Id1 Protein Levels Research Articles

Inhibitor of DNA binding 2 (ID2) regulates the expression of developmental genes and tumorigenesis in ewing sarcoma.

Sarcomas are difficult to treat and the therapy, even when effective, is associated with long-term and life-threatening side effects. In addition, the treatment regimens for many sarcomas, including Ewing sarcoma, rhabdomyosarcoma, and osteosarcoma, are relatively unchanged over the past two decades, indicating a critical lack of progress. Although differentiation-based therapies are used for the treatment of some cancers, the application of this approach to sarcomas has proven challenging. Here, using a CRISPR-mediated gene knockout approach, we show that Inhibitor of DNA Binding 2 (ID2) is a critical regulator of developmental-related genes and tumor growth in vitro and in vivo in Ewing sarcoma tumors. We also identified that homoharringtonine, which is an inhibitor of protein translation and FDA-approved for the treatment of leukemia, decreases the level of the ID2 protein and significantly reduces tumor growth and prolongs mouse survival in an Ewing sarcoma xenograft model. Furthermore, in addition to targeting ID2, homoharringtonine also reduces the protein levels of ID1 and ID3, which are additional members of the ID family of proteins with well-described roles in tumorigenesis, in multiple types of cancer. Overall, these results provide insight into developmental regulation in Ewing sarcoma tumors and identify a novel, therapeutic approach to target the ID family of proteins using an FDA-approved drug.

Low doses of Paclitaxel repress breast cancer invasion through DJ-1/KLF17 signalling pathway.

Paclitaxel (taxol) is an important agent against many tumours, including breast cancer. Ample data documents that paclitaxel inhibits breast cancer metastasis while others prove that paclitaxel enhances breast cancer metastasis. The mechanisms by which paclitaxel exerts its action are not well established. This study focuses on the effect of paclitaxel, particularly the low doses on breast cancer metastasis and the mechanisms that regulate it. Current results show that, paclitaxel exerts significant cytotoxicity even at low doses in both MCF-7 and MDA-MB-231 cells. Interestingly, paclitaxel significantly inhibits cell invasion and migration, decreases Snail and increases E-cadherin mRNA expression levels at the indicated low doses. Furthermore, paclitaxel-inhibiting breast cancer metastasis is associated with down-regulation of DJ-1 and ID-1 mRNA expression level with a concurrent increase in KLF17 expression. Under the same experimental conditions, paclitaxel induces KLF17 and concurrently represses ID-1 protein levels. Our results show for the first time that paclitaxel inhibits breast cancer metastasis through regulating DJ-1/KLF17/ID-1 signalling pathway; repressed DJ-1 and ID-1 and enhanced KLF17 expression.

Changes in the expression of DNA-binding/differentiation protein inhibitors in neurons and glial cells of the gerbil hippocampus following transient global cerebral ischemia.

Inhibitors of DNA-binding/differentiation (ID) proteins bind to basic helix-loop-helix (bHLH) transcription factors, including those that regulate differentiation and cell-cycle progression during development, and regulate gene transcription. However, little is known about the role of ID proteins in the brain under transient cerebral ischemic conditions. In the present study, we examined the effects of ischemia-reperfusion (I-R) injury on the immunoreactivity and protein levels of IDs 1–4 in the gerbil hippocampus proper Cornu Ammonis regions CA1–3 following 5 min of transient cerebral ischemia. Strong ID1 immunoreactivity was detected in the nuclei of pyramidal neurons in the hippocampal CA1–3 regions; immunoreactivity was significantly changed following I-R in the CA1 region, but not in the CA2/3 region. Five days following I-R, ID1 immunoreactivity was not detected in the CA1 pyramidal neurons. ID1 immunoreactivity was detected only in GABAergic interneurons in the ischemic CA1 region. Weak ID4 immunoreactivity was detected in non-pyramidal cells, and immunoreactivity was again only changed in the ischemic CA1 region. Five days following I-R, strong ID4 immunoreactivity was detected in non-pyramidal cells, which were identified as microglia, and not astrocytes, in the ischemic CA1 region. Furthermore, changes in the protein levels of ID1 and ID4 in the ischemic CA1 region studied by western blot were consistent with patterns of immunoreactivity. In summary, these results indicate that immunoreactivity and protein levels of ID1 and ID4 are distinctively altered following transient cerebral ischemia only in the CA1 region, and that the changes in ID1 and ID4 expression may relate to the ischemia-induced delayed neuronal death.

