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ID1 and ID3 are the members of the Inhibitor of DNA Binding (ID) protein family, which negatively regulates the basic helix-loop-helix (bHLH) transcription factors by forming heterodimers, are involved in neurodevelopment, cardiovascular development, and tumor metastasis. We created twoID1/3knockout cell lines from a human embryonic stem cell (hESC) line (H9) by CRISPR/Cas9-mediated gene targeting. These cell lines maintain stem cell morphology, a normal karyotype, and the expression of pluripotent marker genes. Additionally, they retain their in vivo differentiation potential. Thecell lines are valuable tools for studying the roles of ID1/3 in neurodevelopment and cardiovascular diseases., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Introduction: B-cell acute lymphoblastic leukemia (B-ALL) in adults often presents a poor prognosis. ID1 and ID3 genes have been identified as predictors of poor response in Colombian adult B-ALL patients, contributing to cancer development. In various cancer models, these genes have been associated with immune regulatory populations within the tumor immune microenvironment (TIME). B-ALL progression alters immune cell composition and the bone marrow (BM) microenvironment, impacting disease progression and therapy response. This study investigates the relationship between ID1 and ID3 expression, TIME dynamics, and immune evasion mechanisms in adult B-ALL patients., Methods: This exploratory study analysed BM samples from 10 B-ALL adult patients diagnosed at the National Cancer Institute of Colombia. First, RT-qPCR was used to assess ID1 and ID3 expression in BM tumour cells. Flow cytometry characterised immune populations in the TIME. RNA-seq evaluated immune genes associatedwith B-ALL immune response, while xCell and CytoSig analysed TIME cell profiles and cytokines. Pathway analysis, gene ontology, and differential gene expression (DEGs) were examined, with functional enrichment analysis performed using KEGG ontology., Results: Patients were divided into two groups based on ID1 and ID3 expression, namely basal and overexpression. A total of 94 differentially expressed genes were identified between these groups, with top overexpressed genes associated with neutrophil pathways. Gene set enrichment analysis revealed increased expression of genes associated with neutrophil degranulation, immune response-related neutrophil activation, and neutrophil-mediated immunity. These findings correlated with xCell data. Overexpression group showed significant differences in neutrophils, monocytes and CD4+ naive T cells compared to basal group patients. Microenvironment and immune scores were also significantly different, consistent with the flow cytometry results. Elevated cytokine levels associated with neutrophil activation supported these findings. Validation was performed using the Therapeutically Applicable Research to Generate Effective Treatments (TARGET) TCGA B-ALL cohorts., Discussion: These findings highlight significant differences in ID1 and ID3 expression levels and their impact on TIME populations, particularly neutrophil-related pathways. The results suggest a potential role for ID1 and ID3 in immune evasion in adult B-ALL, mediated through neutrophil activation and immune regulation., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Poveda-Garavito, Orozco Castaño, Torres-Llanos, Cruz-Rodriguez, Parra-Medina, Quijano, Zabaleta and Combita.)

Background: Breast cancer is one of the most common cancers in women and is closely associated with obesity. Gremlin-2 (GREM2), an antagonist for bone morphogenetic proteins (BMPs), has been considered an inhibitor of adipogenic differentiation in adipose-derived stromal/stem cells. However, the role of GREM2 in breast cancer cells remains largely unknown, and its signaling mechanism has yet to be clarified., Methods: Bioinformatics analysis was conducted using public databases. Breast cancer cells overexpressing mock or GREM2 were used for in vitro and in vivo studies. Cell viability, colony formation, migration, and animal studies were performed to investigate the role of GREM2 in breast cancer cells. Screening of target genes affected by GREM2 overexpression in breast cancer cells was performed through RNA sequencing (RNA-seq) analysis., Results: The expression level of GREM2 mRNA was significantly reduced in both breast cancer tissues and cell lines. Kaplan-Meier analysis showed that low expression of GREM2 and high methylation of the GREM2 promoter were each associated with poor patient survival. The low mRNA expression of GREM2 in breast cancer cells was increased by the demethylating agent decitabine. Breast cancer cells overexpressing GREM2 decreased cell proliferation when compared to control cells, both in vitro and in vivo. Through comparison of RNA-seq analysis between cell lines and tissue samples, gene ontologies that were consistently upregulated or downregulated by GREM2 in breast cancer were identified. In particular, the expression of inhibitor of DNA-binding-1 (ID1) was repressed by GREM2. BMP2 is one of the upstream regulators that increases the expression of ID1, and the expression of ID1 reduced by GREM2 was restored by overexpression of BMP2. Also, the migration ability of breast cancer cells, which had been suppressed by GREM2, was restored by BMP2 or ID1., Conclusions: Low expression of GREM2 in breast cancer cells is associated with hypermethylation of the GREM2 promoter, which may ultimately contribute to poor patient survival. GREM2 participates in regulating the expression of various genes, including ID1, and is involved in suppressing the proliferation of breast cancer cells. This suggests that GREM2 has the potential to act as a novel tumor suppressor in breast cancer., Competing Interests: Declarations. Ethics approval and consent to participate: All animal experiments were performed in accordance with the guidelines for animal treatment of Soonchunhyang University. All experimental protocols in our study were conducted on protocols approved by the Institutional Animal Care and Use Committee of the Soonchunhyang University (SCH21-0014). Consent for publication: All authors approved submission of the manuscript. Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

Brown and beige adipocytes can be activated by β-adrenergic agonist via cAMP-dependent signaling. Performing RNA-sequencing analysis in human cervical area-derived adipocytes, we found that dibutyryl-cAMP, which can mimic in vivo stimulation of browning and thermogenesis, enhanced the expression of browning and batokine genes and upregulated several signaling pathway genes linked to thermogenesis. We observed that the expression of inhibitor of DNA binding and cell differentiation (ID) 1 and particularly ID3 was strongly induced by the adrenergic stimulation. The degradation of ID1 and ID3 elicited by the ID antagonist AGX51 during thermogenic activation prevented the induction of proton leak respiration that reflects thermogenesis and abrogated cAMP analogue-stimulated upregulation of thermogenic genes and mitochondrial complex I, II, and IV subunits, independently of the proximal cAMP-PKA signaling pathway. The presented data suggests that ID proteins contribute to efficient thermogenic response of adipocytes during adrenergic stimulation., Competing Interests: Declarations Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

Gene expression patterns are very sensitive to external influences and are reflected in phenotypic changes. It was previously described that transferring melanoma cells from a plastic surface to Matrigel led to formation of de novo vascular networks-vasculogenic mimicry-that are characteristic to a stemness phenotype in aggressive tumors. Up to now there was no detailed data about the gene signature accompanying this process. Here, we show that this transfer shortly led to extremely strong epigenetic changes in gene expression in the melanoma cells. We observed that on Matrigel numerous genes controlling ribosome biogenesis were upregulated. However, most of the activated genes were inhibitors of the differentiation genes ( ID1 , ID2 , and ID3 ). At the same time, the genes that control differentiation were downregulated. Both the upregulated and the downregulated genes are simultaneously targeted by different transcription factors shaping sets of co-expressed genes. The specific group of downregulated genes shaping contacts with rDNA genes are also associated with the H3K27me3 mark and with numerous lincRNAs and miRNAs. We conclude that the stemness phenotype of melanoma cells is due to the downregulation of developmental genes and formation of dedifferentiated cells.

Background: The mechanisms enabling dynamic shifts between drug-resistant and drug-sensitive states in cancer cells are still underexplored. This study investigated the role of targeted autophagic protein degradation in regulating ovarian cancer stem cell (CSC) fate decisions and chemo-resistance., Methods: Autophagy levels were compared between CSC-enriched side population (SP) and non-SP cells (NSP) in multiple ovarian cancer cell lines using immunoblotting, immunofluorescence, and transmission electron microscopy. The impact of autophagy modulation on CSC markers and differentiation was assessed by flow cytometry, immunoblotting and qRT-PCR. In silico modeling and co-immunoprecipitation identified ID1 interacting proteins. Pharmacological and genetic approaches along with Annexin-PI assay, ChIP assay, western blotting, qRT-PCR and ICP-MS were used to evaluate effects on cisplatin sensitivity, apoptosis, SLC31A1 expression, promoter binding, and intracellular platinum accumulation in ID1 depleted backdrop. Patient-derived tumor spheroids were analyzed for autophagy and SLC31A1 levels., Results: Ovarian CSCs exhibited increased basal autophagy compared to non-CSCs. Further autophagy stimulation by serum-starvation and chemical modes triggered proteolysis of the stemness regulator ID1, driving the differentiation of chemo-resistant CSCs into chemo-sensitive non-CSCs. In silico modeling predicted TCF12 as a potent ID1 interactor, which was validated by co-immunoprecipitation. ID1 depletion freed TCF12 to transactivate the cisplatin influx transporter SLC31A1, increasing intracellular cisplatin levels and cytotoxicity. Patient-derived tumor spheroids exhibited a functional association between autophagy, ID1, SLC31A1, and platinum sensitivity., Conclusions: This study reveals a novel autophagy-ID1-TCF12-SLC31A1 axis where targeted autophagic degradation of ID1 enables rapid remodeling of CSCs to reverse chemo-resistance. Modulating this pathway could counter drug resistance in ovarian cancer., (© 2024. The Author(s).)

