Increased Id-1 Expression Research Articles

CSIG-09. THERAPEUTIC TARGETING OF PRENATAL PONTINE ID1 SIGNALING IN DIFFUSE MIDLINE GLIOMA

Abstract Diffuse midline gliomas (DMG) are highly invasive brain tumors with rare survival beyond two years past diagnosis. The mechanism behind tumor invasion is currently not well understood. Previous reports demonstrate upregulation of the protein ID1 with H3K27M and ACVR1 mutations in DMG, but this has not been confirmed in human tumors or therapeutically targeted. Whole exome, RNA, and ChIP-sequencing were performed on the ID1 locus in DMG tissue. Scratch-assay migration and transwell invasion assays of cultured cells were performed following shRNA-mediated ID1-knockdown. In vitro and in vivo genetic and pharmacologic [cannabidiol (CBD)] inhibition of ID1 on DMG tumor growth was assessed. Additional in vitro experiments were performed to determine a potential mechanism of action for CBD-mediated effects. Self-reported CBD dosing information was collected from DMG patients. We found that increased ID1 expression in human DMG and in utero electroporation (IUE) murine tumors is associated with H3K27M mutation and brainstem location. ChIP-sequencing indicates a similar epigenetically active state at ID1 regulatory regions in human H3K27M-DMG tumors and prenatal pontine cells. Higher ID1-expressing astrocyte-like DMG cells share a transcriptional program with oligo/astrocyte-precursor cells (OAPCs) from the developing human brain and demonstrate upregulation of the migration regulatory protein SPARCL1. Genetic and pharmacologic (CBD) suppression of ID1 decreases tumor cell migration, tumor growth, and to a lesser extent invasion in both murine IUE and multiple patient-derived in vivo DMG models, improving mouse survival. ID1 knockdown significantly decreases the effect of CBD on migration, tumor growth, and invasion. CBD increases reactive oxygen species production, which also affects DMG cell proliferation in a non-ID1 mediated manner. Overall, we find that H3K27M-mediated reactivation of ID1 in DMG results in a SPARCL1+ migratory transcriptional program that is therapeutically targetable with CBD.

Therapeutic targeting of prenatal pontine ID1 signaling in diffuse midline glioma.

Diffuse midline gliomas (DMG) are highly invasive brain tumors with rare survival beyond two years past diagnosis and limited understanding of the mechanism behind tumor invasion. Previous reports demonstrate upregulation of the protein ID1 with H3K27M and ACVR1 mutations in DMG, but this has not been confirmed in human tumors or therapeutically targeted. Whole exome, RNA, and ChIP-sequencing was performed on the ID1 locus in DMG tissue. Scratch-assay migration and transwell invasion assays of cultured cells were performed following shRNA-mediated ID1-knockdown. In vitro and in vivo genetic and pharmacologic [cannabidiol (CBD)] inhibition of ID1 on DMG tumor growth was assessed. Patient-reported CBD dosing information was collected. Increased ID1 expression in human DMG and in utero electroporation (IUE) murine tumors is associated with H3K27M mutation and brainstem location. ChIP-sequencing indicates ID1 regulatory regions are epigenetically active in human H3K27M-DMG tumors and prenatal pontine cells. Higher ID1-expressing astrocyte-like DMG cells share a transcriptional program with oligo/astrocyte-precursor cells (OAPCs) from the developing human brain and demonstrate upregulation of the migration regulatory protein SPARCL1. Genetic and pharmacologic (CBD) suppression of ID1 decreases tumor cell invasion/migration and tumor growth in H3.3/H3.1K27M PPK-IUE and human DIPGXIIIP* in vivo models of pHGG. The effect of CBD on cell proliferation appears to be non-ID1 mediated. Finally, we collected patient-reported CBD treatment data, finding that a clinical trial to standardize dosing may be beneficial. H3K27M-mediated re-activation of ID1 in DMG results in a SPARCL1+ migratory transcriptional program that is therapeutically targetable with CBD.

