E proteins control the development of NKγδT cells through their invariant T cell receptor.

T cell receptor (TCR) signaling regulates important developmental transitions, partly through induction of the E protein antagonist, Id3. Although normal γδ T cell development depends on Id3, Id3 deficiency produces different phenotypes in distinct γδ T cell subsets. Here, we show that Id3 deficiency impairs development of the Vγ3+ subset, while markedly enhancing development of NKγδT cells expressing the invariant Vγ1Vδ6.3 TCR. These effects result from Id3 regulating both the generation of the Vγ1Vδ6.3 TCR and its capacity to support development. Indeed, the Trav15 segment, which encodes the Vδ6.3 TCR subunit, is directly bound by E proteins that control its expression. Once expressed, the Vγ1Vδ6.3 TCR specifies the innate-like NKγδT cell fate, even in progenitors beyond the normally permissive perinatal window, and this is enhanced by Id3-deficiency. These data indicate that the paradoxical behavior of NKγδT cells in Id3-deficient mice is determined by its stereotypic Vγ1Vδ6.3 TCR complex.

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.

SOX11 expression is restricted to EBV-negative Burkitt lymphoma and is associated with molecular genetic features.

ABSTRACT: SRY-related HMG-box gene 11 (SOX11) is a transcription factor overexpressed in mantle cell lymphoma (MCL), a subset of Burkitt lymphomas (BL) and precursor lymphoid cell neoplasms but is absent in normal B cells and other B-cell lymphomas. SOX11 has an oncogenic role in MCL but its contribution to BL pathogenesis remains uncertain. Here, we observed that the presence of Epstein-Barr virus (EBV) and SOX11 expression were mutually exclusive in BL. SOX11 expression in EBV-negative (EVB-) BL was associated with an IG∷MYC translocation generated by aberrant class switch recombination, whereas in EBV-negative (EBV-)/SOX11-negative (SOX11-) tumors the IG∷MYC translocation was mediated by mistaken somatic hypermutations. Interestingly, EBV- SOX11-expressing BL showed higher frequency of SMARCA4 and ID3 mutations than EBV-/SOX11- cases. By RNA sequencing, we identified a SOX11-associated gene expression profile, with functional annotations showing partial overlap with the SOX11 transcriptional program of MCL. Contrary to MCL, no differences on cell migration or B-cell receptor signaling were found between SOX11- and SOX11-positive (SOX11+) BL cells. However, SOX11+ BL showed higher adhesion to vascular cell adhesion molecule 1 (VCAM-1) than SOX11- BL cell lines. Here, we demonstrate that EBV- BL comprises 2 subsets of cases based on SOX11 expression. The mutual exclusion of SOX11 and EBV, and the association of SOX11 with a specific genetic landscape suggest a role of SOX11 in the early pathogenesis of BL.

Endothelial Cell Flow-Mediated Quiescence Is Temporally Regulated and Utilizes the Cell Cycle Inhibitor p27.

BACKGROUND: Endothelial cells regulate their cell cycle as blood vessels remodel and transition to quiescence downstream of blood flow-induced mechanotransduction. Laminar blood flow leads to quiescence, but how flow-mediated quiescence is established and maintained is poorly understood. METHODS: Primary human endothelial cells were exposed to laminar flow regimens and gene expression manipulations, and quiescence depth was analyzed via time-to-cell cycle reentry after flow cessation. Mouse and zebrafish endothelial expression patterns were examined via scRNA-seq (single-cell RNA sequencing) analysis, and mutant or morphant fish lacking p27 were analyzed for endothelial cell cycle regulation and in vivo cellular behaviors. RESULTS: Arterial flow-exposed endothelial cells had a distinct transcriptome, and they first entered a deep quiescence, then transitioned to shallow quiescence under homeostatic maintenance conditions. In contrast, venous flow-exposed endothelial cells entered deep quiescence early that did not change with homeostasis. The cell cycle inhibitor p27 (CDKN1B) was required to establish endothelial flow-mediated quiescence, and expression levels positively correlated with quiescence depth. p27 loss in vivo led to endothelial cell cycle upregulation and ectopic sprouting, consistent with loss of quiescence. HES1 and ID3, transcriptional repressors of p27 upregulated by arterial flow, were required for quiescence depth changes and the reduced p27 levels associated with shallow quiescence. CONCLUSIONS: Endothelial cell flow-mediated quiescence has unique properties and temporal regulation of quiescence depth that depends on the flow stimulus. These findings are consistent with a model whereby flow-mediated endothelial cell quiescence depth is temporally regulated downstream of p27 transcriptional regulation by HES1 and ID3. The findings are important in understanding endothelial cell quiescence misregulation that leads to vascular dysfunction and disease.