Purpose: Kinase interacting with stathmin (KIS) is a serine/threonine kinase involved in RNA processing and protein phosphorylation. Increasing evidence has suggested its involvement in cancer progression. The aim of this study was to investigate the role of KIS in the development of lung adenocarcinoma (LUAD). Dual luciferase assay was used to explore the relationship between KIS and SOX4, and its effect on ID1/β-catenin pathway., Methods: Real-time qPCR and western blot were used to assess the levels of KIS and other factors. Cell proliferation, migration, and invasion were monitored, and xenograft animal model were established to investigate the biological functions of KIS in vitro and in vivo., Results: In the present study, KIS was found to be highly expressed in LUAD tissues and cell lines. KIS accelerated the proliferative, migratory and invasive abilities of LUAD cells in vitro, and promoted the growth of LUAD in a mouse tumor xenograft model in vivo. Mechanistically, KIS activated the β-catenin signaling pathway by modulating the inhibitor of DNA binding 1 (ID1) and was transcriptionally regulated by SOX4 in LUAD cells., Conclusion: KIS, a target of SOX4, regulates the ID1-mediated enhancement of β-catenin to facilitate LUAD cell invasion and metastasis., (© 2024. The Author(s).)

Although bevacizumab (BVZ), a representative drug for anti-angiogenesis therapy (AAT), is used as a first-line treatment for patients with glioblastoma (GBM), its efficacy is notably limited. Whereas several mechanisms have been proposed to explain the acquisition of AAT resistance, the specific underlying mechanisms have yet to be sufficiently ascertained. Here, we established that inhibitor of differentiation 1 (ID1) high /activin A high glioblastoma cell confers resistance to BVZ. The bipotent effect of activin A during its active phase was demonstrated to reduce vasculature dependence in tumorigenesis. In response to a temporary exposure to activin A, this cytokine was found to induce endothelial-to-mesenchymal transition via the Smad3/Slug axis, whereas prolonged exposure led to endothelial apoptosis. ID1 tumors showing resistance to BVZ were established to be characterized by a hypovascular structure, hyperpermeability, and scattered hypoxic regions. Using a GBM mouse model, we demonstrated that AAT resistance can be overcome by administering therapy based on a combination of BVZ and SB431542, a Smad2/3 inhibitor, which contributed to enhancing survival. These findings offer valuable insights that could contribute to the development of new strategies for treating AAT-resistant GBM., (© 2024. The Author(s).)

Sorafenib resistance greatly restricts its clinical application in patients with hepatocellular carcinoma (HCC). Numerous studies have reported that ID1 exerts a crucial effect in cancer initiation and development. Our previous research revealed an inhibitory role of ID1 in sorafenib resistance. However, the upstream regulatory mechanism of ID1 expression is unclear. Here, we discovered that ID1 expression is negatively correlated with promoter methylation, which is regulated by DNMT3B. Knockdown of DNMT3B significantly inhibited ID1 methylation status and resulted in an increase of ID1 expression. The demethylating agent 5-aza-2'-deoxycytidine (5-aza) remarkably upregulated ID1 expression. The combination of 5-aza with sorafenib showed a synergistic effect on the inhibition of cell viability., (© 2023 The Authors. FEBS Open Bio published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Elimination of cancer stem cells (CSCs) and reinvigoration of antitumor immunity remain unmet challenges for cancer therapy. Tumor-associated macrophages (TAMs) constitute the prominant population of immune cells in tumor tissues, contributing to the formation of CSC niches and a suppressive immune microenvironment. Here, we report that high expression of inhibitor of differentiation 1 (ID1) in TAMs correlates with poor outcome in patients with colorectal cancer (CRC). ID1 expressing macrophages maintain cancer stemness and impede CD8 + T cell infiltration. Mechanistically, ID1 interacts with STAT1 to induce its cytoplasmic distribution and inhibits STAT1-mediated SerpinB2 and CCL4 transcription, two secretory factors responsible for cancer stemness inhibition and CD8 + T cell recruitment. Reducing ID1 expression ameliorates CRC progression and enhances tumor sensitivity to immunotherapy and chemotherapy. Collectively, our study highlights the pivotal role of ID1 in controlling the protumor phenotype of TAMs and paves the way for therapeutic targeting of ID1 in CRC., (© 2023. The Author(s).)

The 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., (© 2023 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

Metastasis is a significant cause of high mortality in lung cancer. Lymph node (LN) metastasis is the most common metastatic pathway in non-small cell lung cancer and the most crucial factor affecting the prognosis of NSCLC. Nevertheless, the molecular mechanism underlying metastasis is unknown. We demonstrated that higher NADK expression suggests worsened survival prognosis, and NADK expression positively correlates with the lymph node metastasis rate and TNM and AJCC stages in NSCLC patients. Moreover, patients with LN metastasis show higher NADK expression than those without LN metastasis. NADK can promote NSCLC progression by enhancing the migration, invasion, lymph node metastasis and growth of NSCLC cells. Mechanistically, NADK inhibits the ubiquitination and degradation of BMPR1A by interacting with Smurf1, further activating the BMPs signalling pathway and promoting ID1 transcription. In conclusion, NADK may be a potential diagnostic indicator and a novel therapeutic target for metastatic NSCLC., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Background: High expression of inhibitor of DNA binding 1 ( ID1 ) correlates with poor prognosis in colorectal cancer (CRC). Aberrant enhancer activation in regulating ID1 transcription is limited., Methods: Immunohistochemistry (IHC), quantitative RT-PCR (RT-qPCR) and Western blotting (WB) were used to determine the expression of ID1 . CRISPR-Cas9 was used to generate ID1 or enhancer E1 knockout cell lines. Dual-luciferase reporter assay, chromosome conformation capture assay and ChIP-qPCR were used to determine the active enhancers of ID1 . Cell Counting Kit 8, colony-forming, transwell assays and tumorigenicity in nude mice were used to investigate the biological functions of ID1 and enhancer E1., Results: Human CRC tissues and cell lines expressed a higher level of ID1 than normal controls. ID1 promoted CRC cell proliferation and colony formation. Enhancer E1 actively regulated ID1 promoter activity. Signal transducer and activator of transcription 3 (STAT3) bound to ID1 promoter and enhancer E1 to regulate their activity. The inhibitor of STAT3 Stattic attenuated ID1 promoter and enhancer E1 activity and the expression of ID1 . Enhancer E1 knockout down-regulated ID1 expression level and cell proliferation in vitro and in vivo., Conclusions: Enhancer E1 is positively regulated by STAT3 and contributes to the regulation of ID1 to promote CRC cell progression and might be a potential target for anti-CRC drug studies., Competing Interests: The authors declare no conflicts of interest.

