic dissemination in experimental models of breast cancer. In this study, we show that ID1 is expressed in rare neoplastic cells within ER-negative breast cancers. To address the function of Id1 expressing cells within tumors, we developed independent murine models of Triple Negative Breast Cancer (TNBC) in which a genetic reporter permitted the prospective isolation of Id1+ cells. Id1+ cells are enriched for self-renewal in tumorsphere assays in vitro and for tumor initiation in vivo. Conversely, depletion of Id1 and Id3 in the 4T1 murine model of TNBC demonstrates that Id1/3 are required for cell proliferation and self-renewal in vitro, as well as primary tumor growth and metastatic colonization of the lung in vivo. Using combined bioinformatic analysis, we have defined a novel mechanism of Id protein function via negative regulation of the Roundabout Axon Guidance Receptor Homolog 1 (Robo1) leading to activation of a Myc transcriptional programme.

Overexpression of ID1 promotes tumor progression in penile squamous cell carcinoma.

Penile squamous cell carcinoma (PSCC) occurs more frequently in developing countries, and is commonly diagnosed at an advanced stage with an unfavorable prognosis. At present, few biomarkers for PSCC have been identified and used in clinical practice. Aberrant expression of inhibitor of DNA binding1 (ID1) has been suggested as a potential regulator of tumor progression in various types cancer. Herein, we evaluated ID1 expression in PSCC and analyzed its association with the clinicopathological parameters of PSCC. Our findings indicated that ID1 overexpression is associated with histological subtype and lymph node metastasis. Kaplan‑Meier survival analysis showed that the overexpression of ID1 is associated with unfavorable cancer‑specific survival. In Cox proportional hazard models, ID1 overexpression was found to be an independent predictor of cancer‑specific survival. Furthermore, we investigated the function of ID1 in PSCC using a PSCC cell line Penl1. Silencing of ID1 expression retarded cell growth, inhibited clonogenesis, and attenuated cell migration and invasion in Penl1 cells. ID1 may regulate key oncogenic and metastasis‑related molecules, as depletion of ID1 expression affected the levels of p‑AKT, p16, PTEN and cleaved caspase‑3, and reduced MMP2/9 secretion in Penl1 cells. Nevertheless, the mRNA expression of p16 and PTEN increased following ID1 knockdown, suggesting that ID1 may repress p16 and PTEN expression in Penl1 cells. Therefore, overexpression of ID1 could serve as a potential prognostic biomarker for the clinical management of PSCC. Strategies targeting ID1‑regulated signaling pathways may have therapeutic benefit in PSCC.

Expression and regulation of inhibitor of DNA binding proteins ID1, ID2, ID3, and ID4 at the maternal-conceptus interface in pigs

Inhibitor of DNA binding (ID) proteins, ID1, ID2, ID3, and ID4 are transcriptional regulators that have a helix-loop-helix (HLH) domain but not a basic DNA binding domain. ID proteins inhibit the functions of basic HLH transcription factors and regulate cell proliferation and differentiation. However, the expression and function of ID1, ID2, ID3, and ID4 at the maternal-conceptus interface are not fully understood in pigs. Therefore, we determined the expressions of ID1, ID2, ID3, and ID4 in porcine endometrium, conceptus, and chorioallantoic tissues. ID1, ID2, ID3, and ID4 mRNAs were expressed in the endometrium, with lower levels of ID1, ID2, and ID4 on Day 12 of pregnancy than during the estrous cycle. ID1, ID2, ID3, and ID4 mRNAs were also detected in conceptus and chorioallantoic tissues during pregnancy. ID2 protein was mainly localized to luminal epithelia and weakly to vascular smooth muscle cells in the endometrium and conceptus trophectoderm. Increasing doses of interleukin-1β decreased levels of ID2 mRNA, while estradiol-17β increased levels of ID3 mRNA in endometrial explants. The expressions of ID2 and ID4 mRNAs were higher in endometria from gilts with somatic cell nuclear transfer-derived conceptuses compared with endometria from gilts carrying conceptuses derived from natural mating on Day 12. These results indicate that the expressions of ID family genes are dynamically regulated at the maternal-conceptus interface, suggesting that ID proteins may play critical roles in the regulation of endometrial epithelial cell function and conceptus development to support the establishment and maintenance of pregnancy in pigs.