Curcumin Inhibits Expression of Inhibitor of DNA Binding 1 in PC3 Cells and Xenografts

Inhibitor of DNA binding 1 (Id1) plays an important role in genesis and metastatic progression of prostate cancer. We previously reported that down regulation of Id1 by small interfering RNA could inhibit the proliferation of PC3 cells and growth of its xenografted tumors. Curcumin, the active ingredient of turmeric, has shown anti-cancer properties via modulation of a number of different molecular regulators. Here we investigated whether Id1 might be involved in the anti-cancer effects of curcumin in vivo and in vitro. We firstly confirmed that curcumin inhibited cell viability in a dose-dependent fashion, and induced apoptosis in PC3 cells, associated with significant decrease in the mRNA and protein expression of Id1. Similar effects of curcumin were observed in tumors of the PC3 xenografted mouse model with introperitoneal injection of curcumin once a day for one month. Tumor growth in mice was obviously suppressed by curcumin during the period of 24 to 30 days. Both mRNA and protein levels of Id1 were significantly down-regulated in xenografted tumors. Our findings point to a novel molecular pathway for curcumin anti-cancer effects. Curcumin may be used as an Id1 inhibitor to modulate Id1 expression.

Gain of 20q11.21 in human embryonic stem cells improves cell survival by increased expression of Bcl-xL

Gain of 20q11.21 is a chromosomal abnormality that is recurrently found in human pluripotent stem cells and cancers, strongly suggesting that this mutation confers a proliferative or survival advantage to these cells. In this work we studied three human embryonic stem cell (hESC) lines that acquired a gain of 20q11.21 during in vitro culture. The study of the mRNA gene expression levels of the loci located in the common region of duplication showed that HM13, ID1, BCL2L1, KIF3B and the immature form of the micro-RNA miR-1825 were up-regulated in mutant cells. ID1 and BCL2L1 were further studied as potential drivers of the phenotype of hESC with a 20q11.21 gain. We found no increase in the protein levels of ID1, nor the downstream effects expected from over-expression of this gene. On the other hand, hESC with a gain of 20q11.21 had on average a 3-fold increase of Bcl-xL (the anti-apoptotic isoform of BCL2L1) protein levels. The mutant hESC underwent 2- to 3-fold less apoptosis upon loss of cell-to-cell contact and were ∼2-fold more efficient in forming colonies from a single cell. The key role of BCL2L1 in this mutation was further confirmed by transgenic over-expression of BCL2L1 in the wild-type cells, leading to apoptosis-resistant cells, and BCL2L1-knock-down in the mutant hESC, resulting in a restoration of the wild-type phenotype. This resistance to apoptosis supposes a significant advantage for the mutant cells, explaining the high frequency of gains of 20q11.21 in human pluripotent stem cells.

Differential Tumor Expression of Inhibitor of Differentiation-1 in Prostate Cancer Patients With Extreme Clinical Phenotypes and Prognostic Implications

BackgroundIn the prostate-specific antigen era, potentially indolent prostate tumors are radically treated, causing overtreatment. Molecular prognostic factors might differentiate indolent from aggressive tumors, allowing avoidance of unnecessary treatment. Patients and MethodsFifty-two prostate cancer patients (20 organ-confined and 32 metastatic) were selected. All formalin-fixed and paraffin-embedded primary biopsies and matched metastases of 15 of them were evaluated for tumor and endothelial cell Id1 protein expression. Seventy-nine additional patients with organ-confined prostate cancer were selected for Id1 mRNA in silico analysis. ResultsAmong metastatic cancer subjects, 48% of primary tumors and 38% of metastases showed Id1 tumor cell expression, and 79% of primary tumors and 81% of metastases showed endothelial immunoreactivity. In the organ-confined group none of them showed Id1 protein tumor cell expression and 50% displayed endothelial expression. In the metastatic patients group, lower levels of Id1 protein predicted a nonsignificant longer overall survival (13 months vs. 7 months; P = .79). In the in silico analysis, however, lower levels of Id1 mRNA predicted a longer disease-free survival (61 months vs. not-reached; P = .018) and the hazard ratio for progression was 0.451 (P = .022) in favor of patients showing lower levels. ConclusionIn our cohort, it seems to be a differential epithelial expression of Id1 protein according to the prognostic features (metastatic/poor prognosis vs. organ-confined/good prognosis). In localized tumors treated with radical prostatectomy, higher Id1 mRNA expression levels might predict a higher hazard ratio for progression and a shorter disease-free survival. Further validation of these results in larger prospective series is warranted.