Silibinin is a suppressor of the metastasis-promoting transcription factor ID3.

BACKGROUND: ID3 (inhibitor of DNA binding/differentiation-3) is a transcription factor that enables metastasis by promoting stem cell-like properties in endothelial and tumor cells. The milk thistle flavonolignan silibinin is a phytochemical with anti-metastatic potential through largely unknown mechanisms. HYPOTHESIS/PURPOSE: We have mechanistically investigated the ability of silibinin to inhibit the aberrant activation of ID3 in brain endothelium and non-small cell lung cancer (NSCLC) models. METHODS: Bioinformatic analyses were performed to investigate the co-expression correlation between ID3 and bone morphogenic protein (BMP) ligands/BMP receptors (BMPRs) genes in NSCLC patient datasets. ID3 expression was assessed by immunoblotting and qRT-PCR. Luciferase reporter assays were used to evaluate the gene sequences targeted by silibinin to regulate ID3 transcription. In silico computational modeling and LanthaScreen TR-FRET kinase assays were used to characterize and validate the BMPR inhibitory activity of silibinin. Tumor tissues from NSCLC xenograft models treated with oral silibinin were used to evaluate the in vivo anti-ID3 effects of silibinin. RESULTS: Analysis of lung cancer patient datasets revealed a top-ranked positive association of ID3 with the BMP9 endothelial receptor ACVRL1/ALK1 and the BMP ligand BMP6. Silibinin treatment blocked the BMP9-induced activation of the ALK1-phospho-SMAD1/5-ID3 axis in brain endothelial cells. Constitutive, acquired, and adaptive expression of ID3 in NSCLC cells were all significantly downregulated in response to silibinin. Silibinin blocked ID3 transcription via BMP-responsive elements in ID3 gene enhancers. Silibinin inhibited the kinase activities of BMPRs in the micromolar range, with the lower IC50 values occurring against ACVRL1/ALK1 and BMPR2. In an in vivo NSCLC xenograft model, tumoral overexpression of ID3 was completely suppressed by systematically achievable oral doses of silibinin. CONCLUSIONS: ID3 is a largely undruggable metastasis-promoting transcription factor. Silibinin is a novel suppressor of ID3 that may be explored as a novel therapeutic approach to interfere with the metastatic dissemination capacity of NSCLC.

ID factors regulate the ability of Müller glia to become proliferating neurogenic progenitor-like cells.

The purpose of this study was to investigate how ID factors regulate the ability of Müller glia (MG) to reprogram into proliferating MG-derived progenitor cells (MGPCs) in the chick retina. We found that ID1 is transiently expressed by maturing MG (mMG), whereas ID4 is maintained in mMG in embryonic retinas. In mature retinas, ID4 was prominently expressed by resting MG, but following retinal damage ID4 was rapidly upregulated and then downregulated in MGPCs. By contrast, ID1, ID2, and ID3 were low in resting MG and then upregulated in MGPCs. Inhibition of ID factors following retinal damage decreased numbers of proliferating MGPCs. Inhibition of IDs, after MGPC proliferation, significantly increased numbers of progeny that differentiated as neurons. In damaged or undamaged retinas inhibition of IDs increased levels of p21Cip1 in MG. In response to damage or insulin+FGF2 levels of CDKN1A message and p21Cip1 protein were decreased, absent in proliferating MGPCs, and elevated in MG returning to a resting phenotype. Inhibition of notch- or gp130/Jak/Stat-signaling in damaged retinas increased levels of ID4 but not p21Cip1 in MG. Although ID4 is the predominant isoform expressed by MG in the chick retina, id1 and id2a are predominantly expressed by resting MG and downregulated in activated MG and MGPCs in zebrafish retinas. We conclude that ID factors have a significant impact on regulating the responses of MG to retinal damage, controlling the ability of MG to proliferate by regulating levels of p21Cip1, and suppressing the neurogenic potential of MGPCs.