RSL3 is a synthetic molecule that inactivates glutathione peroxidase 4 to induce ferroptosis. However, its effect on glioma stem cells (GSC) remains unclear. In this study, we found that RSL3 significantly suppressed GSC proliferation and induced their differentiation into astrocytes, which was accompanied by the downregulation of stemness-related markers, including Nestin and Sox2. Combined transcriptome and proteome analyses further revealed that RSL3 promoted GSC differentiation by suppressing transglutaminase 2 (Tgm2), but not by ferroptosis-related pathways. Tgm2 overexpression in CSC2078 cells rescued the changes in stemness-related markers and differentiation caused by RSL3, which was mediated by inhibitor of DNA binding 1 (ID1) activation. Further studies identified ID1 as a downstream signaling target of Tgm2. Blocking the phosphoinositide-3 kinase (PI3K)/Akt pathway with LY294002 suppressed PI3K, p-Akt, and ID1 levels but not Tgm2. Tgm2 overexpression abrogated the changes in PI3K, p-Akt, and ID1 levels caused by LY294002. Taken together, we demonstrate that RSL3 does not induce ferroptosis; instead, it inhibits GSC proliferation and triggers their differentiation by suppressing the Tgm2/Akt/ID1 signaling axis., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

The regulatory circuits that coordinate epidermal differentiation during development are still not fully understood. Here, we report that the transcriptional regulator ID1 is enriched in mouse basal epidermal progenitor cells and find ID1 expression to be diminished upon differentiation. In utero silencing of Id1 impairs progenitor cell proliferation, leads to precocious delamination of targeted progenitor cells and enables differentiated keratinocytes to retain progenitor markers and characteristics. Transcriptional profiling suggests that ID1 acts by mediating adhesion to the basement membrane while inhibiting spinous layer differentiation. Co-immunoprecipitation reveals ID1 binding to transcriptional regulators of the class I bHLH family. We localize bHLH Tcf3, Tcf4 and Tcf12 to epidermal progenitor cells during epidermal stratification and establish TCF3 as a downstream effector of ID1-mediated epidermal proliferation. Finally, we identify crosstalk between CEBPA, a known mediator of epidermal differentiation, and Id1, and demonstrate that CEBPA antagonizes BMP-induced activation of Id1. Our work establishes ID1 as a key coordinator of epidermal development, acting to balance progenitor proliferation with differentiation and unveils how functional crosstalk between CEBPA and Id1 orchestrates epidermal lineage progression., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2022. Published by The Company of Biologists Ltd.)

Pancreatic Ductal Adenocarcinoma (PDAC) is a deadly disease that has an increasing death rate but no effective treatment to now. Although biological and immunological hallmarks of PDAC have been frequently reported recently, early detection and the particularly aggressive biological features are the major challenges remaining unclear. In the current study, we retrieved multiple scRNA-seq datasets and illustrated the genetic programs of PDAC development in genetically modified mouse models. Notably, the transcription levels of Id1 were elevated specifically along with the PDAC development. Pseudotime trajectory analysis revealed that Id1 was closely correlated with the malignancy of PDAC. The gene expression patterns of human PDAC cells were determined by the comparative analysis of the scRNA-seq data on human PDAC and normal pancreas tissues. ID1 levels in human PDAC cancer cells were dramatically increased compared to normal epithelial cells. ID1 deficiency in vitro significantly blunt the invasive tumor-formation related phenotypes. IPA analysis on the differentially expressed genes suggested that EIF2 signaling was the core pathway regulating the development of PDAC. Blocking EFI2 signaling remarkably decreased the expression of ID1 and attenuated the tumor-formation related phenotypes. These observations confirmed that ID1 was regulated by EIF2 signaling and was the critical determinator of PDAC development and progression. This study suggests that ID1 is a potential malignant biomarker of PDAC in both mouse models and human and detecting and targeting ID1 may be a promising strategy to treat or even rescue PDAC., (© 2022. The Author(s).)

Acute myeloid leukemia (AML) is characterized by the dysregulation of hematopoietic cell proliferation, resulting in the accumulation of immature myeloid cells in bone marrow. 5-Demethylnobiletin (5-demethyl NOB), a citrus 5-hydroxylated polymethoxyflavone, has been reported to exhibit various bioactivities, such as antioxidant, anti-inflammatory and anticancer properties. In this study, we investigated the antileukemic effects of 5-demethyl NOB and its underlying molecular mechanisms in human AML cells. We found that 5-demethyl NOB (20−80 μM) significantly reduced human leukemia cell viability, and the following trend of effectiveness was observed: THP-1 ≈ U-937 > HEL > HL-60 > K562 cells. 5-Demethyl NOB (20 and 40 μM) modulated the cell cycle through the regulation of p21, cyclin E1 and cyclin A1 expression and induced S phase arrest. 5-Demethyl NOB also promoted leukemia cell apoptosis and differentiation. Microarray-based transcriptome, Gene Ontology (GO) and Gene Set Enrichment Analysis (GSEA) of differentially expressed genes (DEGs) analysis showed that the expression of inhibitor of differentiation/DNA binding 1 (ID1), a gene associated with the GO biological process (BP) cell population proliferation (GO: 0008283), was most strongly suppressed by 5-demethyl NOB (40 μM) in THP-1 cells. We further demonstrated that 5-demethyl NOB-induced ID1 reduction was associated with the inhibition of leukemia cell growth. Moreover, DEGs involved in the hallmark gene set NF-κB/TNF-α signaling pathway were markedly enriched and downregulated by 5-demethyl NOB. Finally, we demonstrated that 5-demethyl NOB (20 and 40 μM), combined with cytarabine, synergistically reduced THP-1 and U-937 cell viability. Our current findings support that 5-demethyl NOB dramatically suppresses leukemia cell proliferation and may serve as a potential phytochemical for human AML chemotherapy.

Long non-coding RNA (lncRNA) nuclear-enriched abundant transcript 1 (NEAT1) exerts a pro-oncogenic role in several cancers, whereas its underlying regulatory mechanism in papillary thyroid carcinoma (PTC) progression remains unknown. This research mainly explored the roles of NEAT1 in PTC development. Quantitative real-time polymerase-chain reaction (qRT-PCR) was applied to measure NEAT1, miR-524-5p, and inhibitor of DNA binding 1 (ID1) expression in PTC tissues and cells. Western blot was conducted for detecting the protein levels. MTT, transwell, and flow cytometry assays were applied to assess cell proliferation, metastasis, and apoptosis in PTC cells in vitro . The PTC xenograft tumor model was used for investigating the role of NEAT1 in vivo . Dual-luciferase reporter assay was utilized for confirming the interaction between miR-524-5p and NEAT1 or ID1. In PTC tissues and cells, NEAT1 was significantly up-regulated. NEAT1 silencing blocked cell proliferation, metastasis, and facilitated apoptosis in vitro and impeded xenograft tumor growth in vivo . Bioinformatics prediction revealed the existence of binding sites between NEAT1 and miR-524-5p. Besides, ID1 was confirmed as a direct target to miR-524-5p, and the enhancement of ID1 reversed the regulation of miR-524-5p upregulation on cell progression. In addition, NEAT1 promoted PTC development by regulating ID1 expression via sponging miR-524-5p in PTC. In summary, we demonstrate that NEAT1 advanced the process of PTC by miR-524-5p/ID1 axis, which may enhance our comprehension of PTC pathogenesis.

Since hepatocellular carcinoma (HCC) is a typical hypervascular malignant tumor with poor prognosis, targeting angiogenesis is an important therapeutic strategy for advanced HCC. Involvement of bone morphologic protein 9 (BMP9), a transforming growth factor-beta superfamily member, has recently been reported in the development of liver diseases and angiogenesis. Here, we aimed to elucidate the role of BMP9 signaling in promoting HCC angiogenesis and to assess the antiangiogenic effect of BMP receptor inhibitors in HCC. By analyzing HCC tissue gene expression profiles, we found that BMP9 expression was significantly correlated with angiogenesis-associated genes, including HIF-1α and VEGFR2. In vitro, BMP9 induced HCC cell HIF-1α/VEGFA expression and VEGFA secretion. Silencing of the inhibitor of DNA-binding protein 1 (ID1), a transcription factor targeted by BMP9 signaling, suppressed BMP9-induced HIF-1α/VEGFA expression and VEGFA secretion, resulting in decreased human umbilical vein endothelial cell (HUVEC) lumen formation. BMP receptor inhibitors, which inhibit BMP9-ID1 signaling, suppressed BMP9-induced HIF-1α/VEGFA expression, VEGFA secretion, and HUVEC lumen formation. In vivo, the BMP receptor inhibitor LDN-212854 successfully inhibited HCC tumor growth and angiogenesis by inhibiting BMP9-ID1 signaling. In summary, BMP9-ID1 signaling promotes HCC angiogenesis by activating HIF-1α/VEGFA expression. Thus, targeting BMP9-ID1 signaling could be a pivotal therapeutic option for advanced HCC.