Abstract 3129: COX2 overexpression increases malignant potential of human glioma cells through Id1.

Abstract Increased COX2 expression directly correlates with glioma grade and is also associated with shorter survival in malignant glioma patients. Id1 is similarly overexpressed in higher grade gliomas. Id1 is a member of the helix-loop-helix (HLH) family of transcriptional repressors that act as dominant-negative inhibitors of basic-HLH factors. Recent studies indicate that Id1 may be involved in the genesis/progression of gliomas. Here, we find that COX2 overexpression in human glioma cells correlates with increased Id1 protein levels. We further find that forced expression of COX2 and Id1 enhances soft agar colony formation. In cell invasion assay, both COX2 and Id1 expression in LN229 human glioma cells (LN/COX2 and LN/Id1) increase migration through matrigel. These cells also showed more tumorigenic potential with development of significantly larger subcutaneous tumors compared with controls. Of note, tumors expressing COX2 or Id1 had higher microvessel density than control tumors. Next, the effect of COX2 and Id1 overexpression was assessed in an orthotopic intracranial model. Here, nude mice injected with LN/COX2 or LN/Id1 cells had significantly decreased survival times compared with corresponding controls (Table 1). To assess the role of Id1 induction on the malignant potential of COX2-expressing glioma cells, LN/COX2 cells with shRNA against Id1 were intracranially injected and found to have a statistically significant increase in survival compared to those injected with control shRNA LN/COX2 cells (Table 1). Finally, to test a COX2-targeting therapy, we used the COX2 inhibitor celecoxib in our xenograft model and found inhibition of growth of flank tumors derived from glioma cells overexpressing COX2. In conclusion, COX2 overexpression in human glioma cells results in elevated Id1 protein level, which, in turn, enhances the transformation and invasiveness of human glioma cells, promotes microvessel formation and enhances xenograft tumor growth subcutaneously and intracranially. Table 1. Survival of nude mice injected intracranially with the indicated glioma cells. Expt. 1 Expt. 2 Expt. 3 Cell type N Median Survival (days) N Median Survival (days) N Median Survival (days) LN/Ctrl 10 71 8 78 LN/COX2 10 40 9 53 LN/Id1 9 22 11 21 LN/COX2 shCtrl 10 75 LN/COX2 shId1 10 56 p-value by logrank <0.001 <0.001 0.011 Citation Format: Kaiming Xu, Hui-Kuo G. Shu. COX2 overexpression increases malignant potential of human glioma cells through Id1. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3129. doi:10.1158/1538-7445.AM2013-3129

Abstract 1076: Ids act as downstream effectors in the transforming growth factor-β pathway in prostate cancer cells

