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Hepatitis B virus (HBV) infection is closely related with the occurrence and development of hepatocellular carcinoma (HCC), in which Hepatitis B virus x protein (HBx) and core protein (HBc) play crucial roles. Additionally, inhibitors of differentiation (Id) proteins exhibited significant correlation with liver cancer development. Here, we identified that HBV dramatically inhibited the expression of Id1 and Id3 in both protein and transcriptional levels for the first time, whereas there was little effect of the virus on Id2. Additionally, two HBV coded protein, HBc and HBx, could reduce the expression of Id1 and Id3 distinctly, whereas the other two viral proteins, HBs and HBp were unable to affect Id1 and Id3 proteins. Both the activity inhibitors and activators further confirmed that HBc inhibited the expression of Id1 and Id3 by BMP/Smad signaling pathway. HBx could interact with both Id1 and Id3 at residues 112-136 of HBx protein, and it could inhibit the two Id proteins by accelerating their degradation. This is the first report about HBc and HBx regulating Id1 and Id3, whereas the detailed mechanism associated with above needed further experiments to clarify., Competing Interests: Declaration of Competing Interest The authors declare that they have no potential conflicts of interest., (Copyright © 2020. Published by Elsevier B.V.)

Carbohydrate responsive element binding protein (ChREBP) is a major transcription factor of lipogenesis regulated by glucose status in the liver. However, the function of ChREBP in osteogenic differentiation is unclear. The present study examined the role of ChREBP in osteoblast differentiation in MC3T3-E1 preosteoblast cell line. The mRNA expression of ChREBP, protein phosphatase 2A catalytic subunit-α (PP2A Cα) and the osteogenic genes such as, DNA-binding protein inhibitor (Id1), runt-related transcription factor-2 (Runx2), and alkaline phosphatase (ALP) was measured by qPCR and RT-PCR. Runx2, ChREBP, and PP2A Cα, protein levels were evaluated by Western blotting. ALP staining experiment was carried out to evaluate ALP enzyme activity, and a luciferase reporter assay was performed to analyze Runx2 transcriptional activity. Expression of ChREBP and PP2A Cα did not change during bone morphogenetic protein-2 (BMP2)-induced osteoblast differentiation. Overexpression of ChREBP reduced the osteogenic genes (Runx2 and ALP) expression and ALP activity, while knockdown of ChREBP had the opposite effects. Overexpression of PP2A Cα increased ChREBP expression, while inhibition of PP2A Cα using okadaic acid not only inhibited the expression of ChREBP, but also restored the mRNA and protein expression of Runx2 and activity of ALP enzyme. These results demonstrate that ChREBP inhibits BMP2-induced osteoblast differentiation in a PP2A Cα- dependent manner., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Bone morphogenetic proteins regulate a diverse range of biological processes through their activation of SMAD1, SMAD5, or SMAD8 proteins that, in turn, regulate gene expression. These SMAD transcription factors achieve a layer of functional specificity in different cell types largely through actions with additional transcriptional regulatory molecules. In this study, we demonstrate that the forkhead box C1 (FOXC1) transcription factor can modulate bone morphogenetic protein (BMP) signaling to impair the expression of BMP4-responsive genes and prevent the efficient osteoblast differentiation. We demonstrate that repression occurs downstream of BMP signaling and impacts the ability SMAD1 or SMAD5 to activate gene expression. Repression of SMAD activity requires FOXC1 DNA-binding capacity and the transcriptional inhibitory domain of FOXC1. We report that FOXC1 inhibits BMP4 induction of Id1 expression and identify a motif in the regulatory region of mouse Id1 gene that FOXC1 binds. We determine that this inhibition by FOXC1 binding does not affect SMAD1, SMAD5, or SMAD8 binding to its target sequence in the Id1 gene. Finally, we determine that the elevated expression of FOXC1 can reduces expression osteogenic differentiation genes in mouse embryonic stems directed to the osteoblast lineage through BMP4 treatment. Together, these findings indicate that FOXC1 can negatively regulate certain aspects of BMP4 signaling required for osteoblast differentiation. We propose that FOXC1 acts to attenuate the initial BMP-activated pathways that establish osteoblast differentiation and allow for terminal osteoblast differentiation to conclude., (© 2020 Wiley Periodicals, Inc.)

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).)

Many aspects remain elusive of the mechanisms governing T cell quiescence. Here we show that E protein activity helps to establish a quiescent program in naïve T cells. Decreased E protein activity, as the consequence of enforced expression of an Id1 transgene, led to the accumulation of CD4 + CD44 hi T cells. The naïve CD4 + T cells from this transgenic strain mounted a vigorous proliferative response upon TCR stimulation, as a result of direct inhibition of E protein activity. Transcriptome analyses demonstrated that Id1-tg naïve CD4 + T cells exhibited a transcriptional profile characteristic of activated CD4 + T cells, with particular enrichment in the gene set related to PI3K-AKT signaling. Western blot analysis confirmed low but constitutive activation of this pathway. Moreover, the Id1-tg CD4 + T cells displayed enhanced formation of TCR microcluster. Taken together, these data support that downregulation of E protein activity facilitates the exit of naïve T cells from quiescence., 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 © 2020 Elsevier Inc. All rights reserved.)

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.

ID1 is an oncogenic factor in cancer, but its role in relation to drug sensitivity is unclear. This study aimed to investigate the role of ID1 in drug sensitivity in non-small cell lung cancer (NSCLC). ID1 overexpression in NSCLC cells harboring either EGFR or KRAS mutation was performed and the sensitivity of NSCLC to gefitinib (ZD1839) was measured. A murine orthotopic lung carcinoma model with or without stable ID1 overexpression was developed and treated with gefitinib. Transcriptomic and bioinformatics analyses showed that ID1 overexpression promoted inflammation-related cell death but not apoptosis in gefitinib-treated NSCLC cells. ID1 induced necroptosis by triggering activation of RIP1/RIP3/MLKL pathways. Protein kinase array further suggested that ID1 overexpression maintains Akt activity in gefitinib-treated NSCLC cells, which in turn upregulated FLICE-like inhibitory protein to dissociate the caspase-8-RIP1 complex. The association of RIP1 and RIP3 further activated necroptotic cell death in gefitinib-treated NSCLC. In conclusion, ID1 overexpression in NSCLC induced cellular sensitivity to epidermal growth factor receptor tyrosine kinase inhibitors, regardless of the mutational status of NSCLC. The results may provide scientific evidence for optimizing the treatment outcomes of gefitinib for NSCLC patients., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Copyright © 2020. Published by Elsevier B.V.)