ID3 is a novel target gene of p53 and modulates lung cancer cell metastasis.

The tumor suppressor p53 prevents cancer development by regulating dozens of target genes with diverse biological functions. Although numerous p53 target genes have been identified to date, the dynamics and function of the regulatory network centered on p53 have not yet been fully elucidated. We herein identified inhibitor of DNA-binding/differentiation-3 (ID3) as a direct p53 target gene. p53 bound the distal promoter of ID3 and positively regulated its transcription. ID3 expression was significantly decreased in clinical lung cancer tissues, and was closely associated with overall survival outcomes in these patients. Functionally, ID3 deficiency promoted the metastatic ability of lung cancer cells through its effects on the transcriptional regulation of CDH1. Furthermore, the ectopic expression of ID3 in p53-knockdown cells restored E-cadherin expression. Collectively, the present results demonstrate that ID3 plays a tumor-suppressive role as a downstream effector of p53 and impedes lung cancer cell metastasis by regulating E-cadherin expression.

The nuclear factor ID3 endows macrophages with a potent anti-tumour activity.

Macrophage activation is controlled by a balance between activating and inhibitory receptors1-7, which protect normal tissues from excessive damage during infection8,9 but promote tumour growth and metastasis in cancer7,10. Here we report that the Kupffer cell lineage-determining factor ID3 controls this balance and selectively endows Kupffer cells with the ability to phagocytose live tumour cells and orchestrate the recruitment, proliferation and activation of natural killer and CD8 T lymphoid effector cells in the liver to restrict the growth of a variety of tumours. ID3 shifts the macrophage inhibitory/activating receptor balance to promote the phagocytic and lymphoid response, at least in part by buffering the binding of the transcription factors ELK1 and E2A at the SIRPA locus. Furthermore, loss- and gain-of-function experiments demonstrate that ID3 is sufficient to confer this potent anti-tumour activity to mouse bone-marrow-derived macrophages and human induced pluripotent stem-cell-derived macrophages. Expression of ID3 is therefore necessary and sufficient to endow macrophages with the ability to form an efficient anti-tumour niche, which could be harnessed for cell therapy in cancer.

Investigating the ID3/SLC22A4 as immune-related signatures in ischemic stroke.

BACKGROUND: Ischemic stroke (IS) is a fearful disease that can cause a variety of immune events. Nevertheless, precise immune-related mechanisms have yet to be systematically elucidated. This study aimed to identify immune-related signatures using machine learning and to validate them with animal experiments and single cell analysis. METHODS: In this study, we screened 24 differentially expressed genes (DEGs) while identifying immune-related signatures that may play a key role in IS development through a comprehensive strategy between least absolute shrinkage and selection operation (LASSO) regression, support vector machine (SVM) and immune-related genes. In addition, we explored immune infiltration using the CIBERSORT algorithm. Finally, we performed validation in mouse brain tissue and single cell analysis. RESULTS: We identified 24 DEGs for follow-up analysis. ID3 and SLC22A4 were finally identified as the better immune-related signatures through a comprehensive strategy among DEGs, LASSO, SVM and immune-related genes. RT-qPCR, western blot, and immunofluorescence revealed a significant decrease in ID3 and a significant increase in SLC22A4 in the middle cerebral artery occlusion group. Single cell analysis revealed that ID3 was mainly concentrated in endothelial_2 cells and SLC22A4 in astrocytes in the MCAO group. A CIBERSORT finds significantly altered levels of immune infiltration in IS patients. CONCLUSIONS: This study focused on immune-related signatures after stroke and ID3 and SLC22A4 may be new therapeutic targets to promote functional recovery after stroke. Furthermore, the association of ID3 and SLC22A4 with immune cells may be a new direction for post-stroke immunotherapy.

ID3 regulates progesterone synthesis in bovine cumulus cells through modulation of mitochondrial function.