TAMI-79. THERAPEUTIC REVERSAL OF PRENATAL PONTINE ID1 SIGNALING IN DIPG

Abstract Diffuse intrinsic pontine glioma (DIPG) is a highly aggressive pediatric brain tumor with rare survival beyond two years. This poor prognosis is largely due to the tumor's highly infiltrative and invasive nature. Nearly 80% of DMGs harbor K27M mutation in the genes encoding histone H3.1 (H3F3A) or H3.3 (HISTIH3B), often with concurrent ACVR1 mutation. Inhibitor of DNA-binding (ID) proteins are key transcriptional regulators of genes involved in lineage commitment and are associated with invasiveness and poor clinical outcomes in multiple human cancers. Introduction of H3K27M and ACVR1 mutations increase ID1 expression in cultured astrocytes, but this has not been confirmed in human tumors or targeted therapeutically. We developed an in-utero electroporation (IUE) murine H3K27M-driven tumor model, which demonstrates increased ID1 expression in H3K27M- and ACVR1-mutated tumor cells. Exome and transcriptome sequencing analysis of multi-focal DMG tumors (n=52) and normal brain tissue revealed that increased ID1 expression is associated with H3K27M/ACVR1-mutation and brainstem location, and correlates with poor survival in patients. ChIP-sequencing for H3K27ac and H3K27me3 in multiple DMG tumors (n=5) revealed that the ID1 gene is epigenetically active, which matches the epigenetic state of murine prenatal hindbrain cells. Higher ID1-expressing astrocyte-like DIPG cells share a similar transcriptional program with ID1+/SPARCL1+ positive oligo/astrocyte-precursor (OAPC) cells from the developing human brain and demonstrate upregulation of gene sets involved in regulation of cell migration. Both genetic and pharmacologic [cannabidiol (CBD)] suppression of ID1 result in decreased DIPG cell invasion/migration in vitro and invasion/tumor growth in multiple in vivo models. Mechanistically, CBD reduces proliferation through production of reactive oxygen species. Further, DIPG patients treated off-trial with CBD (n=15) displayed reduced ID1 tumor expression and improved overall survival. In summary, ID1 is upregulated in DIPG through K27M-mediated epigenetic reactivation of a developmental OAPC-like transcriptional state, and ID1-driven invasiveness of DIPG is therapeutically targetable with CBD.

Abstract C15: Neutrophil elastase (NE) induces epithelial-mesenchymal transition (EMT) via upregulation of inhibitor of DNA binding 1 (ID1)

Abstract Neutrophil elastase is a neutrophil-derived serine protease with broad substrate specificity. We have previously shown that NE promotes lung tumor growth in vitro and in vivo and that NE is capable of entering tumor cell endosomes in a clathrin-dependent fashion. Epithelial-mesenchymal transition (EMT) is a fundamental event for primary tumors to metastasize to distant sites. Altered gene and protein expression causes changes in cell morphology and behavior resulting in the loss of attachment to neighboring cells, intravasation, and migration into distant tissue. However, the entities residing within the tumor microenvironment that drive EMT are poorly understood. We identified NE, expressed and released by tumor-associated neutrophils, as such an entity. We found that NE induces EMT-like morphology and behavior in several cancer cell lines as well as upregulates genes (Snail and Twist) and proteins (N-cadherin, α-SMA, Fibronectin and Vimentin) associated with EMT. Furthermore, NE provokes tumor cell migration and invasion in vitro and is required for metastasis formation in vivo. Besides known EMT markers we identified significant upregulation of inhibitor of DNA binding 1 (ID1) in tumor cells in the presence of NE. Increased ID1 expression was previously shown to correlate with tumor progression and metastasis. Clathrin-dependent NE internalization is required for both ID1 expression and the EMT-like phenotype. We localized NE in different compartments of tumor cells and found it predominantly in the nuclear fraction; more specifically, NE is located in the chromatin-bound fraction, indicating a potential role in chromatin remodeling and transcriptional regulation. Additionally, the proteolytic activity of NE was measured in the nuclear and chromatin-bound fractions and is required for induction of ID1 expression and EMT-like behavior. Taken together, these data suggest the importance of NE-induced ID1 expression in metastasis formation. Citation Format: Julia Kargl, Alyssa Gregory, Hye Youn Yang, Stephanie Busch, Heather Metz, A. McGarry Houghton. Neutrophil elastase (NE) induces epithelial-mesenchymal transition (EMT) via upregulation of inhibitor of DNA binding 1 (ID1). [abstract]. In: Proceedings of the AACR Special Conference: Function of Tumor Microenvironment in Cancer Progression; 2016 Jan 7–10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2016;76(15 Suppl):Abstract nr C15.

Inhibition of BMP and of TGFβ receptors downregulates expression of XIAP and TAK1 leading to lung cancer cell death.