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.

Extravillous cytotrophoblasts (EVTs) invade into the uterine wall and remodel spiral arteries for proper placentation. Studies from us and others have demonstrated that the transforming growth factor-β superfamily member bone morphogenetic protein 2 (BMP2) plays important roles in endometrial decidualization and trophoblast cell invasion. However, BMP2 has also been shown to regulate endothelial cell migration and capillary-like tube formation, as has its downstream signaling molecule inhibitor of DNA binding 1 (ID1). Interestingly, insulin-like growth factor binding protein 3 (IGFBP3) also promotes cell migration and angiogenesis in endothelial precursor cell. Moreover, Id1 has a regulatory effect on Igfbp3 expression in rat prostate epithelial cells. However, whether ID1 and IGFBP3 are integrated in BMP2 signaling and involved in the regulation of trophoblast invasive and endovascular differentiation remains unknown. The objective of our study was to examine the effects of BMP2 on ID1 and IGFBP3 expression and their roles in BMP2-regulated human trophoblast invasion and endothelial-like tube formation. Primary and immortalized (HTR8/SVneo) cultures of human trophoblast cells were employed as study models. BMP2 treatment increased ID1 and IGFBP3 mRNA and protein levels in HTR8/SVneo and primary human EVT cells. Intriguingly, ID1 was essential for BMP2-induced IGFBP3 upregulation in both study models, and BMP2-induced trophoblast invasion was attenuated by knockdown of either ID1 or IGFBP3. In addition, BMP2 significantly increased endothelial-like tube formation and knockdown of ID1 and IGFBP3 reduced basal and BMP2-induced tube formation in HTR8/SVneo cells. Similarly, BMP2 increased placenta growth factor (PlGF) production in HTR8/SVneo cells and these effects were attenuated by knockdown of ID1 or IGFBP3. Our results reveal that BMP2 promotes trophoblast cell invasion and endothelial-like tube formation by ID1-mediated IGFBP3 upregulation., (© 2020 Federation of American Societies for Experimental Biology.)

TGFβ is an important tumor suppressor in pancreatic ductal adenocarcinoma (PDA), yet inactivation of TGFβ pathway components occurs in only half of PDA cases. TGFβ cooperates with oncogenic RAS signaling to trigger epithelial-to-mesenchymal transition (EMT) in premalignant pancreatic epithelial progenitors, which is coupled to apoptosis owing to an imbalance of SOX4 and KLF5 transcription factors. We report that PDAs that develop with the TGFβ pathway intact avert this apoptotic effect via ID1. ID1 family members are expressed in PDA progenitor cells and encode components of a set of core transcriptional regulators shared by PDAs. PDA progression selects against TGFβ-mediated repression of ID1 . The sustained expression of ID1 uncouples EMT from apoptosis in PDA progenitors. AKT signaling and mechanisms linked to low-frequency genetic events converge on ID1 to preserve its expression in PDA. Our results identify ID1 as a crucial node and potential therapeutic target in PDA. SIGNIFICANCE: Half of PDAs escape TGFβ-induced tumor suppression without inactivating the TGFβ pathway. We report that ID1 expression is selected for in PDAs and that ID1 uncouples TGFβ-induced EMT from apoptosis. ID1 thus emerges as a crucial regulatory node and a target of interest in PDA. This article is highlighted in the In This Issue feature, p. 1 ., (©2019 American Association for Cancer Research.)

Although inhibitor of differentiation 1 (Id‑1) and NF‑κB have been shown have play a role in tumorigenesis, their mechanisms and exact roles in cervical cancer have not yet been addressed. This study aimed to investigate the clinical significance of Id‑1 and NF‑κB p65 in cervical cancer and to elucidate their roles in malignant properties. Paraffin‑embedded cervical tissue specimens (n=85) were collected for immunohistochemistry and survival analysis. Targeting Id‑1 with lentivirus in vitro allowed the examination of the NF‑κB signaling pathway and cell survival. The results demonstrated the elevated co‑expression of Id‑1 and nuclear NF‑κB p65 was more frequently associated with aggressive cervical cancer behavior and poorer clinical outcomes. Targeting Id‑1 with short hairpin RNA or Id‑1 overexpression lentivirus in SiHa cells demonstrated that Id‑1 is associated with nuclear NF‑κB p65 expression and cell survival capacity. The physical interaction between Id‑1 and NF‑κB p65 was validated in SiHa cells. Moreover, the survival‑promoting or chemoresistant effects of Id‑1 may be attributed to the subsequent activation of NF‑κB signaling. On the whole, the synchronous co‑expression of Id‑1 and nuclear NF‑κB p65 has a prominent role in cervical cancer, suggesting a combined analysis of Id‑1 and NF‑κB may help to predict malignant properties and prognosis. Aside from NF‑κB, Id‑1 may also be developed as a promising therapeutic strategy for cervical cancer.

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 .

Semaphorins are a family of molecular signals that guide cell migration and are implicated in the regulation of cancer cells. In particular, transmembrane semaphorins are postulated to act as both ligands ("forward" mode) and signaling receptors ("reverse" mode); however, reverse semaphorin signaling in cancer is relatively less understood. Here, we identified a previously unknown function of transmembrane semaphorin 4C (Sema4C), acting in reverse mode, to elicit nonconventional TGF-β/BMP receptor activation and selective SMAD1/5 phosphorylation. Sema4C coimmunoprecipitated with TGFBRII and BMPR1, supporting its role as modifier of this pathway. Sema4C reverse signaling led to the increased abundance of ID1/3 transcriptional factors and to extensive reprogramming of gene expression, which suppressed the typical features of the epithelial-mesenchymal transition in invasive carcinoma cells. This phenotype was nevertheless coupled with burgeoning metastatic behavior in vivo, consistent with evidence that Sema4C expression correlates with metastatic progression in human breast cancers. Thus, Sema4C reverse signaling promoted SMAD1/5- and ID1/3-dependent gene expression reprogramming and phenotypic plasticity in invasive cancer cells., (Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

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.)

