Exploring the epigenetic profile of ID4 in breast cancer: bioinformatic insights into methylation patterns and chromatin accessibility dynamics.

PURPOSE: Inhibitor of differentiation 4 (ID4) is a dominant-negative regulator of basic helix-loop-helix (bHLH) transcription factors. The expression of ID4 is dysregulated in various breast cancer subtypes, indicating a potential role for ID4 in subtype-specific breast cancer development. This study aims to elucidate the epigenetic regulation of ID4 within breast cancer subtypes, with a particular focus on DNA methylation and chromatin accessibility. METHODS: Bioinformatic analyses were conducted to assess DNA methylation and chromatin accessibility in ID4 regulatory regions across breast cancer subtypes. Gene Set Enrichment Analysis (GSEA) was conducted to identify related gene sets. Transcription factor binding within ID4 enhancer and promoter regions was explored. In vitro experiments involved ER+ breast cancer cell lines treated with estradiol (E2) and Tamoxifen. RESULTS: Distinct epigenetic profiles of ID4 were observed, revealing increased methylation and reduced chromatin accessibility in luminal subtypes compared to the basal subtype. Gene Set Enrichment Analysis (GSEA) implicated estrogen-related pathways, suggesting a potential link between estrogen signaling and the regulation of ID4 expression. Transcription factor analysis identified ER and FOXA1 as regulators of ID4 enhancer regions. In vitro experiments confirmed the role of ER, demonstrating reduced ID4 expression and increased methylation with estradiol treatment. Conversely, Tamoxifen treatment increased ID4 expression, indicating the potential involvement of ER signaling through ERα in the epigenetic regulation of ID4 in breast cancer cells. CONCLUSION: This study shows the intricate epigenetic regulation of ID4 in breast cancer, highlighting subtype-specific differences in DNA methylation and chromatin accessibility.

Deciphering Potential Molecular Signatures to Differentiate Acute Myeloid Leukemia (AML) with BCR::ABL1 from Chronic Myeloid Leukemia (CML) in Blast Crisis.

Acute myeloid leukemia (AML) with BCR::ABL1 has recently been recognized as a distinct subtype in international classifications. Distinguishing it from myeloid blast crisis chronic myeloid leukemia (BC-CML) without evidence of a chronic phase (CP), remains challenging. We aimed to better characterize this entity by integrating clonal architecture analysis, mutational landscape assessment, and gene expression profiling. We analyzed a large retrospective cohort study including CML and AML patients. Two AML patients harboring a BCR::ABL1 fusion were included in the study. We identified BCR::ABL1 fusion as a primary event in one patient and a secondary one in the other. AML-specific variants were identified in both. Real-time RT-PCR experiments demonstrated that CD25 mRNA is overexpressed in advanced-phase CML compared to AML. Unsupervised principal component analysis showed that AML harboring a BCR::ABL1 fusion was clustered within AML. An AML vs. myeloid BC-CML differential expression signature was highlighted, and while ID4 (inhibitor of DNA binding 4) mRNA appears undetectable in most myeloid BC-CML samples, low levels are detected in AML samples. Therefore, CD25 and ID4 mRNA expression might differentiate AML with BCR::ABL1 from BC-CML and assign it to the AML group. A method for identifying this new WHO entity is then proposed. Finally, the hypothesis of AML with BCR::ABL1 arising from driver mutations on a BCR::ABL1 background behaving as a clonal hematopoiesis mutation is discussed. Validation of our data in larger cohorts and basic research are needed to better understand the molecular and cellular aspects of AML with a BCR::ABL1 entity.

BRCA1 expression, its correlation with clinicopathological features, and response to neoadjuvant chemotherapy in high-grade serous ovarian cancer.