Abstract Transforming growth factor-β (TGFβ) regulates proliferation, differentiation and differentiated function in a wide variety in cells. In prostate cancer cells, TGFβ inhibits proliferation in the earlier stages; however the cancer cells become refractory to growth inhibitory effects in advanced stages where TGFβ promotes cancer progression and metastasis. Intracellular mechanisms involved in these varied effects of TGFβ on prostate cancer cells are poorly understood. Inhibitor of Differentiation (Id) family of closely related proteins (Id1, Id2, Id3, Id4) inhibit binding of bHLH transcription factors to DNA and inhibit their effects on cell proliferation and differentiation. In the present study, we have investigated the involvement of Id1 and Id3 proteins in the effects of TGFβ on prostate cancer cells. Id1 and Id3 isoforms of protein are expressed in DU145 cells, which have previously been shown to be responsive to growth inhibitory effects of TGFβ. TGFβ caused a significant increase of protein levels of Id1 and Id3 as determined by Western blot analyses. This early induction was followed by a late downregulation of Id1, while Id3 induction remained stable. However in PC3 cells, this early induction of Id1 and Id3 proteins remained stable. Proliferation marker p21 was also induced in DU145 cells, while p21 protein was undetected in PC3 cells upon TGFβ1 treatment. Since previous studies have revealed that the loss of Id1 or Id3 is indicative of loss of proliferation in prostate cancer, Id1 effects were further studied between DU145 and PC3 cell lines. Id1 knockdown in DU145 cells resulted in enhanced inhibitory effects of TGFβ on cell proliferation. However, Id1 knockdown in PC3 did not influence TGFβ effects on proliferation. These results indicate that Id1 downregulation by TGFβ may be required for its effects on proliferation in DU145 cancer cells. We next investigated TGFβ effects on migration in PC3 cells, which TGFβ stimulation has been shown to increase its migration properties. Both Id1 and Id3 knockdown in PC3 cells decreased migratory response to TGFβ. This indicated that both Id1 and Id3 are necessary for TGFβ migratory properties of PC3 cells. Taken together, these results show that Id1 and Id3 act as necessary proteins in the TGFβ pathway on both proliferation and migration. Grant support: NIH 1P20MD002285-02 and RCMI G12RR03602 Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1076. doi:1538-7445.AM2012-1076

Abstract 798: Id1 (Inhibitor of differentiation-1) expression demonstrates important translational implications for pancreatic adenocarcinoma

Abstract Introduction: Inhibitor of differentiation-1 (Id1) demonstrates a role in tumor progression in a variety of tumors but scarce data exists for pancreatic adenocarcinoma. Previously, we demonstrated that nicotine, the addicting component of tobacco, induces Id1 expression through a Src-dependent signaling pathway promoting proliferation, invasion, and chemoresistance in pancreatic cancer cells. Conversely, silencing of Id1 re-establishes gemcitabine sensitivity in acquired and innate gemcitabine-resistant cell lines. Therefore, we determined the role of Id1 in tumor progression and chemoresistance in vivo and determined the expression status of Id1 in a large cohort of human pancreatic cancer tissues. Methods: Cell lysate analyses (protein analyses, mRNA expression) were determined in a bio-diverse sample of pancreatic cancer cells by western and RT-PCR. Metastatic pancreatic cancer cells were stably transfected with a luciferase expression vector and stable short-hairpin RNA silencing of Id1 gene expression with controls. Orthotopic nude mouse model utilized intraperitoneal injections of nicotine (1mg/kg, M/W/F) and gemcitabine (250 mg/kg, T/Th) with monitoring of in vivo growth and metastases based on luminescent signal concentration. Confirmation of primary tumor and metastases was confirmed by H&E staining. Tissue microarrays (TMAs) constructed from 100 resected pancreatic adenocarcinoma were stained for Id1, phospho-Src, and total Src using immunohistochemistry techniques. Scores based on intensity, percent of positive cells, and pathological grade (1-4) were assigned to each malignant core. Results: In vivo, nicotine significantly promoted tumor growth and metastases in vivo. While gemcitabine reduced the tumor burden in control mice, nicotine abrogates this inhibitory effect. Additionally, knockdown of Id1 results in a significant decrease in tumor burden with no identifiable liver metastases, regardless of nicotine exposure. Analyses of resected pancreatic tumor TMAs demonstrated a statistically significant correlation between Id1 and tumor grade/differentiation (p =0.0368). The correlation of protein levels of Id1 and phospho-Src was also statistically significant (p =0.0024); Comparison of overall survival and tumor grade was also significant (p = 0.05); the correlation between overall-survival and Id1 expression trended toward statistical significance (p =0.06). Conclusions: Id1, through a Src-dependent-Id1 signaling axis, promotes tumorigenesis and chemoresistance in an in vivo pancreatic cancer model. Id1 expression correlates with phospho-Src levels and tumor differentiation in human pancreatic cancer tissues. These results support the clinical assessment of Id1 expression as a biomarker of prognosis and support investigations toward targeted therapies against Id1 for pancreatic cancer patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 798. doi:1538-7445.AM2012-798