Yes-associated protein 1 (YAP1) transcriptional coactivator has recently been identified to regulate skeletal lineage cell differentiation and bone development. However, the role and molecular mechanisms of YAP1 in the regulation of osteoblastic differentiation remains to be elucidated. In this study, we demonstrated that YAP1 expression was increased during osteogenic differentiation of rat bone mesenchymal stem cells and MC3T3-E1. YAP1 overexpression MC3T3-E1 showed increased expression of osteogenesis markers, such as runt-related transcription factor 2, osteocalcin, and osteopontin, as well as alkaline phosphatase and alizarin red staining. Conversely, YAP1 knockdown significantly suppressed MC3T3-E1 osteoblastic differentiation. Mechanistically, we found that YAP1 overexpression upregulated the mRNA and protein expression of the inhibitor of differentiation/DNA binding 1 (ID1), which was contrary to the results of YAP1-knockdown group. Moreover, the early osteogenic differentiation of MC3T3-E1 cells was enhanced by ID1 overexpression. Furthermore, transient transfection with exogenous ID1 overexpression plasmid completely recaptured the decreased effects of YAP1 knockdown on MC3T3-E1 cell differentiation. In addition, β-catenin and AMP-activated protein kinase signaling pathways participated in YAP1 regulation processes. Taken together, our study suggests that YAP1 is a crucial modulator of osteoblast differentiation in vitro, and provides insight into the mechanism by which YAP1 regulates osteoblast differentiation., (© 2019 Wiley Periodicals, Inc.)

The LKB1 tumor suppressor is often mutationally inactivated in non-small cell lung cancer (NSCLC). LKB1 phosphorylates and activates members of the AMPK family of Ser/Thr kinases. Within this family, the salt-inducible kinases (SIKs) modulate gene expression in part via the inhibitory phosphorylation of the CRTCs, coactivators for CREB (cAMP response element-binding protein). The loss of LKB1 causes SIK inactivation and the induction of the CRTCs, leading to the up-regulation of CREB target genes. We identified CRTC2 as a critical factor in LKB1-deficient NSCLC. CRTC2 is unphosphorylated and therefore constitutively activated in LKB1-mutant NSCLC, where it promotes tumor growth, in part via the induction of the inhibitor of DNA binding 1 (ID1), a bona fide CREB target gene. As ID1 expression is up-regulated and confers poor prognosis in LKB1-deficient NSCLC, our results suggest that small molecules that inhibit CRTC2 and ID1 activity may provide therapeutic benefit to individuals with NSCLC.

Fibrodysplasia ossificans progressiva (FOP) is a genetic disease characterized by heterotopic ossification (HO). The disease is caused by a mutation in the activin receptor type 1 (ACVR1) gene that enhances this receptor's responsiveness to Activin-A. Binding of Activin-A to the mutated ACVR1 receptor induces osteogenic differentiation. Whether Activin-A also affects osteoclast formation in FOP is not known. Therefore we investigated its effect on the osteoclastogenesis-inducing potential of periodontal ligament fibroblasts (PLF) from teeth of healthy controls and patients with FOP. We used western blot analysis of phosphorylated SMAD3 (pSMAD3) and quantitative polymerase chain reaction to assess the effect of Activin-A on the PLF. PLF-induced osteoclast formation and gene expression were studied by coculturing control and FOP PLF with CD14-positive osteoclast precursor cells from healthy donors. Osteoclast formation was also assessed in control CD14 cultures stimulated by macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor kappa-B ligand (RANK-L). Although Activin-A increased activation of the pSMAD3 pathway in both control and FOP PLF, it increased ACVR1, FK binding protein 12 (FKBP12), an inhibitor of DNA binding 1 protein (ID-1) expression only in FOP PLF. Activin-A inhibited PLF mediated osteoclast formation albeit only significantly when induced by FOP PLF. In these cocultures, it reduced M-CSF and dendritic cell-specific transmembrane protein (DC-STAMP) expression. Activin-A also inhibited osteoclast formation in M-CSF and RANK-L mediated monocultures of CD14+ cells by inhibiting their proliferation. This study brings new insight on the role of Activin A in osteoclast formation, which may further add to understanding FOP pathophysiology; in addition to the known Activin-A-mediated HO, this study shows that Activin-A may also inhibit osteoclast formation, thereby further promoting HO formation., (© 2018 Authors. Journal of Cellular Physiology Published by Wiley Periodicals, Inc.)

Non-small cell lung cancer (NSCLC) is the leading cause of cancer death in the world. Inhibitor of differentiation 1 (Id1) is overexpressed in NSCLC and involved in promoting its progression and metastasis. Identifying natural compounds targeting Id1 may have utility in NSCLC treatment. Here, we sought to determine whether the anti-tumor activities of Scutellaria flavonoids (SFs) were related to Id1. We reported that three SFs (baicalin, baicalein and wogonin) exhibited strong antitumor activity in NSCLC cells in vitro and in vivo. Id1 played a pivotal role on blockage of migration and invasion by SFs. Abrogation of invasion and migration mediated by baicalin, baicalein and wogonin were totally abolished by ectopic overexpression of Id1. Mechanistically, baicalin, baicalein and wogonin activated Rap1-GTP binding and dephosphorylated Akt and Src by suppressing a7nAChR, consequently triggering inhibition of Id1. Then attenuation of its downstream mediators, VEGF-A, N-cadherin, vimentin, combined with augment of E-cadherin led to the blockage of proliferation, EMT and angiogenesis of NSCLC. Overall, our data shed light on heretofore-undescribed role of SFs as modulators of Id1, which may be a useful strategy in the treatment of NSCLC., Competing Interests: Competing Interests: The authors have declared that no competing interest exists.

Snail1 plays an important role in epithelial to mesenchymal transition (EMT) during tumor metastasis; however, whether Snai1 potentiates the process of neoangiogenesis is completely unknown. In the present study, tube formation assay was used to evaluate neoangiogenesis in vitro The expression of Snai1 and other pro-neoangiogenic factors was measured by quantitative real time PCR. Tumor derived endothelial cells (TDECs) were stimulated with fibroblast growth factor 1 (FGF1) or VEGF and formed more tubes compared with untreated, whereas cells treated with Sulforaphane had less tube formation. Silencing SNAI1 significantly attenuated tube formation accompanied by decreased CD31, CD34, and VWF expression in TDECs compared with control. In contrast, overexpression of Snai1 led to more CD31, CD34, and VWF expression and tube formation. To determine if the observed effects of SNAI1 on tube formation was a global phenomenon, the same assay was conducted in normal mesenchymal stem cells (MSCs). SNAI1 silencing did not have any effect on tube formation in MSCs. The expression of TIMP2, ENG , and HIF1A was up-regulated 3-fold or higher after silencing SNAI1 , and ID1, VEGFA, PLG, LECT1, HPSE were shown down-regulated. Taken together, our study elucidates an important role of EMT inducer Snai1 in regulating tumor neoangiogenesis, suggesting a potential therapeutic target for overcoming tumor EMT., (© 2019 The Author(s).)