AIM: In high-grade serous ovarian cancers (HG-SOC), BRCA1 mutation is one of the predominant mutations reported by various studies. However, the non-mutational mechanisms of BRCA pathway inactivation in HG-SOC are unclear. We evaluated BRCA1 inactivation by estimating its expression with its repressor, ID4, in primary and neoadjuvant chemotherapy (NACT)-treated HG-SOC tumors with known therapeutic responses. METHODS: We evaluated the expression pattern of BRCA1 protein by immunohistochemistry in 119 cases of HG-SOC from a hospital cohort consisting of primary (N = 69) and NACT-treated (N = 50) tumors. Histological patterns (SET), stromal infiltration by lymphocytes (sTILs), and chemotherapy response score (CRS) were estimated by microscopic examination. Gene expression levels of BRCA1, and its repressor ID4, were estimated by qPCR. The association of BRCA1 protein and mRNA with clinicopathological features was studied. The relevance of the BRCA1/ID4 ratio was evaluated in tumors with different CRS. RESULTS: BRCA1 protein expression was observed in 12% of primary and 19% of NACT-treated HG-SOC tumors. We observed moderate concordance between BRCA1 protein and mRNA expression (AUC = 0.677). High BRCA1 mRNA expression was significantly associated with a more frequent SET pattern (p = 0.024), higher sTILs density (p = 0.042), and increased mitosis (p = 0.028). BRCA1-negative tumors showed higher expression of ID4 though not statistically significant. A higher BRCA1/ID4 ratio was associated with high sTILs density in primary (p = 0.042) and NACT-treated tumors (p = 0.040). CONCLUSION: Our findings show the utility of the BRCA1/ID4 ratio in predicting neoadjuvant therapy response, which needs further evaluation in larger cohorts with long-term outcomes.

Pleiotrophin ameliorates white matter injury of neonatal rats by activating the mTOR/YY1/Id4 signaling pathway.

Brain white matter injury (WMI) is a serious disease of the central nervous system. Pleiotrophin (PTN) promotes the differentiation and myelination of oligodendrocytes (OLs) in vitro. However, the role of PTN in WMI remains unknown. Therefore, this study aimed to investigate the neuroprotective role and potential mechanisms of PTN function in neonatal rats with WMI. The PTN and mammalian target of rapamycin (mTOR) inhibitor everolimus was used to treat a WMI model in postnatal day 3 Sprague-Dawley rats, in which the right common carotid arteries of these rats were isolated, ligated, and exposed to a hypoxic environment (6% O2 + 94% N2 ) for 2 h. OL differentiation and myelination, as well as the spatial learning and memory abilities of the rats were evaluated to examine the effects of PTN. Two proteins of the mTOR signaling pathway, YingYang1 (YY1) and inhibitor of DNA binding 4 (Id4), were detected and were used to explore the potential mechanisms of PTN in rat WMI experiment and oxygen glucose deprivation (OGD) model. We found that the differentiation and myelination of OLs were impaired after WMI. PTN administration rescued this injury by activating mTOR/YY1 and inhibiting Id4. Everolimus administration inhibited mTOR/YY1 and activated Id4, which blocked the neuroprotective role of PTN in WMI. PTN plays a neuroprotective role in neonatal rats with WMI, which could be involved in the mTOR/YY1/Id4 signaling pathway.

Id4 modulates salivary gland homeostasis and its expression is downregulated in IgG4-related disease via miR-486-5p.

Salivary glands are physiologically orchestrated by the coordinated balance between cell differentiation, proliferation, apoptosis, and interactions between epithelial, mesenchymal endothelial, and neuronal cells, and they are frequent sites of manifestations of Sjögren's syndrome (SS) or IgG4-related disease (IgG4-RD). However, little is known about salivary gland homeostasis and its involvement in those diseases. Inhibitor of DNA binding/differentiation 4 (Id4) is an Id protein involved in the transcriptional control of many biological events, including differentiation. Studies of Id4-deficient mice revealed that Id4-deficient submandibular glands were smaller and exhibited accelerated differentiation, compared with those from wild-type littermates. In addition, dry mouth symptoms and Th17 expansion in splenocytes were also observed in the absence of Id4. Furthermore, Id4 levels in the salivary glands of patients with IgG4-RD, but not SS, were significantly decreased compared with those of healthy controls. miRNA-mRNA integrated analysis demonstrated that miR-486-5p was upregulated in IgG4-RD patients and that it might regulate Id4 in the lesion sites. Together, these results provide evidence for the inhibitory role of Id4 in salivary differentiation, and a critical association between Id4 downregulation and IgG4-RD.