Slow-dividing satellite cells retain long-term self-renewal ability in adult muscle

Summary Satellite cells are muscle stem cells that have important roles in postnatal muscle growth and adult muscle regeneration. Although fastand slow-dividing populations in activated satellite cells have been observed, the functional differences between them remain unclear. Here we elucidated the relationship between proliferation behaviour and satellite cell function. To assess the frequency of cell division, satellite cells isolated from mouse EDL muscle were labelled with the fluorescent dye PKH26, stimulated to proliferate and then sorted by FACS. The vast majority of activated satellite cells were PKH26 low fast-dividing cells, whereas PKH26 high slow-dividing cells were observed as a minority population. The fast-dividing cells generated a higher number of differentiated and self-renewed cells compared with the slow-dividing cells. However, cells derived from the slow-dividing population formed secondary myogenic colonies when passaged, whereas those from the fast-dividing population rapidly underwent myogenic differentiation without producing self-renewing cells after a few rounds of cell division. Furthermore, slow-dividing cells transplanted into injured muscle extensively contributed to muscle regeneration in vivo. Id1, a HLH protein, was expressed by all activated satellite cells, but the expression level varied within the slow-dividing cell population. We show that the slow-dividing cells retaining long-term self-renewal ability are restricted to an undifferentiated population that express high levels of Id1 protein (PKH26 high Id1 high population). Finally, genome-wide gene expression analysis described the molecular characteristics of the PKH26 high Id1 high population. Taken together, our results indicate that undifferentiated slow-dividing satellite cells retain stemness for generating progeny capable of long-term self-renewal, and so might be essential for muscle homeostasis throughout life.

Slow-dividing satellite cells retain long-term self-renewal ability in adult muscle

Satellite cells are muscle stem cells that have important roles in postnatal muscle growth and adult muscle regeneration. Although fast- and slow-dividing populations in activated satellite cells have been observed, the functional differences between them remain unclear. Here we elucidated the relationship between proliferation behaviour and satellite cell function. To assess the frequency of cell division, satellite cells isolated from mouse EDL muscle were labelled with the fluorescent dye PKH26, stimulated to proliferate and then sorted by FACS. The vast majority of activated satellite cells were PKH26(low) fast-dividing cells, whereas PKH26(high) slow-dividing cells were observed as a minority population. The fast-dividing cells generated a higher number of differentiated and self-renewed cells compared with the slow-dividing cells. However, cells derived from the slow-dividing population formed secondary myogenic colonies when passaged, whereas those from the fast-dividing population rapidly underwent myogenic differentiation without producing self-renewing cells after a few rounds of cell division. Furthermore, slow-dividing cells transplanted into injured muscle extensively contributed to muscle regeneration in vivo. Id1, a HLH protein, was expressed by all activated satellite cells, but the expression level varied within the slow-dividing cell population. We show that the slow-dividing cells retaining long-term self-renewal ability are restricted to an undifferentiated population that express high levels of Id1 protein (PKH26(high)Id1(high) population). Finally, genome-wide gene expression analysis described the molecular characteristics of the PKH26(high)Id1(high) population. Taken together, our results indicate that undifferentiated slow-dividing satellite cells retain stemness for generating progeny capable of long-term self-renewal, and so might be essential for muscle homeostasis throughout life.