This study aims to explore the effect of an inhibitor of DNA binding-1 (Id-1) on the proliferation and migration of human colon carcinoma cell line SW480 and HT-29. SW480 and HT-29 cells transfected with Id-1-interference sequence were assigned to the experimental groups (inhibition groups 1 and 2), and SW480 and HT-29 cells with blank interference sequence (blank groups) and blank load transfection (blank load groups) were assigned as the control groups. The expression of Id-1 in six groups was detected by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot. Cell proliferation in vitro was assessed by MTT assay. RT-PCR and Western blot results demonstrated that the mRNA and protein expressions of Id-1 in the inhibition group 1 were lower than those in the blank group 1 and blank load group 1. RT-PCR and Western blot results revealed that the mRNA and protein expressions of Id-1 were lower in the inhibition group 2 than in the blank group 2 and blank load group 2. The results of the growth curve revealed that proliferation ability was significantly weaker from the third day in the inhibition groups 1 and 2 than in the blank group and blank load group. Transwell chamber experiment and Matrigel invasion assay revealed that the number of Transwell cells significantly decreased in the inhibition groups 1 and 2 than in the blank groups and blank load groups (P < 0.01). Id-1 significantly promotes the proliferation and migration of human colon carcinoma cell lines SW480 and HT-29., (© 2019 The Authors. The Kaohsiung Journal of Medical Sciences published by John Wiley & Sons Australia on behalf of Kaohsiung Medical University.)

ID genes have an important function in the cell cycle, and ID proteins may help identify aggressive tumors, besides being considered promising therapeutic targets. However, their role in thyroid tumors is still poorly understood. We examined ID expression and their correlation with diagnostic and prognostic features aiming to find a clinical application in differentiated thyroid carcinoma (DTC) cases. mRNA levels of ID1, ID2, ID3, and ID4 genes were quantified and their expression was observed by immunohistochemistry in 194 thyroid samples including 68 goiters, 16 follicular adenomas, 75 classic papillary thyroid carcinomas, 18 follicular variants of papillary thyroid carcinoma, 5 follicular thyroid carcinomas, and 1 anaplastic thyroid cancer, besides 11 normal thyroid tissues. DTC patients were managed according to standard protocols and followed up for M = 28 ± 16 months. ID2, ID3, and ID4 mRNA levels were higher in benign (2.0 ± 1.9; 0.6 ± 0.6; and 0.7 ± 1.0 AU, respectively) than those in malignant nodules (0.30 ± 0.62; 0.3 ± 0.3; and 0.2 ± 0.3 AU, respectively, p < 0.0001 for all three genes) and were associated with no extra thyroid invasion or metastasis at diagnosis. ID3 nuclear protein expression was higher in benign than that in malignant cells (5.2 ± 0.9 vs 3.0 ± 1.8 AU; p < 0.0001). On the contrary, the cytoplasmic expression of ID3 was higher in malignant than that in benign lesions (5.7 ± 1.5 vs 4.0 ± 1.4 AU; p < 0.0001). Our data indicate that ID genes are involved in thyroid tumorigenesis and suggest these genes act impeding the evolution of more aggressive phenotypes. The different patterns of their tissue expression may help identify malignancy and characterize thyroid lesion aggressiveness.

Because of the refractory nature of mutant KRAS lung adenocarcinoma (LUAD) to current therapies, identification of new molecular targets is essential. Genes with a prognostic role in mutant KRAS LUAD have proven to be potential molecular targets for therapeutic development. Here we determine the clinical, functional, and mechanistic role of inhibitor of differentiation-1 (Id1) in mutant KRAS LUAD. Analysis of LUAD cohorts from TCGA and SPORE showed that high expression of Id1 was a marker of poor survival in patients harboring mutant, but not wild-type KRAS . Abrogation of Id1 induced G 2 -M arrest and apoptosis in mutant KRAS LUAD cells. In vivo , loss of Id1 strongly impaired tumor growth and maintenance as well as liver metastasis, resulting in improved survival. Mechanistically, Id1 was regulated by the KRAS oncogene through JNK, and loss of Id1 resulted in downregulation of elements of the mitotic machinery via inhibition of the transcription factor FOSL1 and of several kinases within the KRAS signaling network. Our study provides clinical, functional, and mechanistic evidence underscoring Id1 as a critical gene in mutant KRAS LUAD and warrants further studies of Id1 as a therapeutic target in patients with LUAD. SIGNIFICANCE: These findings highlight the prognostic significance of the transcriptional regulator Id1 in KRAS -mutant lung adenocarcinoma and provide mechanistic insight into how it controls tumor growth and metastasis., (©2018 American Association for Cancer Research.)

Penile squamous cell carcinoma (PSCC) occurs more frequently in developing countries, and is commonly diagnosed at an advanced stage with an unfavorable prognosis. At present, few biomarkers for PSCC have been identified and used in clinical practice. Aberrant expression of inhibitor of DNA binding 1 (ID1) has been suggested as a potential regulator of tumor progression in various types cancer. Herein, we evaluated ID1 expression in PSCC and analyzed its association with the clinicopathological parameters of PSCC. Our findings indicated that ID1 overexpression is associated with histological subtype and lymph node metastasis. Kaplan‑Meier survival analysis showed that the overexpression of ID1 is associated with unfavorable cancer‑specific survival. In Cox proportional hazard models, ID1 overexpression was found to be an independent predictor of cancer‑specific survival. Furthermore, we investigated the function of ID1 in PSCC using a PSCC cell line Penl1. Silencing of ID1 expression retarded cell growth, inhibited clonogenesis, and attenuated cell migration and invasion in Penl1 cells. ID1 may regulate key oncogenic and metastasis‑related molecules, as depletion of ID1 expression affected the levels of p‑AKT, p16, PTEN and cleaved caspase‑3, and reduced MMP2/9 secretion in Penl1 cells. Nevertheless, the mRNA expression of p16 and PTEN increased following ID1 knockdown, suggesting that ID1 may repress p16 and PTEN expression in Penl1 cells. Therefore, overexpression of ID1 could serve as a potential prognostic biomarker for the clinical management of PSCC. Strategies targeting ID1‑regulated signaling pathways may have therapeutic benefit in PSCC.