Hepatitis B virus (HBV) infection is a major risk factor for hepatocellular carcinoma (HCC), which required developing novel therapies targeting the inhibition of HBV transcription and replication due to current limited treatment options. We explored novel target for the development of novel therapies targeting the inhibition of HBV replication and transcription. The expression of Id1 and E2F4 in HCC cells and tissues was detected by qRT-PCR and western blot. We investigated the Id1 and E2F4-mediated transcription of HBV infection by using HepG2.2.15, HepAD38, HepG2-NTCP cell lines and AAV/HBV-infected mice. Interactions between the two host proteins and viral covalently closed circular DNA (cccDNA) were assessed using subcellular localization, protein-protein interaction, chromatin immunoprecipitation, and luciferase assays. Ectopic Id1 significantly reduced HBV transcription and replication in both HBV-expressing cells and AAV/HBV-infected mice. Id1 and E2F4 could form a heterodimer to prevent E2F4 from promoting HBV transcription and replication. E2F4 could directly bind to cccDNA and activate the HBV core promoter in cell lines. Furthermore, in vitro binding experiments confirmed that the sequence 1758'-TTAAAGGTC-1766', which is highly conserved among HBV genotypes, is the target site of the E2F4 homodimer. The findings suggest that E2F4 function as novel cccDNA-binding protein to directly activate HBV transcription by binding to Cp promoter region. Our results highlight the ability that E2F4 represent a pan-potential therapeutic target against HBV transcription and provide more clues to better understand the life cycle of HBV., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Cutaneous wound healing requires intricate synchronization of several key processes. Among them, local nerve regeneration is known to be vitally important for proper repair. However, the underlying mechanisms of local nerve regeneration are still unclear. Fibroblasts are one of the key cell types within the skin whose role in local nerve regeneration has not been extensively studied. In our study, we found skin fibroblasts were in tight contact with regenerated nerves during wound healing, while rare interactions were shown under normal circumstances. Moreover, skin fibroblasts surrounding the nerves were shown to be activated and reprogrammed to exhibit neural cell-like properties by upregulated expressing inhibitor of DNA binding 1 (ID1) and ID3. Furthermore, we identified the regulation of integrin α6 (Itga6) by ID1/ID3 in fibroblasts as the mechanism for axon guidance. Accordingly, transplantation of the ID1/ID3-overexpressing fibroblasts or topical injection of ID1/ID3 lentivirus significantly promoted local nerve regeneration and wound healing following skin excision or sciatic nerve injury. Therefore, we demonstrated a new role for skin fibroblasts in nerve regeneration following local injury by directly contacting and guiding axon regrowth, which might hold therapeutic potential in peripheral nerve disorders and peripheral neuropathies in relatively chronic refractory wounds., (© 2021 The Authors. STEM CELLS TRANSLATIONAL MEDICINE published by Wiley Periodicals LLC on behalf of AlphaMed Press.)

In melanoma, a phenotype switch from proliferation to invasion underpins metastasis, the major cause of melanoma-associated death. The transition from radial to vertical growth phase (invasive) melanoma is characterized by downregulation of both E-cadherin (CDH1) and MITF and upregulation of the key cancer-associated gene TBX3 and the phosphatidylinositol 3 kinase signaling pathway. Yet, whether and how these diverse events are linked remains poorly understood. Here, we show that TBX3 directly promotes expression of ID1, a dominant-negative regulator of basic helix-loop-helix transcription factors, and that ID1 decreases MITF binding and upregulation of CDH1. Significantly, we show that TBX3 activation of ID1 is necessary for TBX3 to enhance melanoma cell migration, and the mechanistic links between TBX3, ID1, MITF, and invasion revealed here are reflected in their expression in human melanomas. Our results reveal that melanoma migration is promoted through a TBX3-ID1-MITF-E-cadherin axis and that ID1-mediated repression of MITF activity may reinforce maintenance of an MITF Low phenotype associated with disease progression and therapy resistance., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

The malignant nature of hepatocellular carcinoma (HCC) is closely related to the presence of cancer stem cells (CSCs). Bone morphologic protein 9 (BMP9), a member of the transforming growth factor-beta (TGF-β) superfamily, was recently reported to be involved in liver diseases including cancer. We aimed to elucidate the role of BMP9 signaling in HCC-CSC properties and to assess the therapeutic effect of BMP receptor inhibitors in HCC. We have identified that high BMP9 expression in tumor tissues or serum from patients with HCC leads to poorer outcome. BMP9 promoted CSC properties in epithelial cell adhesion molecule (EpCAM)-positive HCC subtype via enhancing inhibitor of DNA-binding protein 1 (ID1) expression in vitro. Additionally, ID1 knockdown significantly repressed BMP9-promoted HCC-CSC properties by suppressing Wnt/β-catenin signaling. Interestingly, cells treated with BMP receptor inhibitors K02288 and LDN-212854 blocked HCC-CSC activation by inhibiting BMP9-ID1 signaling, in contrast to cells treated with the TGF-β receptor inhibitor galunisertib. Treatment with LDN-212854 suppressed HCC tumor growth by repressing ID1 and EpCAM in vivo. Our study demonstrates the pivotal role of BMP9-ID1 signaling in promoting HCC-CSC properties and the therapeutic potential of BMP receptor inhibitors in treating EpCAM-positive HCC. Therefore, targeting BMP9-ID1 signaling could offer novel therapeutic options for patients with malignant HCC., (© 2021 The Authors. Molecular Oncology published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Background: ID1 is associated with resistance to the first generation of EGFR tyrosine kinase inhibitors (EGFR-TKIs) in non-small cell lung cancer (NSCLC). However, the effect of ID1 expression on osimertinib resistance in EGFR T790M-positive NSCLC is not clear., Methods: We established a drug-resistant cell line, H1975/OR, from the osimertinib-sensitive cell line H1975. Alterations in ID1 protein expression and Epithelial-mesenchymal transition (EMT)-related proteins were detected with western blot analysis. RT-PCR was used to evaluate the differences of gene mRNA levels. ID1 silencing and overexpression were used to investigate the effects of related gene on osimertinib resistance. Cell Counting Kit-8 (CCK8) was used to assess the proliferation rate in cells with altered of ID1 expression. Transwell assay was used to evaluate the invasion ability of different cells. The effects on the cell cycle and apoptosis were also compared using flow cytometry., Results: In our study, we found that in osimertinib-resistant NSCLC cells, the expression level of the EMT-related protein E-cadherin was lower than that of sensitive cells, while the expression level of ID1 and vimentin were higher than those of sensitive cells. ID1 expression levels was closely related to E-cadherin and vimentin in both osimertinib-sensitive and resistant cells. Alteration of ID1 expression in H1975/OR cells could change the expression of E-cadherin. Downregulating ID1 expression in H1975/OR cells could inhibit cell proliferation, reduce cell invasion, promote cell apoptosis and arrested the cell cycle in the G1/G0 stage phase. Our study suggests that ID1 may induce EMT in EGFR T790M-positive NSCLC, which mediates drug resistance of osimertinib., Conclusions: Our study revealed the mechanism of ID1 mediated resistance to osimertinib in EGFR T790M-positive NSCLC through EMT, which may provide new ideas and methods for the treatment of EGFR mutated NSCLC after osimertinib resistance.

The paradoxical roles of transforming growth factor-β (TGFβ) signaling and nuclear receptor Nur77 in colon cancer development are known but the underlying mechanisms remain obscure. Inhibitor of differentiation 1 (ID1) is a target gene of TGFβ and a key promoter for colon cancer progression. Here, we show that Nur77 enhances TGFβ/Smad3-induced ID1 mRNA expression through hindering Smurf2-mediated Smad3 mono-ubiquitylation, resulting in ID1 upregulation. In the absence of TGFβ, however, Nur77 destabilizes ID1 protein by promoting Smurf2-mediated ID1 poly-ubiquitylation, resulting in ID1 downregulation. Interestingly, TGFβ stabilizes ID1 protein by switching Nur77 interaction partners to inhibit ID1 ubiquitylation. This also endows TGFβ with an active pro-tumorigenic action in Smad4-deficient colon cancers. Thus, TGFβ converts Nur77's role from destabilizing ID1 protein and cancer inhibition to inducing ID1 mRNA expression and cancer promotion, which is highly relevant to colon cancer stemness, metastasis and oxaliplatin resistance. Our data therefore define the integrated duality of Nur77 and TGFβ signaling in regulating ID1 expression and provide mechanistic insights into the paradoxical roles of TGFβ and Nur77 in colon cancer progression.