Id-1 and the p65 subunit of NF-κB promote migration of nasopharyngeal carcinoma cells and are correlated with poor prognosis

Inhibitor of differentiation (Id)-1 and nuclear factor-kappa B (NF-κB) have been detected in many malignant tumors, and their presence has been correlated with the metastatic potential of these tumors. This study was undertaken to investigate the prognostic significance of the expression of Id-1 and the p65 subunit of NF-κB (NF-κB/p65) and the proteins' roles in the invasion process of nasopharyngeal carcinoma (NPC) cells. The messenger RNA (mRNA) and protein levels of Id-1 and NF-κB/p65 in normal nasopharyngeal epithelial cells and NPC cell lines were examined using reverse transcription-PCR and western blot analysis, whereas the mRNA and protein levels of Id-1 and NF-κB/p65 in clinical NPC specimens were determined by reverse transcription-PCR and immunohistochemistry. Short hairpin RNA (shRNA) was used to silence Id-1 and NF-κB/p65 to allow for the examination of matrix metalloproteinase (MMP)-9 expression and migratory capacity changes in CNE-2 cells. Multivariate Cox analysis revealed that elevated Id-1 expression was a significant independent predictor of the 5 year overall survival rate (hazards ratio = 16.720, P = 0.005). Furthermore, elevated expression of both Id-1 and NF-κB/p65 was associated with poor clinical survival (P = 0.049). Targeting Id-1 and NF-κB/p65 mRNA with shRNA in CNE-2 cells inhibited MMP-9 expression and decreased the migratory capacity of CNE-2 cells. In conclusion, Id-1 expression is a novel independent prognostic marker molecule that helps identify NPC patients with a poor prognosis. Additionally, combined analysis of Id-1 and NF-κB/p65 can be useful for identifying patients at risk for unfavorable clinical outcomes. Id-1 or/and NF-κB/p65 enhanced tumor cell migration, which is associated with the secretion of MMP-9.

MicroRNA-29b is involved in the Src-ID1 signaling pathway and is dysregulated in human lung adenocarcinoma

The c-Src kinase regulates cancer cell invasion through inhibitor of DNA binding/differentiation 1 (ID1). Src and ID1 are frequently overexpressed in human lung adenocarcinoma. The current study aimed at identifying microRNAs (miRNAs) involved in the Src-ID1 signaling in lung cancer. Incubation of lung cancer cells with the Src inhibitor saracatinib led to the upregulation of several miRNAs including miR-29b, which was the most highly upregulated miRNA with predicted binding to the ID1 3'-untranslated region (UTR). Luciferase reporter assays confirmed direct binding of miR-29b to the ID1 3'-UTR. Expression of miR-29b suppressed ID1 levels and significantly reduced migration and invasion. Expression of antisense-miR-29b (anti-miR-29b), on the other hand, enhanced ID1 mRNA and protein levels, and significantly increased lung cancer cell migration and invasion, a hallmark of the Src-ID1 pathway. The ectopic expression of ID1 in miR-29b-overexpressing cells was able to rescue the migratory potential of these cells. Both, anti-miR-29b and ID1 overexpression diminished the effects of the Src inhibitors saracatinib and dasatinib on migration and invasion. Saracatinib and dasatinib decreased c-Myc transcriptional repression on miR-29b and led to increased ID1 protein levels, whereas forced expression of c-Myc repressed miR-29b and induced ID1. In agreement, we showed direct recruitment of c-Myc to the miR-29b promoter. miR-29b was significantly downregulated in primary lung adenocarcinoma samples compared with matched alveolar lung tissue, and miR-29b expression was a significant prognostic factor for patient outcome. These results suggest that miR-29b is involved in the Src-ID1 signaling pathway, is dysregulated in lung adenocarcinoma and is a potential predictive marker for Src kinase inhibitors.

Dithranol downregulates expression of Id1 mRNA in human keratinocytes in vitro

The precise causes of psoriasis, a chronic skin disorder characterized by hyperproliferation of keratinocytes and incomplete keratinization, are unclear. It is known that expression of helix-loop-helix transcription factor Id1, which functions as an inhibitor of differentiation, is upregulated in psoriatic skin. We investigated the effect of the antipsoriatic drug dithranol on mRNA and protein expression levels of Id1 in the HaCaT keratinocyte cell line. Cultured HaCaT cells were treated with 0-0.5 μg/mL dithranol for 30 min. After 2 and 4 h, total cellular RNA and total proteins were isolated from HaCaT cells, and quantitative real-time reverse transcriptase (RT-PCR) and Western blot were used to determine the mRNA and protein levels of Id1, respectively. Changes in normalized Id1 mRNA levels were observed only after 4 h of dithranol treatment. There was reduced expression of Id1 mRNA transcripts in the HaCaT cells treated with 0.1 μg/mL dithranol, but the reduction was not significant. The expression of Id1 mRNA was significantly downregulated (almost 50%) when 0.25 or 0.5 μg/mL dithranol was applied to the HaCaT cells. However, the normalized Id1 protein levels were not significantly affected. The molecular mechanisms underlying this finding should be investigated further to help determine the therapeutic action of this drug.