Long non-coding RNAs (lncRNAs) have been reported to play a vital role in non-small-cell lung cancer (NSCLC). ZEB1-AS1 overexpression predicts a poor prognosis in osteosarcoma and colorectal cancers. In the current study, we determined the clinical significance and prognostic value of ZEB1-AS1 in patients with NSCLC. The expression of ZEB1-AS1 and inhibitor of differentiation-1 (ID1) was measured using qRT-PCR and Western blot. Cell growth, migration, and invasion were determined using colony formation assays, Transwell assay, and flow cytometry, respectively. Tumor growth was determined with a xenograft model. ZEB1-AS1 was significantly up-regulated in NSCLC tissues compared with normal samples. ZEB1-AS1 overexpression was significantly associated with advanced tumor, lymph node, and metastases (TNM) stage and tumor size, as well as poorer overall survival. Moreover, ZEB1-AS1 knockdown inhibited NSCLC cell proliferation and migration, and promoted cell apoptosis. In addition, a chromatin immunoprecipitation assay revealed that ZEB1-AS1 interacted with STAT3, thereby repressing ID1 expression. Furthermore, rescue experiments indicated that ZEB1-AS1 functioned as an oncogene partly by repressing ID1 in NSCLC cells. Taken together, our findings indicate that ZEB1-AS1 serves as a promising therapeutic target to predict the prognosis of NSCLC., (© 2019 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Amyloid beta-peptide (Aβ), the neurotoxic component of senile plaques in Alzheimer's disease (AD) brains, is known to trigger cell cycle reentry in post-mitotic neurons followed by apoptosis. However, the underlying mechanisms remain unclear. Recently, we have reported that Aβs stimulate the expression of inhibitor of differentiation-1 (Id1) to induce sonic hedgehog (SHH) (Hung et al., Mol Neurobiol 53(2):793-809, 2016), and both are mitogens capable of triggering cell cycle progression. In this work, we tested the hypothesis that Aβ-induced Id1 and SHH contribute to cell cycle reentry leading to apoptosis in neurons. We found that Aβ triggered cell cycle progression in the post-mitotic neurons, as indicated by the increased expression of two G1-phase markers including cyclin D1 and phosphorylated retinoblastoma protein (pRb), two G2-phase markers such as proliferating cell nuclear antigen (PCNA) and incorporation of 5-bromo-2'-deoxyuridine (BrdU) into newly synthesized DNA, as well as the mitotic marker histone H3 phosphorylated at Ser-10. As expected, Aβ also enhanced caspase-3 cleavage in the cortical neurons. Id1 siRNA, the neutralization antibody against SHH (SHH-Ab), and the cyclin-dependent kinase (CDK)-4/6 inhibitor PD0332991 all attenuated, in part or in full, the Aβ-induced expression of these cell cycle markers. Indeed, exogenous recombinant Id1 protein and the biologically active N-terminal fragment of SHH (SHH-N) were both sufficient to enhance the expression of cell cycle markers independent of Aβ. Taken together, our results revealed the critical roles of Id1 and SHH mediating Aβ-dependent cell cycle reentry and subsequently caspase-dependent apoptosis in the fully differentiated post-mitotic neurons, at least in vitro.

The inhibitor of DNA‑binding (ID) proteins are dominant‑negative modulators of transcription factors with basic helix‑loop‑helix (bHLH) structures, which control a variety of genes in cell cycle regulation. An increasing volume of evidence has demonstrated that the deregulated expression of IDs in several types of malignancy, including breast carcinoma, has been proven to serve crucial regulatory functions in tumorigenesis and the development of breast cancer (BC). The present study evaluated the prognostic values of the ID family members by investigating a set of publicly accessible databases, including Oncomine, bc‑GenExMiner, Kaplan‑Meier plotter and the Human Protein Atlas. The results demonstrated that mRNA levels of distinct IDs exhibited diverse profiles between BC and normal counterparts. The mRNA expression level of ID2 was significantly higher in breast cancer than normal tissues, while the mRNA expression levels of ID1, ID3 and ID4 were significantly lower in breast cancer tissues than in normal tissues. Furthermore, higher mRNA expression levels of ID1 and ID4 were associated with subgroups with lower pathological grades and fewer lymph node metastases. Survival analysis revealed that elevated mRNA levels of ID1 and ID4 predicted an improved survival in all patients with BC. Increased ID1 mRNA levels were associated with higher relapse‑free survival rates in all patients with BC, particularly in those with ER positive and Luminal A subtype tumors. Increased ID4 mRNA expression predicted longer survival times in all patients with BC, particularly in those with hormone receptor‑positive tumors or those treated with endocrine therapy. These results indicated that IDs are essential prognostic indicators in BC. Future studies on the effect of IDs on the pathogenesis and development of BC are warranted.

Sorafenib is the only approved drug for the treatment of advanced hepatocellular carcinoma (HCC). However, its efficacy is limited by the emergence of primary and/or acquired resistance. Senescence-associated secretory phenotype (SASP)-mediated chemo-resistance, which depends on the secreted bioactive molecules, has attracted increasing attention but never revealed in HCC. In this study, we investigated the effect of SASP-related p16/IL6 axis on sorafenib resistance in HCC. Initially, we noticed that HCC cells with a high level of p16/IL6 axis exhibited a low sensitivity to sorafenib. Further in vivo and in vitro studies demonstrated that such a primary resistance resulted from ID1-mediated activation of p16/IL6 axis. Overexpression of ID1 or IL6 blocking in sorafenib-resistant HCC cells could increase the cytotoxicity of sorafenib. Moreover, SASP-related p16/IL6 axis contributed to the formation of acquired resistance in cells received long-term exposure to sorafenib. In acquired sorafenib-resistant cells, ID1 low expression, p16/IL6 axis up-regulation, and AKT phosphorylation activation were observed. A reduced cytotoxicity of sorafenib was detected when sorafenib-sensitive cells incubated with conditioned media from the resistant cells, accompanied by the stimulation of AKT phosphorylation. The reversal of sorafenib resistance could be achieved through ID1 overexpression, IL6 blocking, and AKT pathway inhibition. Our study reveals that SASP-related p16/IL6 axis activation is responsible for sorafenib resistance, which will be a novel strategy to prevent the drug resistance.

