METTL3 orchestrates glycolysis by stabilizing the c-Myc/WDR5 complex in triple-negative breast cancer.

BACKGROUND: The carcinogenic transcription factor c-Myc is the most aggressive oncogene, which drive malignant transformation and dissemination of triple-negative breast cancer (TNBC). Recruitment of many cofactors, especially WDR5, a protein that nucleates H3K4me chromatin modifying complexes, play a pivotal role in regulating c-Myc-dependent gene transcription, a critical process for c-Myc signaling to function in a variety of biological and pathological contexts. For this reason, interrupting the interaction between c-Myc and the transcription cofactor WDR5 may become the most promising new strategy for treating c-Myc driven TNBC. METHODS: Immunoprecipitation and mass spectrometry (IP-MS) is used to screen proteins that bind c-Myc/WDR5 interactions. The interaction of METTL3 with c-Myc/WDR5 in breast cancer tissues and TNBC cells was detected by Co-IP and immunofluorescence. Subsequently, we further analyzed the influence of METTL3 expression on c-Myc/WDR5 protein expression and its interaction stability by Western blot and Co-IP. The correlation between METTL3 and c-Myc pathway was analyzed by ChIP-seq sequencing and METTL3 knockdown transcriptome data. The effect of METTL3 expression on c-Myc transcriptional activity was detected by ChIP-qPCR and Dual Luciferase Reporter. At the same time, the overexpression vector METTL3-MUT (m6A) was constructed, which mutated the methyltransferase active site (Aa395-398, DPPW/APPA), and further explored whether the interaction between METTL3 and c-Myc/WDR5 was independent of methyltransferase activity. In addition, we also detected the changes of METTL3 expression on TNBC's sensitivity to small molecule inhibitors such as JQ1 and OICR9429 by CCK8, Transwell and clonal formation assays. Finally, we further verified our conclusions in spontaneous tumor formation mouse MMTV-PyMT and nude mouse orthotopic transplantation tumor models. RESULTS: METTL3 was found to bind mainly to c-Myc/WDR5 protein in the nucleus. It enhances the stability of c-Myc/WDR5 interaction through its methyltransferase independent mechanism, thereby enhancing the transcriptional activity of c-Myc on downstream glucose metabolism genes. Notably, the study also confirmed that METTL3 can directly participate in the transcription of glucose metabolism genes as a transcription factor, and knockdown METTL3 enhances the drug sensitivity of breast cancer cells to small molecule inhibitors JQ1 and OICR9429. The study was further confirmed by spontaneous tumor formation mouse MMTV-PyMT and nude mouse orthotopic transplantation tumor models. CONCLUSION: METTL3 binds to the c-Myc/WDR5 protein complex and promotes glycolysis, which plays a powerful role in promoting TNBC progression. Our findings further broaden our understanding of the role and mechanism of action of METTL3, and may open up new therapeutic avenues for effective treatment of TNBC with high c-Myc expression.

IGF2BP2-modified UBE2D1 interacts with Smad2/3 to promote the progression of breast cancer.

Recent studies have suggested that ubiquitin-conjugating enzyme E2D1 (UBE2D1) is involved in tumor progression. In this study, we found that UBE2D1 expression was upregulated in breast cancer (BC) and was related to the prognosis of BC patients. Through in vitro and in vivo experiments, we demonstrated the aberrant expression of UBE2D1 promoted the proliferation and migration of BC cells, and the IGF2BP2-mediated N6-methyladenosine (m6A) modification increased the stability of UBE2D1 mRNA. Mechanistically, UBE2D1 expression regulated the activity of TGF-ß signaling through modulating the expression and the phosphorylation level of Smad2/3. Furthermore, UBE2D1 directly bound to Smad2/3 and affected the subsequent binding of Smad2 and Smad3, which is a necessary step for TGF-ß signaling activation. Thus, our study reveals a pro-oncogenic role of UBE2D1 in the progression of BC and may provide novel strategies for BC treatment.

Mettl3 synergistically regulates TGF-ß/SMAD2/3 to promote proliferation and metastasis of gastric cancer.

Transforming Growth factor-ß (TGF-ß)/Smad signaling is a complex regulatory network that both inhibits and promotes tumorigenesis. However, the mechanisms underlying the function of TGF-ß/Smad signaling pathway remain to be fully elucidated. As a methyltransferase, METTL3 is closely related to tumor development, but the role of METTL3 in the proliferation and metastasis of TGF-ß/Smad-activated gastric cancer (GC) is unclear. In this study, we identified TGF-ß/Smad2/3 axis as an important carcinogenic pathway in GC, which significantly promoted the proliferation and metastasis of GC. Furthermore, we found that Smad3 mRNA could be modified by m6A, which was subsequently recognized and stabilized by IGF2BP2, thereby enhancing Smad3 protein expression and promoting the activation of TGF-ß/Smad pathway. Importantly, we also found that METTL3 could combine with p-Smad3 to regulate the transcription of downstream target genes. Therefore, this study revealed a novel mechanism by which METTL3 synergistically regulates TGF-ß/Smad2/3 signaling and provide a new potential therapeutic target for the treatment of GC.