Osteoglycin (OGN) promotes tumorigenesis of pancreatic cancer cell via targeting ID4.

Pancreatic cancer (PC) is the seventh-leading cause of cancer-related mortality, and is associated with limited therapeutic options and poor prognosis. The extracellular matrix (ECM) represents the main component of the tumor microenvironment. Studies have found controversial roles of osteoglycin (OGN), a classical small leucine-rich proteoglycan found in the ECM in human malignancies; however, the significance of OGN in PC has not been determined. Here, the expression profiles of OGN in PC tissues and cell lines were evaluated by Gene Expression Profiling Interactive Analysis (GEPIA) database, immunohistochemistry, western blot, and quantitative PCR. OGN was found to be significantly upregulated in PC tissues and cell lines. Moreover, the expression of OGN was observed to be closely associated with TNM stage, stage III showed a higher OGN expression than that of stages I and II. Survival analysis showed that patients with PC showing high levels of OGN had low survival rates. The effects of OGN on cell proliferation and apoptosis were analyzed using MTT, CCK8, EdU and TUNEL assays. Wound-healing and invasion assays were conducted to test migratory and invasive abilities. Overexpression of OGN was demonstrated to promote proliferation, migration, and invasion, and inhibit apoptosis of PC cells. Further experiments revealed that inhibitor of DNA binding 4 (ID4) was upregulated by OGN. Silencing ID4 by small interfering RNA was shown to partially reverse the tumor-promoting effect of OGN. Collectively, our preliminary results indicate that the elevated expression of OGN may be associated with PC progression and may serve as a potential biomarker for the diagnosis and prognosis of PC. Targeting of OGN/ID4 axis may be a promising strategy in PC therapy.

A novel high throughput screen to identify candidate molecular networks that regulate spermatogenic stem cell functions†.

Spermatogenic regeneration is key for male fertility and relies on activities of an undifferentiated spermatogonial population. Here, a high-throughput approach with primary cultures of mouse spermatogonia was devised to rapidly predict alterations in functional capacity. Combining the platform with a large-scale RNAi screen of transcription factors, we generated a repository of new information from which pathway analysis was able to predict candidate molecular networks regulating regenerative functions. Extending from this database, the SRCAP-CREBBP/EP300 (Snf2-related CREBBP activator protein-CREB binding protein/E1A binding protein P300) complex was found to mediate differential levels of histone acetylation between stem cell and progenitor spermatogonia to influence expression of key self-renewal genes including the previously undescribed testis-specific transcription factor ZSCAN2 (zinc finger and SCAN domain containing 2). Single cell RNA sequencing analysis revealed that ZSCAN2 deficiency alters key cellular processes in undifferentiated spermatogonia such as translation, chromatin modification, and ubiquitination. In Zscan2 knockout mice, while spermatogenesis was moderately impacted during steady state, regeneration after cytotoxic insult was significantly impaired. Altogether, these findings have validated the utility of our high-throughput screening approach and have generated a transcription factor database that can be utilized for uncovering novel mechanisms governing spermatogonial functions.

EGCG attenuates the neurotoxicity of methylglyoxal via regulating MAPK and the downstream signaling pathways and inhibiting advanced glycation end products formation.