As the clinical failure of glioblastoma treatment is attributed by multiple components, including myelin-associated infiltration, assessment of the molecular mechanisms underlying such process and identification of the infiltrating cells have been the primary objectives in glioblastoma research. Here, we adopted radiogenomic analysis to screen for functionally relevant genes that orchestrate the process of glioma cell infiltration through myelin and promote glioblastoma aggressiveness. The receptor of the Nogo ligand (NgR1) was selected as the top candidate through Differentially Expressed Genes (DEG) and Gene Ontology (GO) enrichment analysis. Gain and loss of function studies on NgR1 elucidated its underlying molecular importance in suppressing myelin-associated infiltration in vitro and in vivo. The migratory ability of glioblastoma cells on myelin is reversibly modulated by NgR1 during differentiation and dedifferentiation process through deubiquitinating activity of USP1, which inhibits the degradation of ID1 to downregulate NgR1 expression. Furthermore, pimozide, a well-known antipsychotic drug, upregulates NgR1 by post-translational targeting of USP1, which sensitizes glioma stem cells to myelin inhibition and suppresses myelin-associated infiltration in vivo. In primary human glioblastoma, downregulation of NgR1 expression is associated with highly infiltrative characteristics and poor survival. Together, our findings reveal that loss of NgR1 drives myelin-associated infiltration of glioblastoma and suggest that novel therapeutic strategies aimed at reactivating expression of NgR1 will improve the clinical outcome of glioblastoma patients., (© The Author(s) (2021). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Background: The human liver possesses a remarkable capacity for self-repair. However, liver fibrosis remains a serious medical concern, potentially progressing to end-stage liver cirrhosis and even death. Liver fibrosis is characterized by excess accumulation of extracellular matrix in response to chronic injury. Liver regenerative ability, a strong indicator of liver health, is important in resisting fibrosis. In this study, we provide evidence that ursodesoxycholic acid (UDCA) can alleviate liver fibrosis by promoting liver regeneration via activation of the ID1-WNT2/hepatocyte growth factor (HGF) pathway., Methods: Bile duct ligation (BDL) and partial hepatectomy (PH) mouse models were used to verify the effects of UDCA on liver fibrosis, regeneration, and the ID1-WNT2/HGF pathway. An Id1 knockdown mouse model was also used to assess the role of Id1 in UDCA alleviation of liver fibrosis., Results: Our results demonstrate that UDCA can alleviate liver fibrosis in the BDL mice and promote liver regeneration via the ID1-WNT2/HGF pathway in PH mice. In addition, Id1 knockdown abolished the protection afforded by UDCA in BDL mice., Conclusions: We conclude that UDCA protects against liver fibrosis by proregeneration via activation of the ID1-WNT2/HGF pathway., (© 2021 The Authors. Clinical and Translational Medicine published by John Wiley & Sons Australia, Ltd on behalf of Shanghai Institute of Clinical Bioinformatics.)

Casein and whey being food supplements have been considered to be used in oral health care products. However, the response of oral cells to micellar casein and whey powder remains unclear. Considering that milk contains the growth factor TGF-β, and lactoperoxidase was recently reported to decrease the expression of inhibitor of DNA-binding (ID) proteins, there is a rationale to assume that casein and whey can also provoke these responses in oral cells. To examine the TGF-β activity, gingival fibroblasts were exposed to reconstituted casein and whey powder from food supplement before the expression of TGF-β target genes were analyzed by reverse transcription-quantitative polymerase chain reaction. Immunoassays were performed for interleukin11 (IL11) in the cell culture supernatant and for TGF-β in the reconstituted casein and whey. We blocked TGF-β by neutralizing the antibody and the TGF-β receptor type I kinase with the inhibitor SB431542. We also showed smad3 phosphorylation and smad2/3 nuclear translocation by Western blot and immunostaining, respectively. Moreover, with reconstituted casein and whey powder, ID1 and ID3 expression analysis was evaluated in HSC2 human oral squamous carcinoma cells. We report here that casein and whey powder caused a robust increase of TGF-β target genes interleukin11 (IL11), NADPH oxidase 4 (NOX4) and proteoglycan4 (PRG4) in gingival fibroblasts that was blocked by SB431542 and the neutralizing antibody. Moreover, casein and whey powder increased the phosphorylation of smad3 and nuclear translocation of smad2/3. No changes of proliferation markers Ki67 and cyclinD1 were observed. Furthermore, reconstituted casein and whey powder decreased ID1 and ID3 expression in the HSC2 oral squamous carcinoma cells. These findings suggest that the processing of milk into casein and whey powder maintains the TGF-β activity and its capacity to regulate ID1 and ID3 genes in oral fibroblasts and oral squamous carcinoma cells, respectively. These data increase the scientific knowledge on the biological activity of casein and whey with a special emphasis on oral health.

Deubiquitylating enzyme ubiquitin-specific protease 1 (USP1) has been reported to be aberrantly overexpressed in cancers, and it plays a critical role in regulating various cellular processes, such as cell proliferation, apoptosis, and cell differentiation. However, the role of USP1 in B-cell acute lymphoblastic leukemia (B-ALL) remains largely undefined. USP1 expression in 30 newly diagnosed B-ALL patients was detected by real-time PCR and western blot. We found that USP1 was generally upregulated in the bone marrow cells derived from B-ALL patients. Knockdown of USP1 by siRNA decreased B-ALL cell growth and induced apoptosis. Similarly, pharmacological inhibition of USP1 by SJB3-019A significantly repressed cell proliferation and triggered B-ALL cell apoptosis. Finally, we found that inhibition of USP1 downregulated the expression of ID1 and p-AKT, and upregulated ID1 expression could reverse the suppressive effects of USP1 inhibitor in B-ALL cells. Taken together, these results demonstrate that USP1 promote B-ALL progression at least partially via the ID1/AKT signaling pathway, and USP1 inhibitors might be promising therapeutic application for B-ALL., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Clear cell renal cell carcinoma (ccRCC) is one of the most common cancers in the world. Our aim is to identify prognostic biomarkers that contribute to the progression of early stage ccRCC and clarify the mechanism. Here, the mRNA microarray expression profile of ccRCC samples was obtained from Gene Expression Omnibus (GEO) (GSE68417). 62 differentially expressed genes (DEGs) were gained by R Studio, including 31 upregulated genes and 31 downregulated genes. Pathway enrichment analysis was performed in DAVID database. Then, the protein-protein interaction network was obtained through STRING database and visualized by Cytoscape. Subsequently, among the network, only inhibitor of DNA Binding 1 (ID1) was significant between low-grade and high-grade ccRCC patients in TCGA data set. After analysis of the corresponding clinical information in R Studio, it is shown that low ID1 expression correlated with poor survival, high probability of tumor metastasis, and relatively high serum calcium. Later, functional enrichment of ID1 in GeneMANIA uncovered that regulating DNA binding is a main characteristic of ID1 in ccRCC, which was validated by Kaplan-Meier curve of ID1 associated genes using KM plotter database and R Studio. Immune infiltration analysis performed by Tumor Immune Estimation Resource (TIMER) revealed that CD8+ T cells and macrophages were prognostic factors. Furthermore, Valproic acid was analyzed to be the most convinced target drug of ID1 identified by Comparative Toxicogenomics Database (CTD). Taken together, ID1, a biomarker of clinical outcome in early stage ccRCC patients, has the potential function of preventing deterioration in ccRCC progression and metastasis., Competing Interests: The authors declare no conflicts of interest., (Copyright © 2020 Xiangmin Qiu et al.)

The basic helix-loop-helix (bHLH) transcription factors inhibitor of differentiation 1 ( Id1 ) and inhibitor of differentiation 3 (Id3) (referred to as Id ) have an important role in maintaining the cancer stem cell (CSC) phenotype in the triple-negative breast cancer (TNBC) subtype. In this study, we aimed to understand the molecular mechanism underlying Id control of CSC phenotype and exploit it for therapeutic purposes. We used two different TNBC tumor models marked by either Id depletion or Id1 expression in order to identify Id targets using a combinatorial analysis of RNA sequencing and microarray data. Phenotypically, Id protein depletion leads to cell cycle arrest in the G0/G1 phase, which we demonstrate is reversible. In order to understand the molecular underpinning of Id proteins on the cell cycle phenotype, we carried out a large-scale small interfering RNA (siRNA) screen of 61 putative targets identified by using genomic analysis of two Id TNBC tumor models. Kinesin Family Member 11 ( Kif11 ) and Aurora Kinase A ( Aurka ), which are critical cell cycle regulators, were further validated as Id targets. Interestingly, unlike in Id depletion conditions, Kif11 and Aurka knockdown leads to a G2/M arrest, suggesting a novel Id cell cycle mechanism, which we will explore in further studies. Therapeutic targeting of Kif11 to block the Id1-Kif11 axis was carried out using small molecular inhibitor ispinesib. We finally leveraged our findings to target the Id/Kif11 pathway using the small molecule inhibitor ispinesib in the Id+ CSC results combined with chemotherapy for better response in TNBC subtypes. This work opens up exciting new possibilities of targeting Id targets such as Kif11 in the TNBC subtype, which is currently refractory to chemotherapy. Targeting the Id1-Kif11 molecular pathway in the Id1+ CSCs in combination with chemotherapy and small molecular inhibitor results in more effective debulking of TNBC.