Defining mechanisms that maintain tissue stem cells during homeostasis, stress, and aging is important for improving tissue regeneration and repair and enhancing cancer therapies. Here, we show that Id1 is induced in hematopoietic stem cells (HSCs) by cytokines that promote HSC proliferation and differentiation, suggesting that it functions in stress hematopoiesis. Genetic ablation of Id1 increases HSC self-renewal in serial bone marrow transplantation (BMT) assays, correlating with decreases in HSC proliferation, mitochondrial biogenesis, and reactive oxygen species (ROS) production. Id1 -/- HSCs have a quiescent molecular signature and harbor less DNA damage than control HSCs. Cytokines produced in the hematopoietic microenvironment after γ-irradiation induce Id1 expression. Id1 -/- HSCs display a blunted proliferative response to such cytokines and other inducers of chronic proliferation including genotoxic and inflammatory stress and aging, protecting them from chronic stress and exhaustion. Thus, targeting Id1 may be therapeutically useful for improving HSC survival and function during BMT, chronic stress, and aging., (Copyright © 2018 Elsevier Inc. All rights reserved.)

A family of transcription factors known as Id proteins, or inhibitor of DNA binding and differentiation, is capable of regulating cell proliferation, survival and differentiation, and is often upregulated in multiple types of tumors. Due to their ability to promote self-renewal, Id proteins have been considered as oncogenes, and potential therapeutic targets in cancer models. On the contrary, certain Id proteins are reported to act as tumor suppressors in the development of Burkitt's lymphoma in humans, and hepatosplenic and innate-like T cell lymphomas in mice. The contexts and mechanisms by which Id proteins can serve in such contradictory roles to determine tumor outcomes are still not well understood. In this review, we explore the roles of Id proteins in lymphocyte development and tumorigenesis, particularly with respect to inhibition of their canonical DNA binding partners known as E proteins. Transcriptional regulation by E proteins, and their antagonism by Id proteins, act as gatekeepers to ensure appropriate lymphocyte development at key checkpoints. We re-examine the derailment of these regulatory mechanisms in lymphocytes that facilitate tumor development. These mechanistic insights can allow better appreciation of the context-dependent roles of Id proteins in cancers and improve considerations for therapy.

Inhibitor of DNA binding 1 (Id1) has been shown to be involved in adipogenesis and metabolism, which may contribute to atherosclerotic progression. However, it remains unclear how Id1 regulates endothelial cell functions and atherosclerosis in response to oscillatory shear stress. The current study aims to evaluate the effects of oscillatory shear stress on LDL uptake by endothelial cells and to delineate the roles of Id1 in this process. Using an in vivo ligation model of ApoE -/- mice and applying low and oscillatory shear stress (OSS) in vitro, we found that OSS can effectively promote lipid uptake. In vivo en face staining results showed that OSS down-regulated Id1 expression. In vitro, OSS activated Id1 transiently but eventually inhibited its expression with time. Overexpression of Id1 can abolish OSS-mediated lipid uptake in ECs. In addition, Id1 overexpression and knockdown experiments demonstrated that Id1 can regulate LDLR expression to influence lipid uptake. Immunoprecipitation and subcellular localization results further suggested that Id1 can combine with sterol regulatory element-binding protein1 (SREBP1), which may be related to the Id1-mediated LDLR down-expression. Our study shows a biomechanical role of Id1 in endothelial cells' uptake of lipid by down-regulating LDLR, which could help understand how oscillatory flow affects atherosclerotic development.

Pancreatic ductal adenocarcinoma (PDAC) has poor prognosis and high mortality rate. Inhibitor of differentiation-1 (ID-1) overexpression has already been reported to be associated with low survival, but the detailed roles of ID-1 in PDAC are unclear. As an ID-1 inhibitor, 2-methoxyestradiol (2-ME) has been shown effective in the antitumor therapies, but its effect on PDAC is unknown. In this study, ID-1 overexpression and knockdown stable SW1990 cells were used to examine proliferation, migration and invasion abilities. Effectiveness of 2-ME was estimated in vivo, as well as in vitro by survival analysis, magnetic resonance imaging and tissue analysis. PDAC patients were retrospectively reviewed to assess the ID-1 expression and prognosis. We found that ID-1 was closely associated with in vitro SW1990 cells proliferation, migration and invasion abilities, as well as the expression of HIF-1α, VEGF and MMP-9. 2-ME was shown to be effective on tumor suppression both in vivo and in vitro, perhaps mainly by ID-1 regulation. Moreover, the relevance between high ID-1 expression and poor prognosis was validated in PDAC patients. Our data collectively reveals the roles of ID-1 in PDAC malignancy, and suggests 2-ME treatment may be a potential alternative chemotherapeutic agent for PDAC patients.

4-Hydroxy-3-methoxybenzaldehyde (vanillin), contained in a number of species of plant, has been reported to display beneficial effects against brain injuries. In the present study, the impact of vanillin on scopolamine‑induced alterations in cognition and the expression of DNA binding protein inhibitor ID‑1 (ID1), one of the inhibitors of DNA binding/differentiation proteins that regulate gene transcription, in the mouse hippocampus. Mice were treated with 1 mg/kg scopolamine with or without 40 mg/kg vanillin once daily for 4 weeks. Scopolamine‑induced cognitive impairment was observed from 1 week and was deemed to be severe 4 weeks following the administration of scopolamine. However, treatment with vanillin in scopolamine‑treated mice markedly attenuated cognitive impairment 4 weeks following treatment with scopolamine. ID1‑immunoreactive cells were revealed in the hippocampus of vehicle‑treated mice, and were hardly detected 4 weeks following treatment with scopolamine. However, treatment with vanillin in scopolamine‑treated mice markedly restored ID1‑immunoreactive cells and expression 4 weeks subsequent to treatment. The results of the present study suggested that vanillin may be beneficial for cognitive impairment, by preventing the reduction of ID1 expression which may be associated with cognitive impairment.

Brain arteriovenous malformations (AVMs) which associate with angiogenesis due to local hypertension, chronic cerebral ischaemia and tissue hypoxia usually lead to haemorrhage, however, the therapeutic medicine for the disease is still lacking. 2-methoxyestradiol (2-ME) has been shown effective in the anti-angiogenic treatment. This study was conducted to examine whether and how 2-ME could improve the vascular malformations. Intracranial venous hypertension (VH) model produced in adult male Sprague-Dawley rats and culture of human umbilical vein endothelial cells (HUVECs) at the anoxia condition were used to induce in vivo and in vitro angiogenesis, respectively. Lentiviral vectors of ID-1 and p53 genes and of their siRNA were intracranially injected into rats and transfected into HUVECs to overexpress and down-regulate these molecules. 2-ME treatment not only reduced the in vivo progression of brain tissue angiogenesis in the intracranial VH rats and the in vitro increases in microvasculature formation, cellular migration and HIF-1α expression induced by anoxia in HUVECs but also reversed the up-regulation of ID-1 and down-regulation of p53 in both the in vivo and in vitro angiogenesis models. All of the anti-angiogenesis effects of 2-ME observed in VH rats and anoxic HUVECs were abrogated by ID-1 overexpression and p53 knockdown. Our data collectively suggest that 2-ME treatment inhibits hypoxia/anoxia-induced angiogenesis dependently on ID-1 down-regulation and p53 up-regulation, providing a potential alternative medical treatment for un-ruptured AVM patients., (© 2017 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.)