Methylglyoxal (MGO), a reactive α-oxoaldehyde formed in many foods and beverages during processing and storage, has neurotoxicity. The purpose of this study was to investigate the inhibition mechanism of (-)-epigallocatechin-3-gallate (EGCG) on MGO-induced PC12 cells damage. Cell apoptosis and reactive oxygen species (ROS) level were measured with fluorescent staining methods. Western blotting was used to detect the signal transduction mechanism. The results indicated that EGCG decreased ROS level, inhibited apoptosis and increased the expression of brain-derived neurotrophic factor. Pathways analysis revealed that the neuroprotective mechanism of EGCG might rely on regulating mitogen-activated protein kinase (MAPK) and downstream pathways. Multi-spectroscopy and molecular docking indicated that EGCG inhibited MGO-derived advanced glycation end products (AGEs) formation. Moreover, the neurotoxicity of AGEs could be alleviated by EGCG. These results suggested that EGCG could attenuate MGO-induced nerve damage via regulating MAPK and downstream pathways and inhibiting AGEs formation.

Mesenchymal stem cell-derived exosome mir-342-3p inhibits metastasis and chemo-resistance of breast cancer through regulating ID4.

BACKGROUND: The mesenchymal stem cell-derived exosome (MSCs-exo) carrying microRNAs have been proved to regulate tumor biological activities. Clarifying molecular mechanism and identifying predictive microRNAs will be of great value in anti-tumor therapy improvement. OBJECTIVE: We aimed to investigate the regulatory role of microRNA-342-3p (miR-342-3p) in MSCs-exo on breast cancer. METHODS: Breast cancer tissues and cell lines were used to evaluate miR-342-3p expression in patients with or without lymph node/distal organ metastasis. The impact of MSCs-exo expression on tumor cell chemo-resistance and invasion/migration was measured. Dual-luciferase reporter gene assay was applied to identify binding site. Inhibitor of differentiation 4 (ID4) siRNA and miR-342-3p inhibitor transfection was conducted to further explore the miR-342-3p/ID4 axis on chemo-resistance and metastasis of breast cancer cells. RESULTS: Breast cancer cells revealed significantly lower level of miR-342-3p in patients with metastatic diseases. miR-342-3p suppressed invasive and chemo-resistant behavior of breast cancer tumor cells. Binding site between miR-342-3p and ID4 was proved. ID4 could reverse the influence of miR-342-3p on chemo-resistance. The tumor inhibition effect of IDA siRNA in vivo was also identified. CONCLUSIONS: This study demonstrated that miR-342-3p acted as potential tumor suppressor by inhibiting metastasis and chemo-resistance of breast cancer cells through targeting ID4. This study might provide potential therapy targets for the treatment of breast cancer.

CDC42 as an epigenetic regulator of ID4 in triple-negative breast tumors.

BACKGROUND: Triple-negative (TN) breast cancer represents a subtype of breast cancer that does not express estrogen receptor (ER), progesterone receptor (PR), or human epidermal growth factor receptor 2 (HER-2). Clinically, it is characterized by high invasiveness, high metastatic potential, and poor prognosis. Inhibitor of DNA binding 4 (ID4) has been shown to be overexpressed in these tumors acting as an oncogene responsible for many of its aggressive features. CDC42, a plasma membrane-associated small GTPase, can downregulate ID4 gene expression through hypermethylation of its promoter in colorectal adenocarcinomas. Since ID4 acts as an oncogene and is hypomethylated in TN breast tumors, here we asked whether CDC42 could also epigenetically silence ID4 and in doing so revert aggressive features of this tumor type. METHODS: Gene expression was retrieved from TCGA database using UCSC Xena. Association between overall survival (OS) and gene expression was assessed using Kaplan-Meier plotter. In vitro experiments involved ectopic expression of CDC42 in MDA-MB231and in MDA-MB468 breast cancer cell lines. Gene expression was analyzed by qPCR, western blot and inmunofluorescence assays and methylation by MSP, MS-MLPA, or ddMSP. RESULTS: Data mining analysis revealed that CDC42 expression varies among breast cancer subtypes that in the basal-like subtype there is an inverse correlation between CDC42 and ID4 expression and a positive correlation between CDC42 expression and ID4 methylation. In vitro experiments revealed that CDC42 overexpression induced ID4 methylation through the activation of the EZH2 pathway. ID4 silencing produced an increase in BRCA1 expression and a less aggressive phenotype in the tested cell line. CONCLUSION: We show that CDC42 silences ID4 through methylation in TN breast cancer. Given that ID4 acts as an oncogene in these tumors, we think that finding an epigenetic regulator of ID4 contributes to the research and clinical management of TN breast tumors.