BackgroundBone morphogenetic proteins (BMP) are embryonic proteins that are part of the transforming growth factor (TGFβ) superfamily, which are aberrantly expressed in many carcinomas. Inhibition of BMP receptors with small molecule inhibitors decreases growth and induces death of lung cancer cells, which involves the downregulation of Id1 and Id3 by a Smad dependent mechanism. Developmentally, BMP and TGFβ signaling utilizes Smad-1/5 independent mechanisms to stabilize the expression of X-linked inhibitor of apoptosis protein (XIAP) and activate TGFβ activated kinase 1 (TAK1), which are known to be potent inhibitors of apoptosis. The role of BMP signaling in regulating XIAP and TAK1 in cancer cells is poorly understood. Furthermore, the interaction between the BMP and TGFβ signaling cascades in regulating the activation of TAK1 in cancer cells has not been elucidated.MethodsFeedback regulation between the BMP and TGFβ signaling pathways and their regulation of XIAP, TAK1, and Id1 were examined in lung cancer cells utilizing siRNA and inhibitors targeting BMP type I receptors, inhibitors of BMP and TGFβ type I receptors, and an inhibitor of BMP and TGFβ type I and type II receptors.ResultsWe show that upon inhibition of BMP signaling in lung cancer cells, the TGFβ signaling cascade is activated. Both the BMP and TGFβ pathways activate TAK1, which then increases the expression of Id1. Inhibition of TGFβ signaling increased Id1 expression except when BMP signaling is suppressed, which then causes a dose-related decrease in the expression of Id1. Inhibition of both BMP and TGFβ signaling enhances the downregulation of TAK1. Our data also suggests that the blockade of the BMP type II receptor enhances the downregulation XIAP, which is important in decreasing the activity of TAK1. Knockdown studies demonstrate that both XIAP and TAK1 regulate the survival of lung cancer cells.ConclusionsThis paper highlights that targeting the BMP and TGFβ type I and type II receptors causes a downregulation of XIAP, TAK1, and Id1 leading to cell death of lung cancer cells. Small molecule inhibitors targeting the BMP and TGFβ receptors represents a potential novel means to treat cancer patients.Electronic supplementary materialThe online version of this article (doi:10.1186/s12943-016-0511-9) contains supplementary material, which is available to authorized users.

Abstract A34: Id-1 mediates glioma stem cell chemoresistance to temozolomide

Abstract Introduction: Id-1 (inhibitor of differentiation and DNA binding) is a transcriptional regulator involved in stem cell maintenance. It has previously been shown to regulate self-renewal and confer chemoresistance in colon cancer stem cells. Id-1 has not been studied within the context of glioblastoma (GBM). We hypothesized that Id-1 mediates glioma stem cell (GSC) survival in response to chemotherapy and subsequently promotes tumour recurrence. Methods: To study the role of Id-1 in response to chemotherapy in vitro, three GSC lines were treated with the chemotherapy agent, temozolomide (TMZ), for seven days. Immunoblot and comparative QT-PCR were used to measure Id-1 protein and mRNA expression post-TMZ, respectively. To evaluate Id-1 expression in patient samples, we constructed a tissue microarray (TMA), which included 77 tissue specimens from patients diagnosed with GBM. Detailed clinical information was available for all of the selected samples, including survival times, treatment regimens, and primary versus recurrent tumor status. Immunohistochemistry was performed on the TMA and Id-1 protein expression was subsequently quantified using Panoramic Image Analysis Software Platform. Id-1 expression was correlated to survival outcome, along with other clinically relevant variables. We also employed a bioinformatics approach, exploring survival data from The Cancer Genome Atlas (TCGA) database that were based on Id-1 expression for patients diagnosed with GBM. Results: Id-1 expression in GSCs strongly increased in response to increasing doses of TMZ in vitro (0 uM, 25 uM, 100 uM). In patient tumor samples (n=77), Id-1 expression did not correlate with survival. However, among the recurrent patients who received chemotherapy after primary tumor resection (n=6), patients with increased Id-1 expression post-chemotherapy had a shorter latency to recurrence compared to patients with decreased Id-1 expression post-chemotherapy. Finally, the TCGA data corroborated our TMA patient data, finding comparable overall survival trends between patients with high versus low Id-1 expression. Conclusion: These findings demonstrate that Id-1 levels increase in response to chemotherapy, suggesting its potential role in chemoresistance. Since resistance to chemotherapeutic drugs is one of the major reasons for treatment failure in glioblastoma, Id-1 inactivation may serve as a novel strategy for enhancing therapy and improving outcomes in patients with the disease. Citation Format: Angela Celebre, Megan YiJun Wu, Marc Remke, Michael Taylor, Jason Karamchandani, Sunit Das. Id-1 mediates glioma stem cell chemoresistance to temozolomide. [abstract]. In: Proceedings of the AACR Special Conference: Advances in Brain Cancer Research; May 27-30, 2015; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2015;75(23 Suppl):Abstract nr A34.

