Matrine, a potential c-Myc inhibitor, suppresses ribosome biogenesis and nucleotide metabolism in myeloid leukemia.

Previous studies have shown that matrine, a natural compound extracted from the herb Sophora flavescens, has a good anti-leukemia effect, but its key target and mechanism remains unclear. Here, we found that only c-Myc could respond rapidly to matrine treatment in three myeloid leukemia cell lines, and matrine inhibited both transcription and translation of c-Myc. Ribosome biogenesis and nucleotide metabolism, the key downstream of c-Myc, were significantly suppressed after matrine treatment. Therefore, our results confirmed that matrine is a special c-Myc inhibitor which suppresses ribosome biogenesis and nucleotide metabolism by inhibiting c-Myc in myeloid leukemia. This study provides scientific basis for the development of matrine derivatives to c-Myc-driven cancers.

Notch3 Knockout Suppresses Mouse Mammary Gland Development and Inhibits the Proliferation of 4T1 Murine Mammary Carcinoma Cells via CCL2/CCR4 Axis.

Our previous study found that Notch3 knockout mice exhibit defects in mammary gland development. To elucidate the underlying mechanism, tissue samples were subjected to RNA-seq, GO, and KEGG enrichment analyses and qRT-PCR validation. Of enriched pathways, chemokine signaling pathway and cytokine-cytokine receptor interaction were noticed in both Notch3wt/wt/Notch3wt/- and Notch3wt/wt/Notch3-/- mice, in which the expression of chemokine ligand 2 (CCL2) was sharply reduced in Notch3wt/- and Notch3-/- mammary gland tissues. The Mouse ENCODE transcriptome data reveal that the mammary gland fat pad exhibits a high CCL2, CCR2, and CCR4 expression, indicating that these molecules play important roles during mammary gland development. Specifically, defective mammary glands in Notch3 knockout mice could be partially rescued by CCL2 overexpression lentivirus through intraductal injection. An in vitro study showed that CCL2 overexpression promoted the proliferation, migration, and cancerous acinar formation of 4T1 cells, which could rescue the defective migration of 4T1 cells caused by Notch3 knockdown. We also found that Notch3 transcriptionally regulated the expression of CCL2 in a classical pattern. Our findings illustrated that Notch3-regulating CCL2/CCR4 axis should be an important signaling pathway for mammary gland development and should be a candidate target for breast cancer therapy.

Three-dimensional hierarchical ZnCo2O4@C3N4-B nanoflowers as high-performance anode materials for lithium-ion batteries.

ZnCo2O4 has become one of the most widely used anode materials due to its good specific capacity, cost-efficiency, high thermal stability and environmental benignity. However, its poor conductivity and cycle stability have limited its practical application in lithium-ion batteries. To overcome these issues, we constructed a 3D nanoflower composite material (ZnCo2O4@C3N4-B) by combining ZnCo2O4 as a framework and B-doped g-C3N4 (g-C3N4-B) as a new carbon source material via a simple hydrothermal method. ZnCo2O4@C3N4-B exhibited exceptional specific capacitance of 919.76 mA h g-1 after 500 cycles at 0.2 A g-1 and a long-term capacity retention of 97.8% after 1000 cycles at 2 A g-1. The high reversible capacity, long cycling life and good rate performance could be attributed to the 3D interconnected architecture and doping of g-C3N4-B. This work provides a simple and general strategy to design high-performance anode materials for lithium-ion batteries to meet the needs of practical applications.

Notch3 promotes 3T3-L1 pre-adipocytes differentiation by up-regulating the expression of LARS to activate the mTOR pathway.