Abstract C150: CaMKKβ-mediated phosphorylation of Beclin 1 regulates autophagy and cell differentiation in neuroblastoma cells.

Abstract Autophagy is an evolutionarily conserved degradative pathway by which components of the cells are degraded in the lysosome in response to stress. Beclin 1 plays a central role in autophagy. Pioneering work has indicated that the increase in cytosolic Ca(2+) can induce autophagy via the CaMKKβ-mediated activation of AMPK. Here, we demonstrate a novel signaling pathway in which CaMKKβ regulates autophagy and differentiation. We found that Ca(2+) mobilizing agents (ionomycin and EB1089) induced phosphorylation of Beclin 1 and autophagy through CaMKKβ-dependent pathway. Using In vitro kinase assay we demonstrated that CaMKKβ directly phosphorylated Beclin 1 Ser90. Moreover, autophagy induced by ionomycin or EB1089 contributed to degradation of Id1 (inhibitor of differentiation). The inhibitors of autophagy including Bafilomycin A1 and chloroquine and RNA interference of autophagy-associated gene atg 5 markedly increased the protein levels of Id1 and abrogated neuroblastoma cell differentiation. Taken together, CaMKKβ promoted autophagy by directly phosphorylating Beclin 1 at Ser90, and thus induced cell differentiation in neuroblastoma cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C150.

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.

Abstract 5587: Id1 Regulates Angiogenesis by Determining the Specification and the Timing of Notch Signaling

Id1, a helix-loop-helix transcriptional factor, has emerged as a potent contributor of angiogenesis. We have previously reported that Id1 confers in vivo angiogenic property on human vascular endothelial cells (ECs) and that nucleo-cytoplasmic localization of Id1 might control angiogenesis. However, the downstream targets of Id1 and its interaction with other signaling pathways involved in angiogenesis are grossly unknown. To unveil the issue, we first examined the expression pattern of Id1 in ECs during angiogenesis in vitro and in vivo. We observed that Id1 expression levels were more prominent in sprouting ECs as compared with sheet-forming ECs at the proximal end of the sprouts in murine aortic ring assay. In the sprouts, furthermore, Id1 exhibited a mosaic expression pattern, mixed with weakly or no Id1-expressing ECs. We also identified similar expression pattern immunohistochemically in developing vessels of murine embryonic brain. Because the mosaic pattern of Id1 expression was reminiscent of Notch activation pattern in developing vessels, we next verified a possible crosstalk between Id1 and Notch signaling by RNAi-induced gene knockdown in cultured human ECs. Expectedly, siRNA-induced Id1-knockdown strikingly and specifically enhanced Notch-dependent upregulation of Hey2 (Gridlock) and to a less extent Hey1 but not Hes1. Id1-knockdown also upregulated the expression of a Notch-downstream effector Ephrin B2 in a Hey2-dependent manner. We finally examined how Id1 exhibited the mosaic expression pattern in ECs of developing vessels. Interestingly, Western blot analysis (hourly sampled for 12 hours) showed that total protein levels of Id1 appeared to oscillate periodically in cultured human ECs under confluent condition with no evident relation to cell cycle. This finding partially explains the mosaic expression pattern of Id1 during angiogenesis. Collectively, these results suggest a novel crosstalk between Id1 and Notch signaling, where Id1 might regulate the specification and the timing of Notch/Hey2 activation. We propose that the periodic expression of Id1 may be involved in the control of angiogenesis leading to proper branching morphology.