Betulinic acid inhibits cell proliferation and migration in gastric cancer by targeting the NF-κB/VASP pathway.

Gastric cancer (GC) is one of the most common malignant neoplasms of the digestive system, with China leading in terms of morbidity and mortality rates. Betulinic acid (BA) is a widely-occurring pentacyclic triterpenoid that has been reported to exhibit potent anti-inflammatory, antioxidant, and antitumor activities. BA can combat tumors by inducing apoptosis, regulating cell cycle, and inhibiting autophagy, but its mechanism of action in the context of GC is unclear. A preliminary study found that higher expression of vasodilator-stimulated phosphoprotein (VASP) was correlated with migration in the GC cell line. In this study, BGC-823 cells and MNK45 cells were treated with BA for investigating its effect on the proliferation and migration of cells. Moreover, the expression of VASP and upstream signal molecules were also investigated in this background. The results showed BA could inhibit the proliferation and migration the GC cells. Furthermore, NF-κB acted as a transcription factor to upregulate VASP expression. Moreover, BA could downregulate the expression of VASP at the protein and mRNA level by inhibiting NF-κB activity. In conclusion, these results suggest that BA could inhibit the expression of VASP by negatively regulating NF-κB, thereby inhibiting the proliferation and migration of the GC cells. Our study provides a theoretical basis for exploring the molecular mechanism underlying BA-induced inhibition of proliferation and migration in GC cells.

CREB1/Lin28/miR-638/VASP Interactive Network Drives the Development of Breast Cancer.

Breast cancer is one of the most common malignant tumors worldwide. Metastasis remains the leading cause of death in breast cancer patients. Research on the mechanism of breast cancer metastasis has become a core issue in breast cancer research. Our previous series of studies have shown that VASP, as a key oncogene, plays an important role in the development of various tumors such as breast cancer. In this study, we find that miR-638 can target to inhibit VASP expression, and Lin28 acts as an RNA-binding protein to regulate the processing of miR-638, which inhibits its maturation and promotes the expression of VASP. In addition, we also find that CREB1 acts as a transcription factor that binds to the promoter of Lin28 gene and activates the Lin28/miR-638/VASP pathway. Furthermore, CREB1 can also directly bind to the promoter of VASP, and activate VASP expression, forming a CREB/Lin28/miR-638/VASP interactive network, which plays an important role in promoting cell proliferation and migration in breast cancer. Our study explained the mechanism of CREB1/Lin28/miR-638/VASP network promoting the development of breast cancer, which further elucidated the mechanism of VASP as a key oncogene, and also provided a theoretical basis for expanding new approaches to tumor biotherapy.

Silencing lncRNA SNHG6 suppresses proliferation and invasion of breast cancer cells through miR-26a/VASP axis.

The important role of LncRNA in the development of breast cancer is attracting more and more attention. In the previous study, we found that the expression level of LncRNA SNHG6 in breast cancer tissues and cells was significantly increased, but its mechanism in the development of breast cancer was still unclear. Our study found that knockdown of SNHG6 significantly inhibited the proliferation, migration and invasion of breast cancer cells MCF-7 and MDA-MB-231 cells. Further study showed that knockdown of SNHG6 significantly inhibited the expression level of VASP. More importantly, SNHG6 and VASP both can bind directly to miR-26a, suggesting that SNHG6 could act as a ceRNA to sponge miR-26a, thereby promoting the expression of VASP, which leading to activated proliferation, migration and invasion of breast cancer cells. Taken together, this study revealed the important role of the SNHG6/miR-26a/VASP regulatory network in the development of breast cancer, and provided a reference for exploring new pathogenesis and biomarkers of breast cancer.

Chlorotoxin targets ERα/VASP signaling pathway to combat breast cancer.