Cisplatin (Cis) is a choice chemotherapy approach to cervical cancer by inducing DNA adducts and subsequent apoptosis. We have investigated the effects of Cis on Annexin A1 (ANXA1) and inhibitor of DNA binding 1 (ID1) proteins expression to elucidate further mechanisms of Cis actions. Human cervical tissue samples from twenty-four patients, with Cervical Intraepithelial Neoplasia (CIN, stage I, II and III), were evaluated to quantified ANXA1 and ID1 expressions. In vitro, human epidermoid carcinoma of the cervix (SiHa cell line) were treated with Annexin A1 peptide (ANXA1 2-26 ), Cis or Cis + ANXA1 2-26 to evaluate cell proliferation and migration, cytotoxicity of treatments as well as ANXA1 and ID1 modulations by mRNA and protein expression. Our findings showed expression of ID1 and ANXA1 proteins in tissue samples from Cervical Intraepithelial Neoplasia (CIN) patients, with intense immunological identification of ID1 in the CIN III stage. In SiHa cells, treatments with Cis alone or Cis + ANXA1 2-26 , increase mRNA expressions of the ANXA1 and reduced the ID1. In agreement, Cis + ANXA1 2-26 enhanced ANXA1 protein expression and Cis or Cis + ANXA1 2-26 abolished ID1 protein expression. Cell proliferation was reduced after treatment with ANXA1 2-26 peptide and more significant after Cis or Cis + ANXA1 2-26 treatments. These two last treatments reduced cell viability, by inducing late apoptosis, and impaired cell migration. Together, our data highlight endogenous ANXA1 is involved in Cis therapy for cervical cancer., (Copyright © 2020. Published by Elsevier Masson SAS.)

Although mechanical forces are involved in pressure-overloaded cardiomyopathy, their effects on gene transcription profiles are not fully understood. Here, we used next-generation sequencing (NGS) to investigate changes in genomic profiles after cyclic mechanical stretching of human cardiomyocytes. We found that 85, 87, 32, 29, and 28 genes were differentially expressed after 1, 4, 12, 24, and 48 hours of stretching. Furthermore, 10 of the 29 genes that were up-regulated and 11 of the 28 that were down-regulated after 24 h showed the same changes after 48 h. We then examined expression of the genes that encode serpin family E member 1 (SERPINE1), DNA-binding protein inhibitor 1 (ID1), DNA-binding protein inhibitor 3 (ID3), and CCL2, a cytokine that acts as chemotactic factor in monocytes, in an RT-PCR experiment. The same changes were observed for all four genes after all cyclic stretching durations, confirming the NGS results. Taken together, these findings suggest that cyclical stretching can alter cardiac cell physiology by activating cardiac cell metabolism and impacting cholesterol biosynthesis signaling.

Centrosomes are the primary microtubule-organizing centers that are important for mitotic spindle assembly. Centrosome amplification is commonly observed in human cancer cells and contributes to genomic instability. However, it is not clear how centrosome duplication is dysregulated in cancer cells. Here, we report that ATAD5, a replisome protein that unloads PCNA from chromatin as a replication factor C-like complex (RLC), plays an important role in regulating centrosome duplication. ATAD5 is present at the centrosome, specifically at the base of the mother and daughter centrioles that undergo duplication. UAF1, which interacts with ATAD5 and regulates PCNA deubiquitination as a complex with ubiquitin-specific protease 1, is also localized at the centrosome. Depletion of ATAD5 or UAF1 increases cells with over-duplicated centrosome whereas ATAD5 overexpression reduces such cells. Consistently, the proportion of cells showing the multipolar mode of chromosome segregation is increased among ATAD5-depleted cells. The localization and function of ATAD5 at the centrosomes do not require other RLC subunits. UAF1 interacts and co-localizes with ID1, a protein that increases centrosome amplification upon overexpression. ATAD5 depletion reduces interactions between UAF1 and ID1 and increases ID1 signal at the centrosome, providing a mechanistic framework for understanding the role of ATAD5 in centrosome duplication.

The inhibitor of differentiation (Id) transcription regulators, which are induced in response to oxidative stress, promote cell proliferation and inhibit senescence. Inhibitor of differentiation 1 (Id1) expression is limited to endothelial cells (EC) in the normal mouse kidney and is required for a normal response to injury. Endothelial dysfunction leads to the development of diabetic nephropathy, and so, we hypothesized that endothelial Id1 may help protect against hyperglycemia-induced microvascular injury and nephropathy. Here, we tested this hypothesis by using streptozotocin to induce diabetes in Id1 knockout (KO) mice and WT B6;129 littermates and examining the mice at 3 months. Expression of Id1 was observed to be increased 15-fold in WT kidney EC, and Id1 KO mice exhibited increased mesangial and myofibroblast proliferation, matrix deposition, and albuminuria compared with WT mice. Electron microscopy demonstrated peritubular capillary EC injury and lumen narrowing, and fluorescence microangiography showed a 45% reduction in capillary perfusion area with no reduction in CD31-stained areas in Id1 KO mice. Microarray analysis of EC isolated from WT and KO control and diabetic mice demonstrated activation of senescence pathways in KO cells. Kidneys from KO diabetic mice showed increased histological expression of senescence markers. In addition, premature senescence in cultured KO EC was also seen in response to oxidative stress. In conclusion, endothelial Id1 upregulation with hyperglycemia protects against microvascular injury and senescence and subsequent nephropathy., (© 2020 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.)

Inhibitor of DNA-binding/differentiation (Id) proteins, a family of helix-loop-helix (HLH) proteins that includes four members of Id1 to Id4 in mammalian cells, are critical for regulating cell growth, differentiation, senescence, cell cycle progression, and increasing angiogenesis and vasculogenesis, as well as accelerating the ability of cell migration. Alzheimer's disease (AD), the most common neurodegenerative disease in the adult population, manifests the signs of cognitive decline, behavioral changes, and functional impairment. The underlying mechanisms for AD are not well-clarified yet, but the aggregation of amyloid-beta peptides (Aβs), the major components in the senile plaques observed in AD brains, contributes significantly to the disease progression. Emerging evidence reveals that aberrant cell cycle reentry may play a central role in Aβ-induced neuronal demise. Recently, we have shown that several signaling mediators, including Id1, hypoxia-inducible factor-1 (HIF-1), cyclin-dependent kinases-5 (CDK5), and sonic hedgehog (Shh), may contribute to Aβ-induced cell cycle reentry in postmitotic neurons; furthermore, Id1 and CDK5/p25 mutually antagonize the expression/activity of each other. Therefore, Id proteins may potentially have clinical applications in AD. In this review article, we introduce the underlying mechanisms for cell cycle dysregulation in AD and present some examples, including our own studies, to show different aspects of Id1 in terms of cell cycle reentry and other signaling that may be crucial to alter the neuronal fates in this devastating neurodegenerative disease. A thorough understanding of the underlying mechanisms may provide a rationale to make an earlier intervention before the occurrence of cell cycle reentry and subsequent apoptosis in the fully differentiated neurons during the progression of AD or other neurodegenerative diseases.

Glucagon-like peptide-1 (GLP-1) is a well-known incretin hormone secreted from enteroendocrinal L cells in response to nutrients, such as glucose and dietary fat, and controls glycemic homeostasis. However, the detailed intracellular mechanisms of how L cells control GLP-1 secretion in response to nutrients still remain unclear. Here, we report that bone morphogenetic protein (BMP) signaling pathway plays a pivotal role to control GLP-1 secretion in response to nutrient replenishment in well-established mouse enteroendocrinal L cells (GLUTag cells). Nutrient starvation dramatically reduced cellular respiration and GLP-1 secretion in GLUTag cells. Transcriptome analysis revealed that nutrient starvation remarkably reduced gene expressions involved in BMP signaling pathway, whereas nutrient replenishment rescued BMP signaling to potentiate GLP-1 secretion. Transient knockdown of inhibitor of DNA binding (ID)1, a well-known target gene of BMP signaling, remarkably reduced GLP-1 secretion. Consistently, LDN193189, an inhibitor of BMP signaling, markedly reduced GLP-1 secretion in L cells. In contrast, BMP4 treatment activated BMP signaling pathway and potentiated GLP-1 secretion in response to nutrient replenishment. Altogether, we demonstrated that BMP signaling pathway is a novel molecular mechanism to control GLP-1 secretion in response to cellular nutrient status. Selective activation of BMP signaling would be a potent therapeutic strategy to stimulate GLP-1 secretion in order to restore glycemic homeostasis.