Adipocytes constitute a major component of the tumour microenvironment. Numerous studies have shown that adipocytes promote aggressiveness and invasion by stimulating cancer cells proliferation and modulating their metabolism. Herein, we reported that Notch3 promotes mouse 3T3-L1 pre-adipocytes differentiation by performing the integrative transcriptome and TMT-based proteomic analyses. The results revealed that aminoacyl-tRNA_biosynthesis pathway was significantly influenced with Nocth3 change during 3T3-L1 pre-adipocytes differentiation, and the expression of LARS in this pathway was positively correlated with Notch3. Published studies have shown that LARS is a sensor of leucine that regulates the mTOR pathway activity, and the latter involves in adipogenesis. We therefore supposed that Notch3 might promote 3T3-L1 pre-adipocytes differentiation by up-regulating LARS expression and activating mTOR pathway. CHIP and luciferase activity assay uncovered that Notch3 could transcriptionally regulate the expression of LARS gene. Oil Red staining identified a positive correlation between Notch3 expression and adipocytic differentiation. The activation of mTOR pathway caused by Notch3 overexpression could be attenuated by knocking down LARS expression. Altogether, our study revealed that Notch3 promotes adipocytic differentiation of 3T3-L1 pre-adipocytes cells by up-regulating LARS expression and activating the mTOR pathway, which might be an emerging target for obesity treatment.

The frequency of CpG and non-CpG methylation of Notch3 gene promoter determines its expression levels in breast cancer cells.

Notch3 can act as a tumor suppressor in the breast cancer epithelial cells. Unfortunately, Notch3 expression is decreased or lost, especially in triple-negative breast cancer (TNBC) cells, and the reasons remain unclear. Here, we found Notch3 was upregulated in MDA-MB-231 cells with 5-Aza treatment. Two CpG islands were observed in notch3 promoter. Interestingly, bisulfite sequencing exhibited that large amounts of unconverted cytosines were not only followed by guanine, but also adenine, cytosine and thymine, which implied that there simultaneously existed CpG and non-CpG methylation in notch3 promoter. To better analyze the methylation frequency of non-CpG locus, we designed CpG/non-CpG methylation analysis software. The results showed that the methylation frequency of notch3 gene in different breast cancer cell lines was in order T47D, MCF-7, SKBR3, BT-549 and MDA-MB-231. Furthermore, we identified that DNMT3b, DNMT1, DNMT3L, Mecp2 and EZH2 were important regulators of non-CpG locus of notch3 gene. Immunohistochemistry staining revealed a negative correlation between EZH2 and Notch3 from 22 luminal and 26 TNBC cases. In vitro methylation combined luciferase activity assays showed that non-CpG methylation was still crucial cause leading to notch3 transcriptional repression in TNBC. Our findings provide possible explanation for the downregulation or loss of Notch3 expression in TNBC.

N3ICD with the transmembrane domain can effectively inhibit EMT by correcting the position of tight/adherens junctions.

EMT allows a polarized epithelium to lose epithelial integrity and acquire mesenchymal characteristics. Previously, we found that overexpression of the intracellular domain of Notch3 (N3ICD) can inhibit EMT in breast cancer cells. In this study, we aimed to elucidate the influence of N3ICD or N3ICD combined with the transmembrane domain (TD+N3ICD) on the expression and distribution of TJs/AJs and polar molecules. We found that although N3ICD can upregulate the expression levels of the above-mentioned molecules, TD+N3ICD can inhibit EMT more effectively than N3ICD alone. TD+N3ICD overexpression upregulated the expression of endogenous full-length Notch3 and contributed to correcting the position of TJs/AJs molecules and better acinar structures formation. Co-immunoprecipitation results showed that the upregulated endogenous full-length Notch3 could physically interact with E-ca in MDA-MB-231/pCMV-(TD+N3ICD) cells. Collectively, our data indicate that overexpression of TD+N3ICD can effectively inhibit EMT, resulting in better positioning of TJs/AJs molecules and cell-cell adhesion in breast cancer cells. Abbreviations: EMT: Epithelial-mesenchymal transition; TJs: Tight junctions; AJs: Adherens junctions; aPKC: Atypical protein kinase C; Crb: Crumbs; Lgl: Lethal (2) giant larvae; LLGL2: lethal giant larvae homolog 2; PAR: Partitioning defective; PATJ: Pals1-associated TJ protein.