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.

Abstract LB-87: Analysis of JAZF1 loss-of-function reveals its role in regulation of genes relevant for prostate cancer

Abstract Genome-wide association studies (GWAS) have mapped one of the prostate cancer susceptibility regions to a single nucleotide polymorphism (SNP) rs10486567 within intron 2 of the gene that encodes JAZF1, a putative transcriptional co-factor. In order to translate GWAS findings into clinically relevant information that will ultimately enable us to improve the management of prostate cancer it is essential to identify the biological role of the susceptibility variant in cells and tissues. To date, only a few studies address the biological function of JAZF1 and none in prostate cancer. To investigate the function of JAZF1 we generated expression profiles of several prostate cancer cell lines, in which JAZF1 was silenced. We conducted RNA-Seq of two metastatic prostate cancer cell lines, LNCaP and DU145, in which siRNA-mediated RNAi had been employed to induce JAZF1 loss-of-function (LOF). For this study, three different JAZF1 siRNAs were used, along with a siRNA (siNeg) serving as control for transfection-related changes in gene expression. Approximately 1,500 genes were identified as deregulated in both cell lines. Computational analysis of these genes showed enrichment for genes associated with prostate cancer (p=2.4x10-6, Ingenuity Pathway Analysis), including MAP2K4 and MET. These two genes were up-regulated following JAZF1 LOF in both cell lines, consistent with JAZF1's putative role as a transcriptional repressor. Among the genes down-regulated following siRNA silencing of JAZF1was ID1. ID1 is a negative transcriptional regulator that functions as part of the mitotic checkpoint machinery. One of ID1's target genes is the cell-cycle inhibitor p21. In JAZF1-silenced DU145 cells we observed a decrease in ID1 mRNA expression that was correlated with an increase in CDKN1A expression, the gene encoding p21; an observation confirmed at a protein level. Examination of publically available prostate cancer gene expression data indicated a correlation between JAZF1 and ID1 expression (p<0.001). To further analyze the putative regulation of ID1 by JAZF1, and thus p21, we used lentivirus-mediated delivery of JAZF1-shRNAs to stably transduce DU145 cells,, OPCN-1 non-tumorigenic cells, and a non-metastatic prostate tumor epithelium cell line, OPCT-1. Under conditions of long-term JAZF1 LOF we observed increased ID1 expression as measured by Western Blot analysis and concurrent decreases in, p21 protein levels in all three-cell lines. Although this result appears contradictory to our initial observation of siRNA-mediated JAZF1 silencing, this difference may reflect cell line and temporal specific activity of JAZF1 as a transcriptional activator or repressor of ID1 expression. These findings are consistent though with JAZF1 functioning as a regulator of ID1, a protein reported to be over-expressed in prostate cancer that is associated with cancer cell proliferation and metastasis formation through activation of various oncogenic pathways. Citation Format: Helen Gharwan, Tamara Jones, Wei Tang, Stefan Ambs, Ludmilla Prokunina, Natasha Caplen. Analysis of JAZF1 loss-of-function reveals its role in regulation of genes relevant for prostate cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-87. doi:10.1158/1538-7445.AM2014-LB-87

Id Proteins Regulate Capillary Repair and Perivascular Cell Proliferation following Ischemia-Reperfusion Injury

Acute kidney injury (AKI) results in microvascular damage that if not normally repaired, may lead to fibrosis. The Id1 and 3 proteins have a critical role in promoting angiogenesis during development, tumor growth and wound repair by functioning as dominant negative regulators of bHLH transcription factors. The goal of this study was to determine if Id proteins regulate microvascular repair and remodeling and if increased Id1 expression results in decreased capillary loss following AKI. The effect of changes in Id expression in vivo was examined using Id1−/−, Id3RFP/+ (Id1/Id3 KO) and Tek (Tie2)-rtTA, TRE-lacz/TRE Id1 (TRE Id1) mice with doxycycline inducible endothelial Id1 and β-galactosidase expression. Id1 and 3 were co-localized in endothelial cells in normal adult kidneys and protein levels were increased at day 3 following ischemia-reperfusion injury (IRI) and contralateral nephrectomy. Id1/Id3 KO mice had decreased baseline capillary density and pericyte coverage and increased tubular damage following IRI but decreased interstitial cell proliferation and fibrosis compared with WT littermates. No compensatory increase in kidney size occurred in KO mice resulting in increased creatinine compared with WT and TRE Id1 mice. TRE Id1 mice had no capillary rarefaction within 1 week following IRI in comparison with WT littermates. TRE Id1 mice had increased proliferation of PDGFRβ positive interstitial cells and medullary collagen deposition and developed capillary rarefaction and albuminuria at later time points. These differences were associated with increased Angiopoietin 1 (Ang1) and decreased Ang2 expression in TRE Id1 mice. Examination of gene expression in microvascular cells isolated from WT, Id1/Id3 KO and TRE Id1 mice showed increased Ang1 and αSMA in Id1 overexpressing cells and decreased pericyte markers in cells from KO mice. These results suggest that increased Id levels following AKI result in microvascular remodeling associated with increased fibrosis.