Id2 and Id4 are not the major negative regulators of oligodendrocyte differentiation during early central nervous system development.

Myelin sheathes ensure the rapid conduction of neural impulse and provide nutritional support for neurons. Myelin sheathes are formed by differentiated oligodendrocytes (OLs) in the central nervous system. During OL development, the differentiation of oligodendrocyte progenitor cells (OPCs) into mature OLs is controlled by both positive differentiation factors (drivers) and negative regulatory factors (brakes). Previous studies have suggested Id2 and Id4 as the key negative factors for OL differentiation. However, these conclusions were mainly based on in vitro studies and the reported OL phenotype in Id4 mutants appear to be mild. In this study, we systematically investigated the in vivo function of Id2 and Id4 genes in OL differentiation in their genetic mutants and in embryonic chicken spinal cord. Our results showed that disruption of Id4 has no effect on OL differentiation and maturation, whereas Id2 mutants and Id2/Id4 compound mutants display a mild and transient precocity of OL differentiation. In agreement with these loss-of-function studies, Id2, but not Id4, is weakly expressed in OPCs. Despite their minor roles in OL differentiation, forced expression of Id2 and Id4 in embryonic chicken spinal cords strongly inhibit the differentiation of OPCs. Taken together, our detailed functional and expressional studies strongly suggest that Id2 and Id4 are not the major in vivo repressors of OPC differentiation during animal development, shedding new light on the molecular regulation of early OL development.

miR-9-5p Promotes Lung Adenocarcinoma Cell Proliferation, Migration and Invasion by Targeting ID4.

Objectives Evidence reveals that microRNAs (miRNAs) are abnormally expressed in lung adenocarcinoma (LUAD) tissue and are crucial in LUAD occurrence. Therefore, this study aims to find the miRNA which could regulate LUAD and to further explore its regulatory mechanism, thus providing a potential molecular target for LUAD. Methods miR-9-5p and ID4 expression in LUAD cells were measured by real-time quantitative PCR and western blot. Cell functional assays were conducted to detect the biological functions of LUAD cells. A dual-luciferase reporter assay was utilized to validate the binding relationship between miR-9-5p and ID4. Results miR-9-5p was highly expressed whereas ID4 was lowly expressed in LUAD. miR-9-5p facilitated LUAD cell progression by targeting ID4. Conclusion miR-9-5p promotes LUAD cell progression by modulating ID4 and may become a potential target for LUAD.

Helix-loop-helix protein ID4 expressed in bovine Sertoli cells.

Stage- and cell type-specific biomarkers are important for understanding spermatogenesis in mammalian testis. The present study identified several testicular cell marker proteins in 6- and 24-month old bovine testes. In 6-month old bovine testes, spermatogonia and spermatocytes were detected but complete spermatogenesis occurred in 24-month old testes. The diameters of the seminiferous tubules increased significantly in the 24-month old testes compared with those in the 6-month old testes. Protein Gene Product 9.5 (PGP9.5), also known as the undifferentiated spermatogonium marker, and GATA4 (GATA binding protein 4), vimentin, and SOX9 (SRY-Box Transcription Factor 9) were detected in the basement membrane region. Interestingly, ID4 (inhibitor of DNA binding protein 4; previously known as the undifferentiated cell marker) proteins were located in the basement membrane region but their expression patterns were different from those of PGP9.5. Co-immunohistochemistry results showed that ID4 was detected in the Sertoli cells expressing vimentin and SOX9 in 6- and 24-month old bovine testes. This result indicated that ID4 is a putative biomarker of Sertoli cell in the bovine system, which is different from the rodent models. Thus, these results will contribute in understanding the process of spermatogenesis that is different in bovines compared to other species.