Background : IL-1β is reported to be involved in cancer development and distant metastasis. However, the underlying mechanism of IL-1β upon malignant behaviors remains largely unknown. In this study, we aimed to study whether IL-1β could enhance the stemness traits of tumor cells. Methods : The concentrations of serum IL-1β in head and neck squamous cell carcinoma (HNSCC) and melanoma patients were detected using ELISA assay. The effect and mechanisms of IL-1β on tumor cell growth, migration, invasion and stemness characters were studied using HNSCC cell SCC7 and melanoma cell B16-F10. The underlying mechanisms were further explored. Results : Enhanced concentrations of IL-1β were positively correlated with advanced tumor stage in both HNSCC and melanoma patients. IL-1β treatment led to a significant increase in tumor growth both in vitro and in vivo. IL-1β stimulation promoted cell proliferation, colony formation and tumorigenicity. In addition, IL-1β-stimulated tumor cells gained enhanced capabilities on wounding healing and invasion capabilities. Moreover, IL-1β stimulation promoted the stem-like capabilities of both HNSCC cells and melanoma cells, including the enrichment of aldehyde dehydrogenase + (ALDH + ) cells, up-regulation of stem cell related markers Nanog, OCT4, and SOX2, sphere formation and chemoresistance. Mechanistically, IL-1β treatment promoted the phosphorylation of Smad1/5/8 and activated its downstream target inhibitor of differentiation 1 (ID1). Silencing ID1 abrogated sphere formation and upregulated expression of stemness genes which were induced by IL-1β stimulation. Conclusion : Our data demonstrates that IL-1β promotes the stemness of HNSCC and melanoma cells through activating Smad/ID1 signal pathway., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Investigating mechanisms that regulate endothelial cell (EC) growth and survival is important for understanding EC homeostasis and how ECs maintain stem cell niches. We report here that targeted loss of Id genes in adult ECs results in dilated, leaky sinusoids and a pro-inflammatory state that increases in severity over time. Disruption in sinusoidal integrity leads to increased hematopoietic stem cell (HSC) proliferation, differentiation, migration, and exhaustion. Mechanistically, sinusoidal ECs (SECs) show increased apoptosis because of reduced Bcl2-family gene expression following Id gene ablation. Furthermore, Id1 -/- Id3 -/- SECs and upstream type H vessels show increased expression of cyclin-dependent kinase inhibitors p21 and p27 and impaired ability to proliferate, which is rescued by reducing E2-2 expression. Id1 -/- Id3 -/- mice do not survive sublethal irradiation because of impaired vessel regeneration and hematopoietic failure. Thus, Id genes are required for the survival and regeneration of BM SECs during homeostasis and stress to maintain HSC development., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

BACKGROUND Sorafenib, which is a multitargeted kinase inhibitor, has shown some antitumor effects in patients with non-small cell lung cancer (NSCLC). However, the potential target of sorafenib's antitumor activity is largely unknown. Moreover, definitive predictive biomarkers of benefit have rarely been reported. MATERIAL AND METHODS The alteration in inhibitor of differentiation 1 (ID1) expression in NSCLC cells with sorafenib treatment was detected by western blotting. The sensitivity of NSCLC cells to sorafenib was observed by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium) assay. Loss-of-function and gain-of-function experiments were performed to observe the role of ID1 expression in epithelial to mesenchymal transition (EMT) progression. RESULTS Initially, we observed that ID1 was downregulated in NSCLC cells treated with sorafenib. The response of NSCLC cells to sorafenib was inhibited by the transfection of small interfering RNAs (siRNAs) targeting ID1. In contrast, the transfection of ID1-overexpressing plasmids improved the response of NSCLC cells to sorafenib. Further experiments indicated that ID1 is expressed at high levels in epithelial H460 cells and expressed at low levels in mesenchymal H358 cells. Loss-of-function and gain-of-function experiments suggested that ID1 negatively regulates EMT in NSCLC. CONCLUSIONS The expression of ID1 is dose-dependently inhibited by sorafenib, and the overexpression of ID1 contributes to the antitumor activity of sorafenib by suppressing EMT development. Our results indicate that ID1 might be a potential target for the antitumor activity of sorafenib in NSCLC and that targeting ID1 is a feasible strategy to improve the sensitivity of NSCLC cells to sorafenib.

The inhibitor of DNA binding (Id) proteins are regulators of cell cycle and cell differentiation. Of all Id family proteins, Id1 is mostly linked to tumorigenesis, cellular senescence as well as cell proliferation and survival. Id1 is a stem cell-like gene more than a classical oncogene. Id1 is overexpressed in numerous types of cancers and exerts its promotion effect to these tumors through different pathways. Briefly, Id1 was found significantly correlated with EMT-related proteins, K-Ras signaling, EGFR signaling, BMP signaling, PI3K/Akt signaling, WNT and SHH signaling, c-Myc signaling, STAT3 signaling, RK1/2 MAPK/Egr1 pathway and TGF-β pathway, etc. Id1 has potent effect on facilitating tumorous angiogenesis and metastasis. Moreover, high expression of Id1 plays a facilitating role in the development of drug resistance, including chemoresistance, radiation resistance and resistance to drugs targeting angiogenesis. However, controversial results were also obtained. Overall, Id1 represent a promising target of anti-tumor therapeutics based on its potent promotion effect to cancer. Numerous drugs were found exerting their anti-tumor function through Id1-related signaling pathways, such as fucoidan, berberine, tetramethylpyrazine, crizotinib, cannabidiol and vinblastine., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Milk consumption may modify the risk of squamous cell carcinoma. The role of milk to modulate the gene expression in oral squamous cell carcinoma cells has not been investigated so far. Here, HSC2 oral squamous carcinoma cells were exposed to an aqueous fraction of human milk and a whole-genome array was performed. Among the genes that were significantly reduced by human and cow milk were the DNA-binding protein inhibitor 1 (ID1), ID3 and Distal-Less Homeobox 2 (DLX2) in HSC2 cells. Also, in TR146 oral squamous carcinoma cells, there was a tendency towards a decreased gene expression. Upon size fractionation, lactoperoxidase but not lactoferrin and osteopontin was identified to reduce ID1 and ID3 in HSC2 cells. Dairy products and hypoallergenic infant formula failed to decrease the respective genes. These data suggest that milk can reduce the expression of transcription factors in oral squamous carcinoma cells.

The efficacy of immunotherapies for malignant melanoma is severely hampered by local and systemic immunosuppression mediated by myeloid-derived suppressor cells (MDSC). Inhibitor of differentiation 1 (ID1) is a transcriptional regulator that was shown to be centrally involved in the induction of immunosuppressive properties in myeloid cells in mice, while it was overexpressed in CD11b + cells in the blood of late-stage melanoma patients. Therefore, we comprehensively assessed ID1 expression in PBMC from stage III and IV melanoma patients, and studied ID1 regulation in models for human monocyte differentiation towards monocyte-derived dendritic cells. A highly significant elevation of ID1 was observed in CD33 + CD11b + CD14 + HLA-DR low monocytic MDSC in the blood of melanoma patients compared to their HLA-DR high counterparts, while expression of ID1 correlated positively with established MDSC markers S100A8/9 and iNOS. Moreover, expression of ID1 in monocytes significantly decreased in PBMC samples taken after surgical removal of melanoma metastases, compared to those taken before surgery. Finally, maturation of monocyte-derived DC coincided with a significant downregulation of ID1. Together, these data indicate that increased ID1 expression is strongly associated with expression of phenotypic and immunosuppressive markers of monocytic MDSC, while downregulation is associated with a more immunogenic myeloid phenotype. As such, ID1 may be an additional phenotypic marker for monocytic MDSC. Investigation of ID1 as a pharmacodynamic biomarker or its use as a target for modulating MDSC is warranted.