DNA binding inhibitory factor 3 (ID3) has been shown to have a key role in maintaining proliferation and differentiation. It has been suggested that ID3 may also affect mammalian ovarian function. However, the specific roles and mechanisms are unclear. In this study, the expression level of ID3 in cumulus cells (CCs) was inhibited by siRNA, and the downstream regulatory network of ID3 was uncovered by high-throughput sequencing. The effects of ID3 inhibition on mitochondrial function, progesterone synthesis, and oocyte maturation were further explored. The GO and KEGG analysis results showed that after ID3 inhibition, differentially expressed genes, including StAR, CYP11A1, and HSD3B1, were involved in cholesterol-related processes and progesterone-mediated oocyte maturation. Apoptosis in CC was increased, while the phosphorylation level of ERK1/2 was inhibited. During this process, mitochondrial dynamics and function were disrupted. In addition, the first polar body extrusion rate, ATP production and antioxidation capacity were reduced, which suggested that ID3 inhibition led to poor oocyte maturation and quality. The results will provide a new basis for understanding the biological roles of ID3 as well as cumulus cells.

Inhibitor of DNA binding/differentiation proteins as IDs for pancreatic cancer: Role in pancreatic cancer initiation, development and prognosis.

A family of inhibitors of cell differentiation or DNA-binding proteins, known as ID proteins (ID1-4), function as mighty transcription factors in various cellular processes, such as inhibiting differentiation, promoting cell-cycle progression, senescence, angiogenesis, tumorigenesis, and metastasis in cancer. Pancreatic cancer represents the deadliest cancer with the lowest survival rate of 10% due to the diagnosis at an advanced fatal stage and therapeutic resistance. Modestly, the only curative option for this lethal cancer is surgery but is done in less than 15-20% of patients because of the locally aggressive and early metastatic nature. Finding the earliest biomarkers and targeting the various hallmarks of pancreatic cancer can improve the treatment and survival of pancreatic cancer patients. Therefore, herein in this review, we explore in depth the potential roles of ID proteins function in hallmarks of pancreatic cancer, signaling pathways, and its oncogenic and tumor-suppressive effects. Hence, understanding the roles of dysregulated ID proteins would provide new insights into its function in pancreatic cancer tumorigenesis.

miR-210-3p protects against osteoarthritis through inhibiting subchondral angiogenesis by targeting the expression of TGFBR1 and ID4.

Excessive subchondral angiogenesis is a key pathological feature of osteoarthritis (OA), as it alters the balance of subchondral bone remodeling and causes progressive cartilage degradation. We previously found that miR-210-3p correlates negatively with angiogenesis, though the specific mechanism of miR-210-3p-related angiogenesis in subchondral bone during OA progression remains unclear. This study was conducted to identify the miR-210-3p-modulating subchondral angiogenesis mechanism in OA and investigate its therapeutic effect. We found that miR-210-3p expression correlated negatively with subchondral endomucin positive (Emcn+) vasculature in the knee joints of OA mice. miR-210-3p overexpression regulated the angiogenic ability of endothelial cells (ECs) under hypoxic conditions in vitro. Mechanistically, miR-210-3p inhibited ECs angiogenesis by suppressing transforming growth factor beta receptor 1 (TGFBR1) mRNA translation and degrading DNA-binding inhibitor 4 (ID4) mRNA. In addition, TGFBR1 downregulated the expression of ID4. Reduced ID4 levels led to a negative feedback regulation of TGFBR1, enhancing the inhibitory effect of miR-210-3p on angiogenesis. In OA mice, miR-210-3p overexpression in ECs via adeno-associated virus (AAV) alleviated cartilage degradation, suppressed the type 17 immune response and relieved symptoms by attenuating subchondral Emcn+ vasculature and subchondral bone remodeling. In conclusion, we identified a miR-210-3p/TGFBR1/ID4 axis in subchondral ECs that modulates OA progression via subchondral angiogenesis, representing a potential OA therapy target.

Shifting gears: Id3 enables recruitment of E proteins to new targets during T cell development and differentiation.