DNA methylation regulates the expression of the negative transcriptional regulators ID2 and ID4 during OPC differentiation.

The differentiation of oligodendrocyte precursor cells (OPCs) into myelinating oligodendrocytes is the prerequisite for remyelination in demyelinated disorders such as multiple sclerosis (MS). Epigenetic mechanisms, such as DNA methylation, have been suggested to control the intricate network of transcription factors involved in OPC differentiation. Yet, the exact mechanism remains undisclosed. Here, we are the first to identify the DNA-binding protein inhibitors, Id2 and Id4, as targets of DNA methylation during OPC differentiation. Using state-of-the-art epigenetic editing via CRISPR/dCas9-DNMT3a, we confirm that targeted methylation of Id2/Id4 drives OPC differentiation. Moreover, we show that in the pathological context of MS, methylation and gene expression levels of both ID2 and ID4 are altered compared to control human brain samples. We conclude that DNA methylation is crucial to suppress ID2 and ID4 during OPC differentiation, a process that appears to be dysregulated during MS. Our data do not only reveal new insights into oligodendrocyte biology, but could also lead to a better understanding of CNS myelin disorders.

Targeted Disruption of the Inhibitor of DNA Binding 4 (Id4) Gene Alters Photic Entrainment of the Circadian Clock.

Inhibitor of DNA binding (Id) genes comprise a family of four helix-loop-helix (HLH) transcriptional inhibitors. Our earlier studies revealed a role for ID2 within the circadian system, contributing to input, output, and core clock function through its interaction with CLOCK and BMAL1. Here, we explore the contribution of ID4 to the circadian system using a targeted disruption of the Id4 gene. Attributes of the circadian clock were assessed by monitoring the locomotor activity of Id4-/- mice, and they revealed disturbances in its operation. Id4-mutant mice expressed a shorter circadian period length, attenuated phase shifts in responses to continuous and discrete photic cues, and an advanced phase angle of entrainment under a 12:12 light:dark cycle and under short and long photoperiods. To understand the basis for these properties, suprachiasmatic nucleus (SCN) and retinal structures were examined. Anatomical analysis reveals a smaller Id4-/- SCN in the width dimension, which is a finding consistent with its smaller brain. As a result of this feature, anterograde tracing in Id4-/- mice revealed retinal afferents innovate a disproportionally larger SCN area. The Id4-/- photic entrainment responses are unlikely to be due to an impaired function of the retinal pathways since Id4-/- retinal anatomy and function tested by pupillometry were similar to wild-type mice. Furthermore, these circadian characteristics are opposite to those exhibited by the Id2-/- mouse, suggesting an opposing influence of the ID4 protein within the circadian system; or, the absence of ID4 results in changes in the expression or activity of other members of the Id gene family. Expression analysis of the Id genes within the Id4-/- SCN revealed a time-of-day specific elevated Id1. It is plausible that the increased Id1 and/or absence of ID4 result in changes in interactions with bHLH canonical clock components or with targets upstream and/or downstream of the clock, thereby resulting in abnormal properties of the circadian clock and its entrainment.

ID4 Is Required for Normal Ependymal Cell Development.

Ependymal cells are radial glia-derived multiciliated cells lining the lateral ventricles of the brain and spinal cord. Correct development and coordinated cilia beating is essential for proper cerebrospinal fluid (CSF) flow and neurogenesis modulation. Dysfunctions of ependymal cells were associated with transcription factor deregulation. Here we provide evidence that the transcriptional regulator ID4 is involved in ependymal cell development and maturation. We observed that Id4-deficient mice display altered ventricular cell cytoarchitecture, decreased ependymal cell number and enlarged ventricles. In addition, absence of ID4 during embryonic development resulted in decreased ependymal cell number and delayed maturation. Our findings open the way for a potential role of ID4 in ependymal cell development and motor cilia function.