Id1 Promotes Obesity by Suppressing Brown Adipose Thermogenesis and White Adipose Browning.

Obesity results from increased energy intake or defects in energy expenditure. Brown adipose tissue (BAT) is specialized for energy expenditure, a process called adaptive thermogenesis. Peroxisome proliferator–activated receptor γ coactivator 1α (PGC1α) controls BAT-mediated thermogenesis by regulating the expression of Ucp1. Inhibitor of differentiation 1 (Id1) is a helix-loop-helix transcription factor that plays an important role in cell proliferation and differentiation. We demonstrate a novel function of Id1 in BAT thermogenesis and programming of beige adipocytes in white adipose tissue (WAT). We found that adipose tissue–specific overexpression of Id1 causes age-associated and high-fat diet–induced obesity in mice. Id1 suppresses BAT thermogenesis by binding to and suppressing PGC1α transcriptional activity. In WAT, Id1 is mainly localized in the stromal vascular fraction, where the adipose progenitor/precursors reside. Lack of Id1 increases beige gene and Ucp1 expression in the WAT in response to cold exposure. Furthermore, brown-like differentiation is increased in Id1-deficient mouse embryonic fibroblasts. At the molecular level, Id1 directly interacts with and suppresses Ebf2 transcriptional activity, leading to reduced expression of Prdm16, which determines beige/brown adipocyte cell fate. Overall, the study highlights the existence of novel regulatory mechanisms between Id1/PGC1α and Id1/Ebf2 in controlling brown fat metabolism, which has significant implications in the treatment of obesity and its associated diseases, such as diabetes.

Florigen-Encoding Genes of Day-Neutral and Photoperiod-Sensitive Maize Are Regulated by Different Chromatin Modifications at the Floral Transition.

The activity of the maize (Zea mays) florigen gene ZEA CENTRORADIALIS8 (ZCN8) is associated with the floral transition in both day-neutral temperate maize and short-day (SD)-requiring tropical maize. We analyzed transcription and chromatin modifications at the ZCN8 locus and its nearly identical paralog ZCN7 during the floral transition. This analysis was performed with day-neutral maize (Zea mays ssp. mays), where flowering is promoted almost exclusively via the autonomous pathway through the activity of the regulatory gene indeterminate1 (id1), and tropical teosinte (Zea mays ssp. parviglumis) under floral inductive and noninductive photoperiods. Comparison of ZCN7/ZCN8 histone modification profiles in immature leaves of nonflowering id1 mutants and teosinte grown under floral inhibitory photoperiods reveals that both id1 floral inductive activity and SD-mediated induction result in histone modification patterns that are compatible with the formation of transcriptionally competent chromatin environments. Specific histone modifications are maintained during leaf development and may represent a chromatin signature that favors the production of processed ZCN7/ZCN8 messenger RNA in florigen-producing mature leaf. However, whereas id1 function promotes histone H3 hyperacetylation, SD induction is associated with increased histone H3 dimethylation and trimethylation at lysine-4. In addition, id1 and SD differently affect the production of ZCN7/ZCN8 antisense transcript. These observations suggest that distinct mechanisms distinguish florigen regulation in response to autonomous and photoperiod pathways. Finally, the identical expression and histone modification profiles of ZCN7 and ZCN8 in response to floral induction suggest that ZCN7 may represent a second maize florigen.

The helix-loop-helix protein id1 controls stem cell proliferation during regenerative neurogenesis in the adult zebrafish telencephalon.

The teleost brain has the remarkable ability to generate new neurons and to repair injuries during adult life stages. Maintaining life-long neurogenesis requires careful management of neural stem cell pools. In a genome-wide expression screen for transcription regulators, the id1 gene, encoding a negative regulator of E-proteins, was found to be upregulated in response to injury. id1 expression was mapped to quiescent type I neural stem cells in the adult telencephalic stem cell niche. Gain and loss of id1 function in vivo demonstrated that Id1 promotes stem cell quiescence. The increased id1 expression observed in neural stem cells in response to injury appeared independent of inflammatory signals, suggesting multiple antagonistic pathways in the regulation of reactive neurogenesis. Together, we propose that Id1 acts to maintain the neural stem cell pool by counteracting neurogenesis-promoting signals.