Breast cancer is one of the most common malignant tumors among women worldwide. About 70-75% of primary breast cancers belong to estrogen receptor (ER)-positive breast cancer. In the development of ER-positive breast cancer, abnormal activation of the ERα pathway plays an important role and is also a key point leading to the failure of clinical endocrine therapy. In this study, we found that the small molecule peptide chlorotoxin (CTX) can significantly inhibit the proliferation, migration and invasion of breast cancer cells. In in vitro study, CTX inhibits the expression of ERα in breast cancer cells. Further studies showed that CTX can directly bind to ERα and change the protein secondary structure of its LBD domain, thereby inhibiting the ERα signaling pathway. In addition, we also found that vasodilator stimulated phosphoprotein (VASP) is a target gene of ERα signaling pathway, and CTX can inhibit breast cancer cell proliferation, migration, and invasion through ERα/VASP signaling pathway. In in vivo study, CTX significantly inhibits growth of ER overexpressing breast tumor and, more importantly, based on the mechanism of CTX interacting with ERα, we found that CTX can target ER overexpressing breast tumors in vivo. Our study reveals a new mechanism of CTX anti-ER-positive breast cancer, which also provides an important reference for the study of CTX anti-ER-related tumors.

Upregulated IQUB promotes cell proliferation and migration via activating Akt/GSK3ß/ß-catenin signaling pathway in breast cancer.

Breast cancer was the highest incidence of tumor in women, which seriously threaten women's health. Our previous study found that the expression of IQUB (IQ motif and ubiquitin domain containing) was significantly increased in the development of breast cancer by transcriptome sequencing. However, there were no studies on the mechanism of IQUB in tumorigenesis. Further study showed that IQUB expression was significantly increased in breast cancer, which had a significantly positive correlation with pathological differentiation of breast cancer by tissue microarray analysis. Furthermore, we also discovered that IQUB overexpression could obviously promote the proliferation and migration of MCF-7 cells and increase the proportion of MCF-7 cells in S and G2/M phase in vitro study, while knockdown of IQUB caused inhibition of cell proliferation and migration in MDA-MB-231 cells and increased the proportion of MDA-MB-231 cells in G1 phase. Furthermore, IQUB overexpression or knockdown combined with treatment of Licl or MG-132 showed that IQUB activated Akt to promote GSK3ß phosphorylation, which in turn activated Wnt/ß-catenin signaling pathway in breast cancer cells. Taken together, these results indicated that upregulated IQUB promoted breast cancer cell proliferation and migration via activating Akt/GSK3ß/ß-catenin signaling pathway, which played an important part in the tumorigenesis and development of breast cancer.

KN-93, a specific inhibitor of CaMKII inhibits human hepatic stellate cell proliferation in vitro.

AIM: To investigate the effects of KN-93, a CaMKII selective inhibitor on cell proliferation and the expression of p53 or p21 protein in human hepatic stellate cells. METHODS: Human hepatic stellate cells (LX-2) were incubated with various concentrations (0-50 micromol/L) of KN-93 or its inactive derivative, KN-92. Cell proliferation was measured by CCK-8 assay, and the expression of two cell cycle regulators, p53 and p21, was determined by SDS-PAGE and Western blotting. RESULTS: KN-93 (5-50 micromol/L) decreased the proliferation of human hepatic stellate cells in a dose-dependent manner from 81.76% (81.76% +/- 2.58% vs 96.63% +/- 2.69%, P < 0.05) to 27.15% (27.15% +/- 2.86% vs 96.59% +/- 2.44%, P < 0.01) after 24 h treatment. Incubation of 10 micromol/L KN-93 induced the cell growth reduction in a time-dependent manner from 78.27% at 8 h to 11.48% at 48 h. However, KN-92, an inactive derivative of KN-93, did not inhibit cell proliferation effectively. Moreover, analysis of cell cycle regulator expression revealed that KN-93 rather than KN-92 reduced the expression of p53 and p21. CONCLUSION: KN-93 has potent inhibitory effect on proliferation of LX-2 cells by modulating the expression of two special cell cycle regulators, p53 and p21.

Transcriptional diversity of DMRT1 (dsx- and mab3-related transcription factor 1) in human testis.

Recent advances in the evolutionary genetics of sex determination indicate that the only molecular similarity in sex determination found so far among phyla is between the fly doublesex, worm mab-3 and vertebrate DMRT1(dsx- and mab3-related transcription factor 1) /DMY genes. Each of these factors encodes a zinc-finger-like DNA-binding motif, DM domain. Insights into the evolution and functions of human DMRT1 gene could reveal evolutionary mechanisms of sexual development. Here we report the identification and characterization of multiple isoforms of human DMRT1 in the testis. These transcripts encode predicted proteins with 373, 275 and 175 amino acids and they were generated by alternative splicing at 3' region. Expression level of DMRT1a is higher than those of both DMRT1b and c, and the DMRT1c expression was the lowest in testis, based on comparisons of mean values from real-time fluorescent quantitative RT-PCR analysis. Both DMRT1b and c result from exonization of intronic sequences, including the exonization of an Alu element. A further search for Alu elements within the DMRT1 gene demonstrated that all 99 Alu elements are non-randomly distributed among the non-coding regions on both directions. These new characteristics of DMRT1 would have an important impact on the evolution of sexual development mechanisms.