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.

GMFG Has Potential to Be a Novel Prognostic Marker and Related to Immune Infiltrates in Breast Cancer.

A growing amount of evidence has indicated immune genes perform a crucial position in the development and progression of breast cancer microenvironment. The purpose of our study was to identify immunogenic prognostic marker and explore potential regulatory mechanisms for breast cancer. We identified the genes related to ImmuneScore using ESTIMATE algorithm and WGCNA analysis, and we identified the differentially expressed gene (DEGs). Then, Glia maturation factor γ (GMFG) was determined as a predictive factor by intersecting immune-related genes with DEGs and survival analysis. We found the expression of GMFG was lower in breast cancer tissues compared with normal breast tissues, which was further verified by immunohistochemical (IHC). Moreover, the decreased expression of GMFG was significantly related to the poor prognosis. Besides, the expression of GMFG was related to the age, ER status, PR status, HER2 status and tumor size, which further suggested that the expression of GMFG was correlated with the subtype and the growth of tumor. The univariate and multivariate Cox regression analyses revealed that age, stage, the expression level of GMFG and radiotherapy were independent factors for predicting the prognosis of breast cancer patients. Subsequently, a prognostic model to predict the 3-year, 5-year and 10-year overall survival rate was developed based on the above four variables, and visualized as a nomogram. The values of area under the curve of the nomogram at 3-year, 5-year and 10-year were 0.897, 0.873 and 0.922, respectively, which was higher than stage in prognostic accuracy. In addition, we also found that GMFG expression level was correlated with sensitivity of some breast cancer chemotherapy drugs. Furthermore, the results of GSEA indicated immune-related pathways were mainly enriched in GMFG-high-expression group. CIBERSORT analysis for the proportion of tumor-infiltrating immune cells (TIICs) suggested that expression of GMFG was positively association with multiple kinds T-cell in BC. Among them, CD8+ T cells had the strongest correlation with GMFG expression, which revealed that GMFG might has an antitumor effect by increasing the infiltration of CD8+ T cells in breast cancer. Accordingly, GMFG has the potential to become a novel immune biomarker for the diagnosis and treatment of breast cancer.

RNA N6-Methyladenosine Regulators Contribute to Tumor Immune Microenvironment and Have Clinical Prognostic Impact in Breast Cancer.

Purpose: This study aims to reveal the relationship between RNA N6-methyladenosine (m6A) regulators and tumor immune microenvironment (TME) in breast cancer, and to establish a risk model for predicting the occurrence and development of tumors. Patients and methods: In the present study, we respectively downloaded the transcriptome dataset of breast cancer from Gene Expression Omnibus (GEO) database and The Cancer Genome Atlas (TCGA) database to analyze the mutation characteristics of m6A regulators and their expression profile in different clinicopathological groups. Then we used the weighted correlation network analysis (WGCNA), the least absolute shrinkage and selection operator (LASSO), and cox regression to construct a risk prediction model based on m6A-associated hub genes. In addition, Immune infiltration analysis and gene set enrichment analysis (GSEA) was used to evaluate the immune cell context and the enriched gene sets among the subgroups. Results: Compared with adjacent normal tissue, differentially expressed 24 m6A regulators were identified in breast cancer. According to the expression features of m6A regulators above, we established two subgroups of breast cancer, which were also surprisingly distinguished by the feature of the immune microenvironment. The Model based on modification patterns of m6A regulators could predict the patient's T stage and evaluate their prognosis. Besides, the low m6aRiskscore group presents an immune-activated phenotype as well as a lower tumor mutation load, and its 5-years survival rate was 90.5%, while that of the high m6ariskscore group was only 74.1%. Finally, the cohort confirmed that age (p < 0.001) and m6aRiskscore (p < 0.001) are both risk factors for breast cancer in the multivariate regression. Conclusion: The m6A regulators play an important role in the regulation of breast tumor immune microenvironment and is helpful to provide guidance for clinical immunotherapy.