A novel role of Id-1 in regulation of epithelial-to-mesenchymal transition in bladder cancer

Background and objectiveInhibitor of differentiation or DNA binding -1 (Id-1) has been shown to be increased in several types of advanced cancer, and to be associated with aggressive and metastatic abilities of cancer cells. Recently, more and more evidence indicates that epithelial-to-mesenchymal transition (EMT) is an important mechanism taking place during tumor invasion and metastasis, but the molecular pathways underlying EMT have not been clearly established. This study was to investigate the expression of Id-1 in bladder cancer and its association with EMT. Materials and methodsA total of 169 tissues, consisting of 147 primary bladder cancers and 22 adjacent normal tissues were included in this study. Id-1, E-cadherin, and β-catenin were examined immunohistochemically in paraffin sections. The pBabe-Id-1 expression retroviral vector and retroviral vectors containing an Id-1-specific small interfering RNA oligonucleotides (si-Id-1) were transfected into 2 bladder cancer cell lines respectively. Then, we used Western blotting and immunofluorescent staining to detect the cellular expression of epithelial markers and mesenchymal markers. The invasion and migration ability of bladder cancer cells were identified by type I collagen invasion assay and wound closure assay. ResultsWe demonstrated that increased Id-1 expression was associated with advanced tumor stage and grade. In addition, the increased Id-1 expression in bladder tumors was also correlated with decreased membranous E-cadherin and β-catenin expression. In vitro, studies showed that inactivation of the Id-1 gene conferred morphologic transition of bladder cancer cells from a fibroblastic to epithelial appearance, and overexpression of Id-1 could lead to acquisition of a fibroblastic spindle cell phenotype accompanied by loss of cell-to-cell contacts. By Western blotting and immunofluorescent staining, we showed that the expression level of Id-1 was correlated with the expression of mesenchymal markers but was inversely correlated with the expression of epithelial markers. Moreover, results of collagen invasion and wound closure assays showed ectopic Id-1 expression led to increased ability of invasion and migration. ConclusionsOur results suggest that Id-1 may play roles in tumor progression and EMT activation in bladder cancer.

Abstract P4-06-05: Silencing of Cyclin D1 Increases Breast Cancer Cell Migration through Increased Id1 Expression and Induction of Epithelial-Mesenchymal Transition

Abstract Backround: The oncogene cyclin D1 is overexpressed in many breast cancers and despite proliferation activating properties, has been linked to a less malignant phenotype. We have previously shown that down-regulation of cyclin D1 in actively cycling breast cancer cells significantly increased their migratory capacity whilst also decreasing proliferation. Gene expression analysis and animal modeling have highlighted an increase in the inhibitor of differentiation-1 (Id1) gene following cyclin D1 gene (CCND1) silencing. Notably, increased Id1 is associated not only with enhanced migratory and invasive features of breast cancer cells but also with prevention of cell differentiation and induction of an epithelial-mesenchymal-like (EMT) phenotype. Here we sought to determine if the increase in cell motility following cyclin D1 silencing was through increased Id1 expression and induction of an EMT phenotype, and confirm our findings in a large patient cohort. Materials and Methods: Expression of cyclin D1, Id1 and SNAI2 was determined in MDA-MB-231 and ZR75 cells by qPCR and western blot, whilst cell migration was monitored by transwell assay. Gene expression of CCND1, ID1 and EMT markers was analysed from a pooled cohort of batch-mean centered publically available data sets, with data analysis performed in SPSS. Results: Silencing of cyclin D1 increased cell migration 1.38 and 1.72 fold in MDA-MB-231 (P<0.001), and ZR75 (P<0.001) cells respectively, an effect abolished by silencing of Id1. qPCR analysis confirmed an increase in Id1 (1.73) and SNAI2 (1.50) expression following siRNA treatment of cyclin D1, an effect reversed following siRNA treatment of Id1. CCND1 gene exression was negatively, while Id1 was positively, associated with the EMT markers Vimentin (P<0.001), SNAI2 (P<0.001) and TWIST1 (P<0.001) in 1,100 breast tumors. In ER+ patients CCND1 was negatively correlated to Id1 expression (0.050) and in CCND1 low patients increasing Id1 expression was predictive of worse cumulative survival (0.027). Conclusion: Decreased CCND1 expression enhances MDA-MB-231 and ZR75 cell migration through increased Id1 expression and induction of EMT. Moreover, both genes are correlated to EMT markers in breast cancer patients and in a subset of the cohort, confer a worse survival. These results may explain the conflicting data regarding cyclin D1 expression in breast carcinomas and solidifies the rationale of the protein as a clinical target. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P4-06-05.