The helix-loop-helix transcriptional regulator Id4 is required for terminal differentiation of luminal epithelial cells in the prostate.

Inhibitor of differentiation 4 (Id4), a member of the helix-loop-helix family of transcriptional regulators has emerged as a tumor suppressor in prostate cancer. In this study we investigated the effect of loss of Id4 (Id4-/-) on mouse prostate development. Histological analysis was performed on prostates from 25 days, 3 months and 6 months old Id4-/- mice. Expression of Amacr, Ck8, Ck18, Fkbp51, Fkbp52, androgen receptor, Pten, sca-1 and Nkx3.1 was investigated by immunohistochemistry. Results were compared to the prostates from Nkx3.1-/- mice. Id4-/- mice had smaller prostates with fewer and smaller tubules. Subtle PIN like lesions were observed at 6mo. Decreased Nkx3.1 and Pten and increased stem cell marker sca-1, PIN marker Amacr and basal cell marker p63 was observed at all ages. Persistent Ck8 and Ck18 expression suggested that loss of Id4 results in epithelial commitment but not terminal differentiation in spite of active Ar. Loss of Id4 attenuates normal prostate development and promotes hyperplasia/ dysplasia with PIN like lesions. The results suggest that loss of Id4 maintains stem cell phenotype of "luminal committed basal cells", identifying a unique prostate developmental pathway regulated by Id4.

CircDDX42 Accelerates the Development of Pancreatic Cancer via miR-613/ID4/PI3K/AKT Axis.

BACKGROUND: Pancreatic cancer (PC) is one of the fatal cancers globally. CircDEAD-box helicase 42 (circDDX42) has been reported to play an oncogenic role in many cancers. The purpose of our study was to explore the relationship between circDDX42 and PC development and the potential mechanism by which circDDX42 modulating the progression of PC. METHODS: The enrichment of circDDX42, miR-613 and inhibitor of DNA binding 4 (ID4) was determined by quantitative real-time polymerase chain reaction (qRT-PCR) in PC tissues and cells. The proliferation, apoptosis and metastasis of PC cells were examined by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), Western blot, flow cytometry and transwell migration and invasion assays, respectively. The binding sites between miR-613 and circDDX42 or ID4 were predicted by Starbase bioinformatic software, and dual-luciferase reporter assay was conducted to verify the combination between miR-613 and circDDX42 or ID4. Western blot was carried out to detect the abundance of ID4, p-phosphatidylinositol 3-kinase (p-PI3K), PI3K, p-AKT serine/threonine kinase (p-AKT) and AKT in PC cells. The in vivo role of circDDX42 was verified through using murine xenograft model. RESULTS: The level of circDDX42 was enhanced in PC tissues and cells compared with that in matching normal tissues and HPDE cells. CircDDX42 promoted the proliferation and metastasis and suppressed the apoptosis of PC cells. CircDDX42 could sponge miR-613, and miR-613 was negatively regulated by circDDX42 in PC cells. MiR-613 suppressed the progression of PC. ID4 was a direct target of miR-613. ID4 was inversely modulated by miR-613 and positively regulated by circDDX42 in PC cells. ID4 played an oncogenic role in the tumorigenesis of PC. CircDDX42/miR-613/ID4 axis regulated the activation of PI3K/AKT pathway in PC cells. ID4 facilitated the progression of PC via activating PI3K/AKT signal pathway. CircDDX42 promoted the tumor growth of PC in vivo. CONCLUSION: CircDDX42 accelerated the proliferation and metastasis while impeded the apoptosis of PC cells via circDDX42/miR-613/ID4/PI3K/AKT axis. This axis might be a promising target for PC therapy.

ID4 Promotes Breast Cancer Chemotherapy Resistance via CBF1-MRP1 Pathway.