Differential regulation of Id1 expression by TGF-β1 in HepG2 cells

Introduction: In early stages of hepatocellular carcinoma (HCC) TGF-β is proposed to have a tumour suppressive function whereas in advanced HCC it acts as a tumor promoter [1]. However, the relevance of a potential TGF-β1 target gene, i.e. Id1, is discussed controversial during HCC development and progression [2 – 4]. In HCC cell lines expressing various types of TGF-β receptors and being growth inhibited by TGF-β1, Id1 is suppressed by this ligand [5]. In order to refine the response pattern to TGF-β1 and identify putative regulatory circuits in HCC cells, we analysed the gene activation pattern in the TGF-β responsive HepG2 cell line. Methods and results: The time dependent mRNA expression in response to TGF-β1 was analysed by microarray analysis. 166 candidate genes were found to be differentially regulated during the time course. These were grouped in five clusters according to their expression kinetics. Candidate genes were validated by qRT-PCR and the corresponding protein expression and activation of signalling intermediates was confirmed by Western blot analysis. Most genes were localized in the immediate early cluster including pathway inhibitors, i.e. Smad6, Smad7, Skil, and Id1. The latter showed a biphasic expression, being immediate early induced (2 hrs) and thereafter repressed (4 hrs), an expression pattern that was also found in hepatic stellate cells. The function of Id1 was elucidated by transient overexpression in HepG2 and found to influence Connective Tissue Growth Factor protein expression and Smad activation. Induction and repression were direct responses since cycloheximide failed to abrogate the observed effects on Id1 expression. In contrast the ALK5 inhibitor SB-431542 partially blocked induction and repression of Id1. Conclusion: Id1 is an important target gene of TGF-β1-signalling in hepatoma cells which are sensitive to TGF-β1 and express ALK5 since this receptor is crucial for the complex regulation of this target gene.

ID1 regulates U87 human cell proliferation and invasion

Despite therapeutic advances, the prognosis of patients diagnosed with malignant glioma has not improved in recent years. In particular, the molecular mechanisms that mediate glioma invasion remain poorly understood. The importance of ID1 in promoting tumor invasion and metastasis has recently emerged and a role for ID1 as a possible molecular marker of tumor aggressiveness has been proposed. To investigate the biological function of ID1 in glioblastomas, ID1-silenced U87 glioblastoma multiforme (GBM) cells were constructed using a small hairpin RNA (shRNA) sequence. The effect of the knockdown of ID1 on proliferation and invasion in these cells was analyzed using the 5-bromo-2′-deoxy-uridine cell proliferation, Transwell invasion, scratch and cell adhesion assays. Compared with the controls, the U87 cells expressing ID1-shRNA exhibited a significantly decreased proliferation and invasion capacity (P<0.05), as well as increased cell adhesion. Furthermore, silencing ID1 reduced the expression of c-Myc, cyclin D1 and β-catenin, while increasing E-cadherin expression in U87 cells. This study showed that ID1 regulates the metastatic potential of GBM cells by controlling the epithelial-mesenchymal transition. Therefore, ID1 is a potential prognostic indicator and therapeutic target in glioblastomas.

Effect of Id1 knockdown on formation of osteolytic bone lesions by prostate cancer PC3 cells in vivo

The formation of osteolytic bone lesions is a key process for osteolytic cancer to metastasize to the bone and is under the control of a set of transcription factors. Recently, the inhibitor of differentiation 1 (Id1) has been linked with angiogenesis, tumorigenesis, metastasis and bone formation. However, the function of Id1 during the process of bone destruction caused by cancer in vivo has not yet been elucidated. We, therefore, examined whether and how Id1 affects the ability of cancer to form osteolytic lesion in vivo. The study used a lentiviral vector overexpressing short hairpin RNA (shRNA) targeting Id1 gene. PC3 cells, a prostate cancer cell line, were transduced with Id1 shRNA or negative control (NC) shRNA before implantation in BALB/c mice. Cells were implanted in a tibial injection model. Tumor formation in bone was monitored by X-ray. The relationship between parathyroid hormone-related protein (PTHrP), an osteolytic factor, and Id1 was analyzed by using immunohistochemistry in tissue sections from osteolytic lesion of the BALB/c mice. Our results showed that Id1 shRNA delivery to PC3 cells by lentivirus caused efficient and stable Id1 gene silencing. In the intratibial model, PC3 cells produced primarily osteolytic lesions in the bone. Eleven of 14 mice in Id1 shRNA group but only 4 of 14 mice in the NC shRNA group developed osteolytic lesions with cortical destruction at 4th week. Mice treated with Id1 shRNA had larger tumor volume in the bone and larger cortical destruction. The expression of PTHrP protein in PC3 cells was not affected by Id1 knockdown in vivo. These results indicate that Id1 may down-regulate the ability of PC3 cells to form osteolytic lesions in vivo and the signal pathway needs to be further investigated.