Id-1 expression in androgen-dependent prostate cancer is negatively regulated by androgen through androgen receptor

This study discovered that Id-1 expression in androgen-dependent prostate cancer decreased immediately after androgen deprivation but increased after longer androgen deprivation both in vivo and in vitro. Id-1 expression in androgen-independent LNCaP cells was about 6 fold as that in their parental cells. As was the case with LNCaP cells, when androgen receptor (AR) was introduced into AR-negative PC-3 cells, dihydrotestosterone inhibited while flutamide increased Id-1 expression. Thus, Id-1 expression in androgen-dependent prostate cancer was negatively regulated by androgen in a receptor-dependent way. The re-increased Id-1 might partially contribute to the emergence of androgen-independent prostate cancer after longer androgen deprivation therapy.

Increased Id-1 expression is significantly associated with poor survival of patients with prostate cancer

The levels of Id-1 (inhibitor of DNA binding or inhibitor of cell differentiation) expression in a series of prostate cell lines and in an archival set of prostate tissues were examined. Western blot analysis showed that the level of Id-1 expressed in the androgen sensitive cell line LNCaP was 1.2 +/- 0.2 times that detected in the benign cell line PNT-2. The level of Id-1 increased further to 1.8 +/- 0.2 and 2.9 +/- 0.3 in the androgen-insensitive cell lines Du-145 and PC-3, respectively. Immunohistochemical staining with Id-1 antibody performed on 113 cases of prostate tissues showed that among the 7 normal cases, 6 (86%) stained either negative or weakly positive whereas only 1 (14%) stained moderately positive. Among the 36 benign prostatic hyperplasia (BPH) samples, 34 (94%) stained either negative or weakly positive; only 1 (3%) stained moderately and 1 (3%) stained strongly. Of the 70 carcinomas, 8 (11.5%) stained weakly, 34 (48.5%) stained moderately, and 28 (40%) stained strongly positive. The intensity of Id-1 staining in carcinomas was significantly stronger than that detected in the normal prostate and BPH (chi(2) test, P < .001) and it was significantly increased as the increasing malignancy of carcinomas measured by Gleason score (chi(2) test, P < .001). The intensity of Id-1 staining was partially associated with the levels of prostate-specific antigen, but not related to the level of androgen receptor. Kaplan-Meier survival curve analysis showed that, similar to Gleason scores, overexpression of Id-1 was significantly associated with the reduced length of patient survival (log-rank test, P = .01). These results suggest that Id-1 is a useful prognostic marker to predict the outcomes of patients with prostate cancer.

Inactivation of Id‐1 in prostate cancer cells: A potential therapeutic target in inducing chemosensitization to taxol through activation of JNK pathway

Resistance to anticancer drugs is the major problem in the treatment of many advanced cancers, including androgen-independent prostate cancer. Recently, increased expression of Id-1, a basic helix-loop-helix protein, is reported in several types of advanced cancer. It is suggested that high expression of Id-1 may provide an advantage for cancer cell survival and inactivation of Id-1 may be able to increase cancer cells' susceptibility to apoptosis. To test this hypothesis, in this study, by using RNA interfering technology, we inactivated the Id-1 gene in 2 androgen-independent prostate cancer cell lines, DU145 and PC3, and investigated whether downregulation of Id-1 could lead to increased sensitivity to a commonly used anticancer drug, taxol. By using colony forming assay and MTT assay, we found that inactivation of Id-1 resulted in both decreased colony forming ability and cell viability in prostate cancer cells, after taxol treatment. In addition, the si-Id-1-induced sensitization to taxol was associated with activation of apoptosis pathway, which is demonstrated by increased apoptotic index, DNA laddering, sub-G1 phase of the cell cycle, as well as cleaved-PARP and Caspase 3. Furthermore, c-Jun N-terminal kinase (JNK), one of the common pathways responsible for taxol-induced apoptosis, was also activated in the si-Id-1 transfected cells. Inhibition of JNK activity by a specific inhibitor, SP600125, blocked the si-Id-1-induced sensitivity to taxol. These results indicate that increased Id-1 expression in prostate cancer cells may play a protective role against apoptosis, and downregulation of Id-1 may be a potential target to increase sensitivity of taxol-induced apoptosis in prostate cancer cells.