The best characterized signaling pathway downstream of transforming growth factor β (TGF-β) is through SMAD2 and SMAD3. However, TGF-β also induces phosphorylation of SMAD1 and SMAD5, but the mechanism of this phosphorylation and its functional relevance is not known. Here, we show that TGF-β-induced SMAD1/5 phosphorylation requires members of two classes of type I receptor, TGFBR1 and ACVR1, and establish a new paradigm for receptor activation where TGFBR1 phosphorylates and activates ACVR1, which phosphorylates SMAD1/5. We demonstrate the biological significance of this pathway by showing that approximately a quarter of the TGF-β-induced transcriptome depends on SMAD1/5 signaling, with major early transcriptional targets being the ID genes. Finally, we show that TGF-β-induced epithelial-to-mesenchymal transition requires signaling via both the SMAD3 and SMAD1/5 pathways, with SMAD1/5 signaling being essential to induce ID1. Therefore, combinatorial signaling via both SMAD pathways is essential for the full TGF-β-induced transcriptional program and physiological responses., Competing Interests: AR, PV, DD, PC, JV, KR, PM, AH, GS, CH No competing interests declared, (© 2018, Ramachandran et al.)

Purpose: Normal stem cells tightly control self-renewal and differentiation during development, but their neoplastic counterparts, cancer stem cells (CSCs), sustain tumorigenicity both through aberrant activation of stemness and evasion of differentiation. Although regulation of CSC stemness has been extensively studied, the molecular mechanisms suppressing differentiation remain unclear. Experimental Design: We performed in silico screening and in vitro validation studies through Western blotting, qRT-PCR for treatment of WNT and SHH signaling inhibitors, and BMP signaling inducer with control and ID1-overexpressing cells. We also performed in vivo drug treatment assays with Balb/c nude mice. Results: Inhibitor of differentiation 1 (ID1) abrogated differentiation signals from bone morphogenetic protein receptor (BMPR) signaling in glioblastoma stem cells (GSCs) to promote self-renewal. ID1 inhibited BMPR2 expression through miRNAs, miR-17 and miR-20a, which are transcriptional targets of MYC. ID1 increases MYC expression by activating WNT and SHH signaling. Combined pharmacologic blockade of WNT and SHH signaling with BMP treatment significantly suppressed GSC self-renewal and extended survival of tumor-bearing mice. Conclusions: Collectively, our results suggested that ID1 simultaneously regulates stemness through WNT and SHH signaling and differentiation through BMPR-mediated differentiation signaling in GSCs, informing a novel therapeutic strategy of combinatorial targeting of stemness and differentiation. Clin Cancer Res; 24(2); 383-94. ©2017 AACR ., (©2017 American Association for Cancer Research.)

Background/aims: The underlying molecular mechanisms involved in sphingosine kinase 1 (SphK1)/sphingosine 1-phosphate (S1P) mediation of platelet-derived growth factor (PDGF)-induced pulmonary arterial smooth muscle cell (PASMC) proliferation are still unclear, and the present study aims to address this issue., Methods: Small interfering RNA (siRNA) and microRNA inhibitor transfection was performed to block the expression of SphK1, bone morphogenetic protein receptor II (BMPRII) and microRNA-21 (miR-21). Gene expression levels of SphK1, BMPRII and inhibitor of DNA binding 1 (Id1) were detected by immunoblotting, miR-21 expression level was examined with qRT-PCR, and S1P production was measured by ELISA. Additionally, PASMC proliferation was determined by BrdU incorporation assay., Results: Our results indicated that PDGF increased the expression of SphK1 protein and S1P production, up-regulated miR-21 expression, reduced BMPRII and Id1 expression, and promoted PASMCs proliferation. Pre-silencing of SphK1 with siRNA reversed PDGF-induced S1P production, miR-21 up-regulation, BMPRII and Id1 down-regulation, as well as PASMC proliferation. Pre-inhibition of miR-21 also blocked BMPRII and Id1 down-regulation as well as PASMC proliferation caused by PDGF. Knockdown of BMPRII down-regulated Id1 expression in PASMCs. We further found that inhibition of PI3K/Akt and ERK signaling pathways, particularly ERK cascade, suppressed PDGF-induced above changes., Conclusion: Our study indicates that SphK1/S1P pathway plays an important role in PDGF-induced PASMC proliferation via miR-21/BMPRII/Id1 axis and targeting against SphK1/S1P axis might be a novel strategy in the prevention and treatment of pulmonary arterial hypertension (PAH)., (© 2018 The Author(s). Published by S. Karger AG, Basel.)