Shifting levels of E proteins and Id factors are pivotal in T cell commitment and differentiation, both in the thymus and in the periphery. Id2 and Id3 are two different factors that prevent E proteins from binding to their target gene cis-regulatory sequences and inducing gene expression. Although they use the same mechanism to suppress E protein activity, Id2 and Id3 play very different roles in T cell development and CD4 T cell differentiation. Id2 imposes an irreversible choice in early T cell precursors between innate and adaptive lineages, which can be thought of as a railway switch that directs T cells down one path or another. By contrast, Id3 acts in a transient fashion downstream of extracellular signals such as T cell receptor (TCR) signaling. TCR-dependent Id3 upregulation results in the dislodging of E proteins from their target sites while chromatin remodeling occurs. After the cessation of Id3 expression, E proteins can reassemble in the context of a new genomic landscape and molecular context that allows induction of different E protein target genes. To describe this mode of action, we have developed the "Clutch" model of differentiation. In this model, Id3 upregulation in response to TCR signaling acts as a clutch that stops E protein activity ("clutch in") long enough to allow shifting of the genomic landscape into a different "gear", resulting in accessibility to different E protein target genes once Id3 decreases ("clutch out") and E proteins can form new complexes on the DNA. While TCR signal strength and cytokine signaling play a role in both peripheral and thymic lineage decisions, the remodeling of chromatin and E protein target genes appears to be more heavily influenced by the cytokine milieu in the periphery, whereas the outcome of Id3 activity during T cell development in the thymus appears to depend more on the TCR signal strength. Thus, while the Clutch model applies to both CD4 T cell differentiation and T cell developmental transitions within the thymus, changes in chromatin accessibility are modulated by biased inputs in these different environments. New emerging technologies should enable a better understanding of the molecular events that happen during these transitions, and how they fit into the gene regulatory networks that drive T cell development and differentiation.

Id3 expression identifies CD4+ memory Th1 cells.

Memory CD4+ T cells play a pivotal role in mediating long-term protective immunity, positioning them as an important target in vaccine development. However, multiple functionally distinct helper CD4+ T-cell subsets can arise in response to a single invading pathogen, complicating the identification of rare populations of memory precursor cells during the effector phase of infection and memory CD4+ T cells following pathogen clearance and the contraction phase of infection. Furthermore, current literature remains unclear regarding whether a single CD4+ memory T-cell lineage gives rise to secondary CD4+ T helper subsets or if there are unique memory precursor cells within each helper lineage. A majority of T follicular helper (Tfh) cells, which have established memory potential, express Id3, an inhibitor of E protein transcription factors, following acute viral infection. We show that expression of Id3 definitively identified a subset of cells within both the CD4+ Tfh and T helper 1 (Th1) lineages at memory time points that exhibited memory potential, with the capacity for significant re-expansion in response to secondary infection. Notably, we demonstrate that a subset of Th1 cells that survive into the memory phase were marked by Id3 expression and possessed the potential for enhanced expansion and generation of both Th1 and Tfh secondary effector cell populations in a secondary response to pathogen. Additionally, these cells exhibited enrichment of key molecules associated with memory potential when compared with Id3lo Th1 cells. Therefore, we propose that Id3 expression serves as an important marker to indicate multipotent potential in memory CD4+ T cells.

Circadian Period 2 (Per2) downregulate inhibitor of differentiation 3 (Id3) expression via PTEN/AKT/Smad5 axis to inhibits glioma cell proliferation.

In this study, we employed multiple laboratory techniques to acknowledge the biological activities and processes of Per2 and Id3 in glioma. We analyzed TCGA and CGGA databases for seeking association among Per2, Id3, and clinical features in glioma. Immunohistochemistry and Western blot were used to detect protein expression levels. CCK-8 assay, colony formation assay, Transwell assay, the wound healing assay, flow cytometric, and Xenograft nude mice were used to acknowledge the impact of Per2 and Id3 on biological behavior of glioma. The results showed that the Per2 mRNA expression was negatively correlated with the WHO grade, while the Id3 mRNA expression was positively correlated with the WHO grade in patients with glioma in TCGA and CGGA databases. Per2 and Id3 maintained separate prognostic abilities and had a negative connection in human glioma. In the clinical sample study, Per2 and Id3 were validated at the protein level with the same results compared to the mRNA expression level in TCGA and CGGA. By using a wide range of functional examples, overexpression of Per2 restrains malignant biological behaviors in glioma cells by many ways, while Id3 promotes malignant biological behaviors in glioma cells. Furthermore, overexpression of Per2 can inhibit Id3 expression via regulating PTEN/AKT/Smad5 signaling pathway and thereby abolish malignant biological behaviors that are caused by Id3 overexpression. These results suggested that Per2 inhibits glioma cell proliferation through regulating PTEN/AKT/Smad5/Id3 signaling pathway, which may be a viable therapeutic target for glioma.

MicroRNA-24-3p promotes skeletal muscle differentiation and regeneration by regulating HMGA1.