Inhibitor of DNA binding 1 (Id1) induces differentiation and proliferation of mouse embryonic carcinoma P19CL6 cells

The inhibitor of DNA binding (Id) family of genes encodes negative regulators of basic helix–loop–helix transcription factors and has been implicated in such diverse cellular processes as differentiation, proliferation, apoptosis and migration. Id knockout mouse embryos display multiple cardiac defects but the specific role of Id1 in cardiac differentiation is unclear. In the present study, we investigated the function of Id1 in DMSO-induced P19CL6 cells, a widely-accepted cell model of cardiac differentiation.We found that Id1 was upregulated during the cardiac differentiation of P19CL6 cells. The expression of cardiac specific marker genes, Gata4, α-MHC and ISL1, was upregulated in P19CL6 cells stably transfected with Id1 (P19CL6-Id1) during cardiac differentiation. The overexpression of Id1 reduced the number of cells in G1 phase and increased the cell population in G2, M and S phases, while knockdown of Id1 increased the number of cells in G1 phase from 48.6±2.51% to 62.2±1.52% at day 0 of cardiac induction, and from 52.5±3.41% to 63.7±1.02% at day 3 after cardiac induction, indicating that Id1 promoted proliferation of P19CL6 cells. Luciferase assays showed that the activity of TOP flash was higher in P19CL6-Id1 cells than wildtype P19CL6 cells, while Id1 expression was also upregulated in P19CL6 cells treated with Wnt3a or LiCl. This indicates that there may be positive feedback between Id1 and Wnt signaling which plays an important role in cardiac differentiation.

Id1 induces tubulogenesis by regulating endothelial cell adhesion and cytoskeletal organization through β1-integrin and Rho-kinase signalling

The inhibitor of differentiation 1 (Id1) protein is required for tubulogenesis, but the molecular signalling pathways remain unclear. Overexpression (Id1-t) or down-regulation (si-Id1) of Id-1 in cell lines, were used to study the function of Id1. The expression of Id1 and β1-integrin was assessed by Western blotting. Up-regulation of Id1 in human umbilical vascular endothelial cells (HUVECs) activated the expression of β1-integrin and promoted cell adhesion and spreading. Conversely, down-regulation of Id1 suppressed β1-integrin expression and inhibited tubulogenesis. By using a β1-integrin antibody to inhibit β1-integrin function, we demonstrated that Id1-induced cell adhesion and tubulogenesis were mediated by β1-integrin. In addition, HUVECs overexpressing Id1 were able to promote capillary tube formation through cytoskeleton reorganization and cell contraction. Finally, the Rho-kinase inhibitor Y27632 inhibited tubulo-genesis induced by Id1. Our findings provide evidence that Id1 regulates tubulogenesis in vitro through β1-integrin and Rho-kinase signalling.

Id1-induced inhibition of p53 facilitates endothelial cell migration and tube formation by regulating the expression of beta1-integrin

The Id1 protein is critical for endothelial cell angiogenesis, and this function is particularly relevant to cancer development, cardiovascular disease, and wound healing. We hypothesized that Id1 enhanced migration and tubulogenesis by controlling the expression and function of p53. In this study, we examined cell migration following Id1 overexpression and silencing endothelial cells. The results showed that overexpression of Id1 enhanced cell migration and increased beta1-integrin expression, but inhibition of beta1-integrin blocked motility even in clones overexpressing Id1, suggesting that Id1 regulated motility through beta1-integrin. Further analysis revealed that p53, whose expression and distribution is regulated by Id1, was critical for cell migration, and may be involved in regulating the expression of beta1-integrin. Inhibiting p53 function using PFT-α, a functional inhibitor of p53, increased the expression of beta1-integrin and promoted cell migration even in Id1-silencing endothelial cells, demonstrating that the Id1 knockdowns induced inhibition of endothelial cell migration and the expression of beta1-integrin were controlled by p53. In addition, Id1-p53 pathway regulated the cytoskeleton formation and tubulogenesis. These results demonstrate that Id1-induced beta1-integrin expression in endothelial cells and the function of Id1 in cell migration and tubulogenesis are dependent on p53.