The inhibitor of DNA binding and cell differentiation (Id) proteins are dominant negative regulators of the helix-loop-helix transcription factor family and play a key role during development as well as in vascular disorders and cancer. In fact, impairing the Id-protein activity in cancer cells reduces cell growth and even chemoresistance. Recently, we have shown that a synthetic Id-protein ligand (1Y) consisting of a cyclic nonapeptide can reduce the viability of the two breast cancer cell lines MCF-7 and T47D and of the bladder cancer cells T24 to about 50% at concentrations ≥100μM. Moreover, the cyclopeptide displays both proapoptotic and antiproliferative effects on MCF-7 cells. Herein, we show that the cyclopeptide does not induce cell death at the dose of 5μΜ, but it still inhibits MCF-7 and T24 cell proliferation, which correlates with an increased protein level of the cell-cycle regulator p27 Kip1 . Furthermore, 1Y-pretreated MCF-7, T47D, and T24 cells are more susceptible than untreated cells to the phototoxic effects of the three photosensitizers meta-tetra(hydroxyphenyl)chlorin, porfimer sodium, and hypericin, which are applied in photodynamic therapy (PDT). The combination of the Id-protein ligand with each of the light-activated photosensitizers shows synergistic effects on the reduction of cell viability. In conclusion, an Id-protein ligand with moderate cancer cell killing activity at concentrations ≥100μM can be applied at a 20-fold lower and barely toxic dose to raise the sensitivity of cancer cells towards phototoxicity associated with photodynamic treatment. This suggests the potential benefit of targeting the Id proteins in combined drug approaches for cancer therapy., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Survival of cochlear sensory epithelial cells may be regulated by inhibitor of differentiation-1 (Id1) and the N-methyl-D-aspartic acid (NMDA) receptor. However, it is unclear whether Id1 and the NMDA receptor are involved in the radiation-mediated survival of rat cochlear sensory epithelial cells. Here, we show that the percentage of apoptotic cells increased, the percentage of cells in the S phase decreased, Id1 mRNA and protein expression decreased and the NMDA receptor subtype 2B (NR2B) mRNA and protein level increased in OC1 cells after radiation. Cells infected with the Id1 gene exhibited higher Id1 mRNA and protein levels and lower NR2B mRNA and protein levels than the control cells. In contrast, after transfection of the Id1 siRNA into OC1 cells, Id1 mRNA and protein expression decreased and NR2B mRNA and protein expression increased relative to that of the control group. Additionally, treatment with ifenprodil for 24 h before radiation reduced apoptosis and increased the percentage of cells in the S phase. Our results suggest that Id1 and NR2B might regulate the survival of OC1 cells following radiation., (© 2018 S. Karger AG, Basel.)

Background: Numerous studies have shown that Id-1 (Inhibitor of differentiation 1) is upregulated in several cancers and associated with tumor malignant characters. However, the clinical significance and biological role of Id-1 in non-small cell lung cancer (NSCLC) remains unclear., Methods: We used RT-PCR, Western blot and Immunohistochemistry to measure Id-1 expression in NSCLC tissues and matched adjacent noncancerous tissues. The expression pattern of Id-1 in NSCLC tissues was determined by scoring system of immunohistochemical analysis. The Kaplan-Meier method was used to calculate the survival curve, and log-rank test to determine statistical significance. The Id-1 gene was overexpressed or downreuglated with Lentiviral vectors in NSCLC cells. And, the migration ability of NSCLC cells was tested in a Transwell Boyden Chamber., Results: We found that Id-1 is generally expressed higher in NSCLC tissues compared with matched adjacent noncancerous tissues. We also found that high Id-1 expression in tumor tissues is significantly correlated with tumor progression and poor survival in NSCLC patients. Furthermore, our experimental data revealed that knockdown of Id-1 significantly suppressed the proliferation, migration and invasion of NSCLC cells, whereas ectopic expression of Id-1 promoted the malignant phenotype of NSCLC cells. Mechanistic study showed that NF-κB signaling pathway contributed to the effects of Id-1 in NSCLC cells. Moreover, blocking the NF-κB pathway significantly inhibited the tumor-promoting actions of Id-1 in NSCLC cells., Conclusions: We identified a tumorigenic role of Id-1 in NSCLC and provided a novel therapeutic target for NSCLC patients.

The fundamental role of the brain-specific myelin transcription factor 1-like (MYT1L) gene in cases of intellectual disability and in the etiology of neurodevelopmental disorders is increasingly recognized. Yet, its function remains under-investigated. Here, we identify a network of helix-loop-helix (HLH) transcriptional regulators controlled by MYT1L, as indicated by our analyses in human neural stem cells and in the human brain. Using cell-based knockdown approaches and microarray analyses we found that (1) MYT1L is required for neuronal differentiation and identified ID1, a HLH inhibitor of premature neurogenesis, as a target. (2) Although MYT1L prevented expression of ID1, it induced expression of a large number of terminal differentiation genes. (3) Consistently, expression of MYT1L in the human brain coincided with neuronal maturation and inversely correlated with that of ID1 and ID3 throughout the lifespan. (4) Genetic polymorphisms that reduced expression of MYT1L in the hippocampus resulted in increased expression of ID1 and ID3, decreased levels of the proneural basic HLH (bHLH) transcriptional regulators TCF4 and NEUROD6 and decreased expression of genes involved in long-term potentiation and synaptic transmission, cancer and neurodegeneration. Furthermore, our neuroimaging analyses indicated that MYT1L expression associated with hippocampal volume and activation during episodic memory recall, as measured by blood-oxygen-level-dependent (BOLD) signals. Overall, our findings suggest that MYT1L influences memory-related processes by controlling a neuronal proliferation/differentiation switch of ID-bHLH factors.

Background: Drug resistance is one of the major concerns in the treatment of hepatocellular carcinoma (HCC). The aim of the present study was to determine whether aberrant high expression of the inhibitor of differentiation 1(ID1) confers oxaliplatin-resistance to HCC by activating the pentose phosphate pathway (PPP)., Methods: Aberrant high expression of ID1 was detected in two oxaliplatin-resistant cell lines MHCC97H-OXA(97H-OXA) and Hep3B-OXA(3B-OXA). The lentiviral shRNA or control shRNA was introduced into the two oxaliplatin-resistant cell lines. The effects of ID1 on cell proliferation, apoptosis and chemoresistance were evaluated in vitro and vivo. The molecular signaling mechanism underlying the induction of HCC proliferation and oxaliplatin resistance by ID1 was explored. The prognostic value of ID1/G6PD signaling in HCC patients was assessed using the Cancer Genome Atlas (TCGA) database., Results: ID1 was upregulated in oxaliplaitin-resistant HCC cells and promoted HCC cell proliferation and oxaliplatin resistance. Silencing ID1 expression in oxaliplaitin-resistant HCC cell lines inhibited cell proliferation and sensitized oxaliplaitin-resistant cells to death. ID1 knockdown significantly decreased the expression of glucose-6-phosphate dehydrogenase (G6PD), a key enzyme of the PPP. Silencing ID1 expression blocked the activation of G6PD, decreased the production of PPP NADPH, and augmented reactive oxygen and species (ROS), thus inducing cell apoptosis. Study of the molecular mechanism showed that ID1 induced G6PD promoter transcription and activated PPP through Wnt/β-catenin/c-MYC signaling. In addition, ID1/G6PD signaling predicted unfavorable prognosis of HCC patients on the basis of TCGA., Conclusions: Our study provided the first evidence that ID1 conferred oxaliplatin resistance in HCC by activating the PPP. This newly defined pathway may have important implications in the research and development of new more effective anti-cancer drugs.