Numerous studies have established the critical roles of microRNAs in regulating post-transcriptional gene expression in diverse biological processes. Here, we report on the role and mechanism of miR-24-3p in skeletal muscle differentiation and regeneration. miR-24-3p promotes myoblast differentiation and skeletal muscle regeneration by directly targeting high mobility group AT-hook 1 (HMGA1) and regulating it and its direct downstream target, the inhibitor of differentiation 3 (ID3). miR-24-3p knockdown in neonatal mice increases PAX7-positive proliferating muscle stem cells (MuSCs) by derepressing Hmga1 and Id3. Similarly, inhibition of miR-24-3p in the tibialis anterior muscle prevents Hmga1 and Id3 downregulation and impairs regeneration. These findings provide evidence that the miR-24-3p/HMGA1/ID3 axis is required for MuSC differentiation and skeletal muscle regeneration in vivo.

Expression of Id3 represses exhaustion of anti-tumor CD8 T cells in liver cancer.

Id3, an inhibitor of DNA binding protein, plays important roles in the function and homeostasis of effector and memory T cells. Recent evidence has shown that Id3 is also implicated in CD8 T cell exhaustion. However, whether and how Id3 might regulate effector function or exhaustion of CD8 T cells, especially in the tumor setting, is still unknown. Here, we first showed that Id3 expression was impaired in tumor-infiltrating CD8 T cells as liver cancer progressed, especially in PD-1 +Tim-3 + exhausted CD8 T cells. Enforced expression of Id3 in CD8 T cells resulted in repressed development of anti-tumor CTLs exhaustion, which offered better tumor control. And partially depletion of Id3 in CD8 T cells promoted the development of exhausted CD8 T cells. Furthermore, Id3hi CD8 T cells could respond to PD-1 blockade. Collectively, Id3 exerts protective functions in CD8 T cells for liver 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.

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.

Prosurvival IL-7-Stimulated Weak Strength of mTORC1-S6K Controls T Cell Memory via Transcriptional FOXO1-TCF1-Id3 and Metabolic AMPKα1-ULK1-ATG7 Pathways.

CD8+ memory T (TM) cells play a critical role in immune defense against infection. Two common γ-chain family cytokines, IL-2 and IL-7, although triggering the same mTORC1-S6K pathway, distinctly induce effector T (TE) cells and TM cells, respectively, but the underlying mechanism(s) remains elusive. In this study, we generated IL-7R-/and AMPKα1-knockout (KO)/OTI mice. By using genetic and pharmaceutical tools, we demonstrate that IL-7 deficiency represses expression of FOXO1, TCF1, p-AMPKα1 (T172), and p-ULK1 (S555) and abolishes T cell memory differentiation in IL-7R KO T cells after Listeria monocytogenesis rLmOVA infection. IL-2- and IL-7-stimulated strong and weak S6K (IL-2/S6Kstrong and IL-7/S6Kweak) signals control short-lived IL-7R-CD62L-KLRG1+ TE and long-term IL-7R+CD62L+KLRG1- TM cell formations, respectively. To assess underlying molecular pathway(s), we performed flow cytometry, Western blotting, confocal microscopy, and Seahorse assay analyses by using the IL-7/S6Kweak-stimulated TM (IL-7/TM) and the control IL-2/S6Kstrong-stimulated TE (IL-2/TE) cells. We determine that the IL-7/S6Kweak signal activates transcriptional FOXO1, TCF1, and Id3 and metabolic p-AMPKα1, p-ULK1, and ATG7 molecules in IL-7/TM cells. IL-7/TM cells upregulate IL-7R and CD62L, promote mitochondria biogenesis and fatty acid oxidation metabolism, and show long-term cell survival and functional recall responses. Interestingly, AMPKα1 deficiency abolishes the AMPKα1 but maintains the FOXO1 pathway and induces a metabolic switch from fatty acid oxidation to glycolysis in AMPKα1 KO IL-7/TM cells, leading to loss of cell survival and recall responses. Taken together, our data demonstrate that IL-7-stimulated weak strength of mTORC1-S6K signaling controls T cell memory via activation of transcriptional FOXO1-TCF1-Id3 and metabolic AMPKα1-ULK1-ATG7 pathways. This (to our knowledge) novel finding provides a new mechanism for a distinct IL-2/IL-7 stimulation model in T cell memory and greatly impacts vaccine development.