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 4994: Downstream targets of Id1 transcription factor in non-small cell lung cancer

Abstract Id1 (Inhibitor of DNA binding/Differentiation) is a helix loop helix (HLH) protein, regulates transcription by binding to bHLH transcription factors and preventing their binding to the DNA. Id1 is especially known to mediate repression of E box proteins and Ets transcription factors. Id1 gene is overexpressed in lung, breast, pancreatic and prostate cancer and it is thought to promote the progression and metastasis of these tumors. At the same time, not much is known about its role in non-small cell lung carcinoma.Preliminary studies from our lab shows that Nicotine as well as EGF induces Id1 in both adenocarcinoma and squamous carcinoma cells and is notably upregulated in metastatic lung cancers. Depletion of Id1 also prevents the proliferation and invasion of NSCLS cells. Attempts were made to understand the downstream effectors of Id1 function in lung cancer progression and metastasis. As a step in this direction, A549 (K-Ras mutant, EGFR wild-type) and H1650 (K-Ras wild-type, EGFR mutant) cells were transfected with short interfering RNA (siRNA) for Id1and stimulated with 1μm Nicotine or 100ng/ml EGF. The samples were subjected to microarray analysis to identify various genes upregulated and downregulated in the Id1 siRNA transfected cell lines. 200 genes were upregulated 2 fold or more by Nicotine and 150 by EGF. Few genes such as stathmin like-3 (STMN3), GSPT1 (G1 to S phase transition), TPD52 (tumor protein D52) were downregulated in the absence of Id1 suggesting those are Id1 regulated genes. The microarray results were validated using Real-time PCR for the above genes. Further depletion of these three genes resulted in decreased cell proliferation, migration and invasion. Indeed STMN3 promoter could be induced by Id1 and Id1 was necessary for Nicotine to induce this gene. These results might reveal the mechanisms by which Id1 promotes tumor progression and metastasis. STMN3 is a microtubule destabilizing phosphoprotein which is overexpressed in adenocarcinoma as well as squamous cell carcinoma and induced tumor cell proliferation, migration and matrix invasion in respective cell lines. GSPT1, which is also known as eukaryotic translation release factor eRF3 and TPD52 are also reported to be over expressed in various kinds of cancer. 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 4994. doi:10.1158/1538-7445.AM2011-4994

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

Identification of a novel function of Id-1 in mediating the anticancer responses of SAMC, a water-soluble garlic derivative, in human bladder cancer cells

Studies have shown that the expression of inhibitor of differentiation (Id-1) is increased in bladder cancer and is associated with drug resistance. S-allylmercaptocysteine (SAMC), a water-soluble component of garlic, is known to have a potent therapeutic effect on human cancer. The aim of this study was to investigate whether Id-1 expression mediates SAMC-induced cell death in bladder cancer cells. After generating stable Id-1-expressing and si-Id-1 transfectants in various bladder cancer cell lines, cell sensitivity to SAMC was compared by colony formation and MTT assays. The results indicated that Id-1 overexpression reduced the positive effect of SAMC on cell survival, while the inactivation of Id-1 increased cellular susceptibility to SAMC. Using DAPI staining, the apoptosis of bladder cancer cells induced by SAMC was shown to be negatively regulated by Id-1 expression. The expression of apoptosis-related proteins analyzed by Western blotting further supported the negative role of Id-1 in SAMC-induced apoptosis. Furthermore, by wound closure and type I collagen invasion assays, the inhibitory effect of SAMC on the invasion and migration of bladder cancer cells was found to be associated with the down-regulation of Id-1. Our results demonstrated that SAMC-induced apoptosis is associated with the Id-1 pathway, and that the inactivation of Id-1 enhances the ability of SAMC to inhibit the survival, invasion and migration of bladder cancer cells. These findings may lead to the development of novel therapeutic strategies for the treatment of bladder cancer.