Hypomethylation at non-CpG/CpG sites in the promoter of HIF-1α gene combined with enhanced H3K9Ac modification contribute to maintain higher HIF-1α expression in breast cancer.

HIF-1α has a broad impact on tumors, including enhanced utilization of glucose, tumor cell stemness, migration, metastasis and so on. In pilot study, we found that the expression of HIF-1α significantly increased in breast cancer cell lines and tissue samples with higher malignant behaviors and decreased in luminal subtype breast cancer cells and tissue samples. We analyzed and found there is one large CpG island in HIF-1α promoter around transcription start site, and the hypermethylation occurred at these CpGs and their surrounding non-CpGs sites. Epigenetic events driving tumorigenesis has been characterized. However, knowledge is lacking on the non-CpGs methylation of HIF-1α promoter in breast cancer cells. We validated that non-CpGs methylation can directly regulate HIF-1α expression by luciferase activity assay. We also found DNMT3a and Mecp2 play vital role in methylation at non-CpGs and CpGs sites. In addition, we noticed that H3K9ac modification could promote the transcription of HIF-1α in MDA-MB-231 cells by binding to the region contained hypomethylated non-CpG and CpG sites. Taken together, the hypomethylation status at non-CpG and CpG loci in HIF-1α promoter and H3K9ac modification together contribute to maintain higher HIF-1αactivity in invasive breast cancer cells when compared with the non-invasive breast cancer cells, which may establish a tissue-specific epigenetic modification pattern and point to the new directions for future understanding breast cancer therapy.

The EZH2- H3K27me3-DNMT1 complex orchestrates epigenetic silencing of the wwc1 gene, a Hippo/YAP pathway upstream effector, in breast cancer epithelial cells.

It is well known that epithelial-mesenchymal transition (EMT) can confer cancer cells with invasive and migratory capabilities associated with distant metastasis. As a key upstream factor in the Hippo pathway, Kibra (wwc1 gene) has been shown to suppress EMT in breast cancer cells, and we have found that its expression is reduced or lost completely in both human breast cancer cell lines and clinical tissue samples, particularly in triple negative breast cancer (TNBC). Unfortunately, the molecular mechanisms underlying this progression-associated event remain to be elucidated. Epigenetic gene silencing is one of the most common causes of suppressed expression of tumor suppressor genes. Furthermore, recent studies have demonstrated that EZH2 can recruit DNA methyltransferases, resulting in DNA methylation and subsequent gene silencing in certain circumstances. Thus, we hypothesized that there may exist a link between EZH2 and DNA methylation in association with wwc1 silencing in breast cancer. To test this hypothesis, we performed bisulfite sequencing, shRNA, co-IP, ChIP, MeDIP and ChIP-qPCR. As expected, RG108 or 5-Aza treatment improved the wwc1 gene transcription and Kibra protein expression. Both bisulfite sequencing and MeDIP demonstrated higher CpG methylation of the wwc1 promoter the TNBC cells (MDA-MB-231) than in luminal breast cancer cells (MCF7). It is noteworthy that ChIP and co-IP assays showed that EZH2, H3K27me3 and DNMT1 are enriched at the wwc1 promoter, and there exist physiologically relevant protein-protein interactions between them. We also found that EZH2 knockdown leads to a partial increase in Kibra expression and a considerable reduction in H3K27 and DNMT1 trimethylation. Moreover, ChIP-qPCR revealed more DNA fragments containing the wwc1 promoter in MDA-MB-231 than in MCF7 cells after immunoprecipitation with EZH2, DNMT1 and H3K27me3 antibodies. Collectively, our results reveal crosstalk between H3K27me3 inhibition catalyzed by EZH2 and CpG island methylation mediated by DNMT1 within the wwc1 promoter, which synergistically silence wwc1 gene expression in TNBC. Based on these results, we conclude that EZH2 shows promise as a potential anti-tumor target.