MODULATION OF MITOGEN-ACTIVATED PROTEIN KINASE CASCADES BY DIFFERENTIATION-1 PROTEIN: ACQUIRED DRUG RESISTANCE OF HORMONE INDEPENDENT PROSTATE CANCER CELLS

The inhibitor of differentiation-1 protein (Id-1) is over expressed in multidrug resistance prostate cancer cells. We determined the effect of Id-1 expression and its underlying pathways on the development of multidrug resistance in prostate cancer. AT3 cells were transfected with the Id-1 gene or a blank vector. Id-1 mRNA expression was determined by reverse transcriptase-polymerase chain reaction and Id-1 protein content was detected by immunoblot and flow cytometry. Cellular cytotoxicity was determined by MTT (microculture 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay (Sigma Chemical Co., St. Louis, Missouri). The activation and expression of mitogen-activated protein kinase (MAPK) were measured by transactivation assay and Western blotting, respectively. Id-1 overproduction drove AT3 cells to become resistant to chemotherapeutic agents but did not induce mdr-1 gene expression. The p38MAPK and c-jun N-terminal kinase (JNK) pathways were suppressed, which correlated with increased Id-1 expression. No significant change in extracellular signal-regulated kinase (ERK) activation was observed in Id-1 transfectants compared with that of AT3 or vector control. Treatment of Id-1 expressing cells with p38MAPK and JNK inhibitors resulted in decreased doxorubicin induced apoptosis. In contrast, Id-1 expressing cells treated with ERK inhibitor made cells more sensitive to drug induced apoptosis. Up-regulation of Id-1 was found in prostate cancer multidrug resistant cells. Sustained ERK activation, and JNK and p38MAPK inhibition by Id-1 in cells may confer drug resistance. These changes in MAPKs could be a mechanism for the acquisition of multidrug resistance in prostate cancer.

Overexpressed Id‐1 is associated with a high risk of hepatocellular carcinoma development in patients with cirrhosis without transcriptional repression of p16

Inhibitor of differentiation/DNA binding protein 1 (Id-1) plays a pivotal role in the regulation of cell proliferation and carcinogenesis via inhibiting basic helix-loop-helix (HLH) transcription factors. Recently, Id-1 was found to repress p16 in tumorous tissue specimens including hepatocellular carcinoma (HCC), but its relevance in precancerous liver tissues is unknown. Id-1 expression in the liver tissue specimens of 112 patients with cirrhosis without HCC was studied by immunohistochemical analysis. Correlations were investigated between Id-1 expression and clinicopathologic features, the status of p16, and the risk of HCC occurrence. A high expression of Id-1 was observed in 42 patients (38%). The level of Id-1 expression was not associated with clinical standard parameters or the status of p16 in cirrhotic tissue specimens. The cumulative incidence of HCC development was significantly higher in a group of patients with high Id-1 expression (P = 0.0008). Multivariate analysis revealed that increased Id-1 expression is an independent significant factor for the risk of HCC development in patients with cirrhosis (relative risk = 2.75, P = 0.003). The results of the current study suggested that increased expression of Id-1 may play an important role in the early step of hepatocarcinogenesis, and might serve as a useful marker for determining patients with cirrhosis with a high risk of HCC occurrence.

Evidence of increased Id-1 expression and its role in cell proliferation in nasopharyngeal carcinoma cells.