Chemo-resistance is considered a key problem in triple negative breast cancer (TNBC) chemotherapy and as such, an urgent need exists to identify its exact mechanisms. Inhibitor of DNA binding factor 4 (ID4) was reported to play diverse roles in different breast cancer molecular phenotypes. In addition, ID4 was associated with mammary carcinoma drug resistance however its functions and contributions remain insufficiently defined. The expression of ID4 in MCF-7, MCF-7/Adr and MDA-MB-231 breast cancer cell lines and patients' tissues were detected by RT-PCR, western blot and immunohistochemistry. Furthermore, TCGA database was applied to confirm these results. Edu and CCK8 assay were performed to detect the proliferation and drug resistance in breast cancer cell lines. Transwell and scratch migration assay were used to detected metastasis. Western blot, TCGA database, Immunoprecipitation (IP), Chromatin Immunoprecipitation (ChIP) and Luciferase reporter assay were used to investigate the tumor promotion mechanisms of ID4. In this study, we report that the expression levels of ID4 appeared to correlate with breast cancers subtype differentiation biomarkers (including ER, PR) and chemo-resistance related proteins (including MRP1, ABCG2, P-gp). Down-regulation of ID4 in MCF-7/Adr and MDA-MB-231 breast cancer cell lines significantly suppressed cell proliferation and invasion, however enhanced Adriamycin sensitivity. We further demonstrated that the oncogenic and chemo-resistant effects of ID4 could be mediated by binding to CBF1 promoter region though combination with MyoD1, and then the downstream target MRP1 could be activated. We reveal for the first time that ID4 performs its function via a CBF1-MRP1 signaling axis, and this finding provides a novel perspective to find potential therapeutic targets for breast cancer chemotherapy.

Expression and Prognostic Value of Id-4 in Patients with Esophageal Squamous Cell Carcinoma.

BACKGROUND: Our previous study demonstrated that Id-1 may promote the tumorigenicity of esophageal squamous cell carcinoma (ESCC). Id-4 is another member of Id family, which is rare to be studied in ESCC. In this study, we investigated the expression of Id-4 in human ESCC specimens and determined whether Id-4 expression was associated with the clinicopathologic characteristic and the prognosis of ESCC patients. METHODS: We examined Id-4 expression using immunohistochemistry in 92 ESCC tissues and adjacent normal tissues. The association between Id-4 expression and clinical parameters and survival was evaluated by statistical analysis. Cox regression analyses were conducted to identify prognostic factors associated with overall survival (OS). In addition, we explored the functional mechanism of Id-4 in ESCC. RESULTS: Id-4 expression was significantly downregulated in ESCC tissues compared with adjacent normal tissues. The expression of Id-4 was associated negatively with pT stage (p=0.002), AJCC stage (p=0.008) and histologic differentiation (p<0.001). OS was more unfavorable in patients with low expression of Id-4 than those with high expression of ESCC patients (p=0.007). In subgroup analysis, low expression of Id-4 could reveal unfavorable OS of patients with pT1b/T2 stage (p=0.024) or with pN0/N1 stage (p=0.004). By univariate analysis, pT stage and Id-4 expression showed statistically significant associations with OS (p=0.025, p=0.01, respectively). By multivariate analysis, Id-4 expression was an independent prognostic factor in ESCC (p =0.038). In addition, we observed that Id-4 could decrease the levels of the p-Smad2, p-Smad3 and TGF-ß1 in both Eca109 and TE1 cells, indicating Id-4 may inactivate the TGF-ß signaling pathway. CONCLUSION: Low expression of Id-4 suggested unfavorable prognosis for ESCC patients and could identify the prognosis in patients of early-stage tumors. The potential mechanism for Id-4's tumor suppressor role in ESCC may be related to its inhibitory effect on TGF-ß signaling pathway. Thus, we believe that Id-4 may be a promising prognostic marker and a therapeutic target in ESCC.