Induction of ID1 expression and apoptosis by the histone deacetylase inhibitor (trichostatin A) in human acute myeloid leukaemic cells

ID1, founding member of the inhibitor of differentiation (ID) family, is involved in cell population growth, apoptosis and tumourigenesis. We investigated mRNA levels of ID1 in human myeloid leukaemic cell lines and in specimens of patients with acute myeloid leukaemia (AML), using semiquantitative reverse transcription-polymerase chain reaction, and protein levels of ID1 in human myeloid leukaemic cell lines using Western blot analysis. Six of seven AML cell lines and 12 of 15 AML patient samples were found to have barely detectable ID1 mRNA. All of these cell lines showed the same levels of protein in proportion to levels of mRNA. Two of the AML cell lines with low ID1 expression, KG1 and KG-1a, were chosen for treatment with either the DNA demethylation reagent, 5-aza-2'-deoxycytidine (DAC), or the histone deacetylase (HDAC) inhibitor, trichostatin A (TSA). These treatments were alone or in combination, and ID1 expression was induced by both DAC and TSA. No hypermethylated ID1 gene promoter was detected in the majority of the cell lines and patient specimens, by methylation-specific polymerase chain reaction, suggesting that induction of ID1 in KG1 and KG-1a was not due to direct demethylation of the ID1 gene promoter. Chromatin immunoprecipitation showed that accumulation of acetyl-histone H3 and release of HDAC1 were correlated with ID1 induction by these drugs. Flow cytometric assay demonstrated more apoptosis induced by TSA or TSA in combination with DAC, in both KG-1 and KG-1a cell lines. Increase of intracellular reactive oxygen species was observed when treated with TSA. Most AML cell lines and human AML samples have very low levels of expression of ID1. TSA or TSA in combination with DAC is able to restore ID1 expression in low ID1-expressing AML cell lines by re-activating the aberrantly deacetylated promoter, and this also results in more apoptotic cell death, in which ID1 and the redox pathway may be involved.

Peptide-conjugated antisense oligonucleotides for targeted inhibition of a transcriptional regulator in vivo

Transcription factors are important targets for the treatment of a variety of malignancies but are extremely difficult to inhibit, as they are located in the cell's nucleus and act mainly by protein-DNA and protein-protein interactions. The transcriptional regulators Id1 and Id3 are attractive targets for cancer therapy as they are required for tumor invasiveness, metastasis and angiogenesis. We report here the development of an antitumor agent that downregulates Id1 effectively in tumor endothelial cells in vivo. Efficient delivery and substantial reduction of Id1 protein levels in the tumor endothelium were effected by fusing an antisense molecule to a peptide known to home specifically to tumor neovessels. In two different tumor models, systemic delivery of this drug led to enhanced hemorrhage, hypoxia and inhibition of primary tumor growth and metastasis, similar to what is observed in Id1 knockout mice. Combination with the Hsp90 inhibitor 17-(allylamino)-17-demethoxygeldanamycin yielded virtually complete growth suppression of aggressive breast tumors.

Id1 expression is transcriptionally regulated in radial growth phase melanomas

Id genes have been demonstrated to be upregulated in a wide variety of human malignancies and their expression has been correlated with disease prognosis; however, little is known about the mechanisms of Id gene activation in tumors. We have previously shown that the helix-loop-helix transcription factor, Id1, is highly expressed in primary human melanomas during the radial growth phase and that Id1 is a transcriptional repressor of the familial melanoma gene CDKN2A. Here we use a series of melanoma cell lines that recapitulate the phenotypic characteristics of melanomas at varying stages of malignant progression to evaluate the expression levels of Id1 in this model system and determine the mechanism of Id1 dysregulation in these tumor cells. We find elevated protein levels of Id1 to be present consistently in radial growth phase tumor cells in accordance with our primary tumor data. Id1 transcript levels were also found to be elevated in these radial growth phase melanoma cells without any appreciable evidence of gene amplification and Id1 promoter activity was found to correlate with Id expression levels. We therefore conclude that Id1 expression is primarily regulated at the transcriptional level in radial growth phase melanomas and expect that therapies that target Id1 gene expression may be useful in the treatment of Id-associated malignancies.