Inhibitor of differentiation or DNA binding (Id-1), a helix-loop-helix transcription factor, has recently been shown to inactivate the retinoblastoma (RB)/p16(INK4a) pathway through down-regulation of p16(INK4a) and increasing phosphorylation of RB in certain cell types. Nasopharyngeal carcinoma (NPC) is a common cancer in Hong Kong, and inactivation of the tumor suppressor RB at transcription level is a rare event in NPC. The objective of this study was to investigate the role of Id-1 in NPC cell proliferation and its expression in NPC samples. An NPC cell line, CNE1, was transfected with a retroviral vector containing a full-length Id-1 cDNA, and six stable transfectant clones were isolated with differential Id-1 expression levels. The effect of ectopic Id-1 expression on serum-independent cell growth, cell-cycle distribution, and expression of proteins associated with RB pathway was studied. The Id-1 expression in five NPC samples was also investigated using immunohistochemistry. Ectopic Id-1 expression in CNE1 cells resulted in an increase in serum-independent cell growth, percentage of cells in S phase, and phosphorylation of RB and cyclin-dependent kinase 2 proteins. In addition, immunohistochemical studies on NPC samples showed that expression of Id-1 was present in NPC cells but absent in normal tissues. This study demonstrates that Id-1 plays an important role in cell proliferation in NPC cells, and our results provide evidence for the first time of the significance of Id-1 expression in NPC cells and suggest a possible role of Id-1 expression in the inactivation of RB and development of NPC.

Id1 expression is associated with histological grade and invasive behavior in endometrial carcinoma

Basic helix–loop–helix (bHLH) DNA-binding proteins have been reported to regulate tissue-specific transcription of cellular differentiation within multiple cell lineages. The Id family of helix–loop–helix proteins does not possess a basic DNA-binding domain and functions as a negative regulator of bHLH proteins by forming high-affinity heterodimers with bHLH proteins. Id proteins were originally characterized as inhibitors of DNA binding and cell differentiation. Thus, overexpression of Id proteins correlates with cell proliferation and arrested differentiation in many cell lineages. To elucidate the involvement of Id1 in endometrial carcinogenesis, we analyzed serial frozen sections for Id1 protein expression in 20 cases of endometrial carcinoma and 20 cases of normal endometria by fluorescent immunohistochemistry. We analyzed the relationship between the percentages of Id1-stained cells and the patient's characteristics, including histological grade, clinical stage, presence of invasion to >1/2 myometrium, and clinical outcome. In normal endometria, Id1 was not detected in either the proliferative or the secretory phase. There was, however, abundant Id1 immunoreactivity in the endometrial carcinoma cells. Moreover, Id1 was strongly expressed in the inflammatory cells. Scoring on the basis of the percentage of positive cells indicated that Id1 expression is significantly associated with histological grade ( P<0.05) and the presence of invasion to >1/2 myometrium ( P<0.05). Our results demonstrate that increased Id1 expression in endometrial carcinoma correlates with the malignant potential of this tumor.

Helix-loop transcription factor, Id1, is overexpressed in medullary thyroid cancer

Abstract Background The helix-loop transcription factor (Id1) protein inhibits differentiation, enhances cell proliferation and is required for cell-cycle progression. Id1 is highly expressed in a variety of tumour-derived cell lines, increases following mitogenic stimuli and is overexpressed in some human neoplasms. It was therefore reasoned that the Id1 gene may play a role in medullary thyroid carcinogenesis. Methods Id1 protein expression in human medullary thyroid cancer (MTC) and corresponding normal thyroid tissue was determined by Id1 immunohistochemistry. Calcitonin immunohistochemistry was used to determine the cellular localization of Id1 immunostaining. In a well characterized human MTC cell line (TT), the effects of growth stimulation (with serum) and redifferentiation (with phorbol esters) on Id1 expression were determined by Northern blot analysis. Results Id1 immunostaining intensity in MTC (n = 9; six sporadic MTCs, two familial MTCs and one multiple endocrine neoplasia type 2A MTC) was moderate to strong whereas in the corresponding normal thyroid tissues (n = 9) it was absent or faint in the C cells and follicular cells. Id1 immunostaining was significantly higher in MTC than the corresponding normal thyroid tissue based on the percentage of positive cells and immunostaining intensity (P = 0·002, rank sum test). The 1·2-kilobase Id1 transcript was expressed in the TT cell line under basal culture conditions. Interestingly, Id1 messenger RNA (mRNA) expression was markedly upregulated (4·0 fold) following growth stimulation with 10 per cent fetal bovine serum. In addition, phorbol ester (which is known to induce redifferentiation of the TT cell line) downregulated Id1 mRNA expression in the TT cell line. Conclusion Id1 is overexpressed in MTC. Furthermore, increased Id1 expression is associated with an enhanced proliferative potential in MTC. The inappropriate proliferation and dedifferentiation of the C cells that lead to MTC, at least in part, may be regulated by the Id1 gene.