One neurotoxic mechanism of amyloid-beta peptide (Aβ), the major component of senile plaques in the brains of Alzheimer's disease (AD) patients, is to trigger cell cycle reentry in fully differentiated neurons. However, the detailed underlying mechanisms remain unclear. Using Aβ25-35-treated primary rat cortical neurons as the experimental system, in the present study we tested whether Aβ-induced inhibitor of differentiation-1 (Id1)/hypoxia-inducible factor-1alpha (HIF-1α) and cyclin-dependent kinase-5 (CDK5) contribute to cell cycle reentry in fully differentiated post-mitotic neurons. We found that Id1-induced HIF-1α mediated Aβ25-35-dependent expression of the cell cycle markers cyclin D1 and proliferating cell nuclear antigen (PCNA), both colocalized with microtubule-associated protein-2 (MAP-2) + cells, indicative of cell cycle reentry in the mature neurons. Aβ25-35 also enhanced p35 cleavage to p25 without affecting CDK5 expression. The CDK5 inhibitor roscovitine and the siRNA targeting CDK5 both suppressed Aβ25-35-dependent HIF-1α expression and cell cycle reentry. Intriguingly, Aβ25-35-induced Id1 repressed p25 production while CDK5/p25 reciprocally inhibited Id1 expression, despite the observation that both Id1 and CDK5/p25 acted upstream of HIF-1α. These results demonstrated that both Id1/HIF-1 and CDK5/HIF-1 contribute to Aβ-induced cell cycle reentry in post-mitotic neurons; furthermore, Id1 and CDK5/p25 reciprocally suppress expression of each other., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Chemoresistance is one of the major reasons leading to ovarian cancer high mortality and poor survival. Studies have shown that the alteration of cellular autophagy is associated with cancer cell chemoresistance. Here, we investigated whether the ovarian cancer chemoresistance is associated with the autophagy induced by the inhibitor of DNA binding 1 (ID1). By using gene overexpression or silencing, luciferase assay and human specimens, we show that ID1 induces high autophagy and confers cancer cell chemoresistance. The mechanistic study demonstrates that ID1 first activates the NF-κB signaling through facilitating the nuclear translocation of NF-κB p65, which strengthens the expression and secretion of IL-6 from cancer cells to subsequently activate the signal transducer and activator of transcription 3 (STAT3) through the protein phosphorylation at Y705. We further identified that STAT3 functions to promote the transcription of the activating transcription factor 6 (ATF6), which induces endoplasmic reticulum stress to promote cellular autophagy, granting cancer cell resistance to both cisplatin and paclitaxel treatment. Moreover, we found a significant correlation between the expression of ID1 and ATF6 in 1104 high grade serous ovarian cancer tissues, and that patients with the high expression of ID1 or ATF6 were resistant to platinum treatment and had the poor overall survival and progression-free survival. Thus, we have uncovered a mechanism in which ID1 confers cancer cell chemoresistance largely through the STAT3/ATF6-induced autophagy. The involved molecules, including ID1, STAT3, and ATF6, may have a potential to be targeted in combination with chemotherapeutic agents to improve ovarian cancer survival.

Background: Percutaneous coronary intervention (PCI) treatment can benefit patients, but also cause irreversible mechanical damage to the vascular endothelium, ultimately leading to restenosis of the target vessel. Thus, achieving rapid re-endothelialization and restoring the integrity of the vascular endothelium and function plays an important role in inhibiting neointimal hyperplasia and preventing restenosis. Id1 (inhibitor of DNA binding/differentiation factor 1) plays an important role in promoting cell proliferation and angiogenesis., Study Objective: This study aims to investigate the relationship between Id1 and NFκB/survivin signaling pathways and their role in injured vascular repair by establishing a rat carotid balloon injury model., Methods: The carotid artery model of rat balloon injury was established. The injured common carotid artery was obtained at different time points after vascular injury. RNA and protein were extracted and the mRNA and protein expression levels of Id1, NFκB and survivin were detected in vascular injury. The NFκB blocker BAY 11-7082 and survivin blocker YM155 were used and the effects of Id1, NFκB, survivin mRNA and protein expression, revascularization of blood vessels and neointimal responsiveness after vascular injury were observed in the vascular tissues of Ad-Id1 transfected balloon injury., Results: Id1, NFκB and survivin were expressed in injured rat carotid arteries. Overexpression of Id1 promoted re-endothelialization of injured vessels through NFκB/survivin signaling pathway, inhibited early vascular endometrial reactive hyperplasia; blocked NFκB the/survivin signaling pathway attenuates the re-endothelialization of Ad-Id1 and the early endothelium of Ad-Id1. Blocking the NFκB/survivin signaling pathway attenuates the re-endothelialization and early reactive hyperplasia of vascular intima of Ad-Id1., Conclusion: NF-kappa B/survivin signaling pathway may play an important role in Id1 promoting vascular re-endothelialization, inhibiting neointimal hyperplasia and preventing vascular restenosis., Competing Interests: The authors report no conflicts of interest in this work., (© 2019 Li et al.)

Id helix-loop-helix (HLH) proteins (Id1-4) bind E protein bHLH transcription factors, preventing them from forming active transcription complexes that drive changes in cell states. Id proteins are primarily expressed during development to inhibit differentiation, but they become re-expressed in adult tissues in diseases of the vasculature and cancer. We show that the genetic loss of Id1/Id3 reduces ocular neovascularization in mouse models of wet age-related macular degeneration (AMD) and retinopathy of prematurity (ROP). An in silico screen identifies AGX51, a small-molecule Id antagonist. AGX51 inhibits the Id1-E47 interaction, leading to ubiquitin-mediated degradation of Ids, cell growth arrest, and reduced viability. AGX51 is well-tolerated in mice and phenocopies the genetic loss of Id expression in AMD and ROP models by inhibiting retinal neovascularization. Thus, AGX51 is a first-in-class compound that antagonizes an interaction formerly considered undruggable and that may have utility in the management of multiple diseases., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

This study aimed to investigate the role of prostate cancer associated transcript 1 (PCAT1) underlying the molecular mechanisms of prostate cancer. Using GSE29886 data set downloaded from Gene Expression Omnibus database, we screened the differentially expressed genes (DEGs) in PCAT1-siRNA interfering (PCAT1-siRNA) LNCaP cells compared with control-siRNA cells. Transcription factor (TF) and tumor-associated genes database were used to obtain oncogenes and tumor suppressor genes. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis were used to investigate the function and pathways of DEGs. Subnetwork was further analyzed using BioNet. A total of 93 DEGs were identified. KEGG analysis showed downregulated TF genes (ID1 and ID3) were enriched in transforming growth factor- β pathway, whereas upregulated genes were involved in pathways associated with immune system, environmental sensing, and metabolism. GO analysis showed that downregulated genes were primarily enriched in cell cycle-related biological functions and upregulated DEGs were related to immune response, exogenous genetic material response, and viral response. Centromere protein F (CENPF) was identified as the central node of the regulatory subnetwork. In the PCAT1 knockdown LNCaP cells, the CENPF, ID1, and ID3 were obviously decreased based on the RT-PCR (quantitative real-time reverse transcription PCR) analysis. PCAT1 may be involved in cell cycle and proliferation of prostate cancers by mediating the expression of CENPF , ID1 , and ID3 .

Glioblastoma is the most common primary brain tumor in adults. While the introduction of temozolomide chemotherapy has increased long-term survivorship, treatment failure and rapid tumor recurrence remains universal. The transcriptional regulatory protein, inhibitor of DNA-binding-1 (ID1), is a key regulator of cell phenotype in cancer. We show that CRISPR-mediated knockout of ID1 in glioblastoma cells, breast adenocarcinoma cells, and melanoma cells dramatically reduced tumor progression in all three cancer systems through transcriptional downregulation of EGF, which resulted in decreased EGFR phosphorylation. Moreover, ID1-positive cells were enriched by chemotherapy and drove tumor recurrence in glioblastoma. Addition of the neuroleptic drug pimozide to inhibit ID1 expression enhanced the cytotoxic effects of temozolomide therapy on glioma cells and significantly prolonged time to tumor recurrence. Conclusively, these data suggest ID1 could be a promising therapeutic target in patients with glioblastoma. SIGNIFICANCE: These findings show that the transcriptional regulator ID1 is critical for glioblastoma initiation and chemoresistance and that inhibition of ID1 enhances the effect of temozolomide, delays tumor recurrence, and prolongs survival., (©2019 American Association for Cancer Research.)