Hypoxia-Associated Prognostic Markers and Competing Endogenous RNA Co-Expression Networks in Breast Cancer.

Objective: Many primary tumors have insufficient supply of molecular oxygen, called hypoxia. Hypoxia is one of the leading characteristics of solid tumors resulting in a higher risk of local failure and distant metastasis. It is quite necessary to investigate the hypoxia associated molecular hallmarks in breast cancer. Materials and Methods: According to the published studies, we selected 13 hypoxia related gene expression signature to define the hypoxia status of breast cancer using ConsensusClusterPlus package based on the data from The Cancer Genome Atlas (TCGA). Subsequently, we characterized the infiltration of 24 immune cell types under different hypoxic conditions. Furthermore, the differentially expressed hypoxia associated microRNAs, mRNAs and related signaling pathways were analyzed and depicted. On this basis, a series of prognostic markers related to hypoxia were identified and ceRNA co-expression networks were constructed. Results: Two subgroups (cluster1 and cluster2) were identified and the 13 hypoxia related gene signature were all up-regulated in cluster1. Thus, we defined the cluster1 as "hypoxic subgroup" compared with cluster2. The infiltration of CD8+ T cell and CD4+ T cell were lower in cluster1 while the nTreg cell and iTreg cell were higher, indicating that there was immunosuppressive status in cluster1. We observed widespread hypoxia-associated dysregulation of microRNAs and mRNAs. Next, a risk signature for predicting prognosis of breast cancer patients was established based on 12 dysregulated hypoxia associated prognostic genes. Two microRNAs, hsa-miR-210-3p and hsa-miR-190b, with the most significant absolute logFC value were related to unfavorable and better prognosis, respectively. Several long non-coding RNAs were predicted to be microRNA targets and positively correlated with two selected mRNAs, CPEB2 and BCL11A. Predictions based on the SNHG16-hsa-miR-210-3p-CPEB2 and LINC00899/PSMG3-AS1/PAXIP-AS1-hsa-miR-190b-BCL11A ceRNA regulation networks indicated that the two genes might act as tumor suppressor and oncogene, respectively. Conclusion: Hypoxia plays an important role in the initiation and progression of breast cancer. Our research provides potential mechanisms into molecular-level understanding of tumor hypoxia.

Analysis of N6-Methyladenosine Methyltransferase Reveals METTL14 and ZC3H13 as Tumor Suppressor Genes in Breast Cancer.

OBJECTIVES: Recently, an increasing number of studies have revealed that N6-methyladenosine (m6A) functions as a significant post-transcriptional modification which plays a critical role in the occurrence and progression of enriched tumors by regulating coding and non-coding RNA biogenesis. However, the biological function of m6A in breast cancer remains largely unclear. MATERIALS AND METHODS: In this study, we used a series of bioinformatic databases and tools to jointly analyze the expression of m6A methylation transferases (METTL3, METTL14, WTAP, RBM15, RBM15B and ZC3H13) and investigate the prognostic value of METTL14 and ZC3H13 in breast cancer. Besides, we analyzed the downstream carcinogenic molecular mechanisms related to METTL14 and ZC3H13 and their relationship with immune infiltration in breast tumor tissues. RESULTS: The results showed that METTL14 and ZC3H13 were the down-regulated m6A methylation transferases in breast cancer. Survival outcome analysis suggested that abnormally low expression of METTL14 and ZC3H13 could predict unfavorable prognosis in four breast cancer subtypes. Moreover, their down-regulation was associated with ER-, PR- and triple-negative breast cancer patients, as well as tumor progression (increased Scarff, Bloom and Richardson grade status and Nottingham Prognostic Index classification). Co-expression analysis revealed that METTL14 and ZC3H13 had a strong positive correlation with APC, an antagonist of the Wnt signaling pathway, indicating they might cooperate in regulating proliferation, invasion, and metastasis of tumor cells. METTL14, ZC3H13, and APC expression levels had significant positive correlation with infiltrating levels of CD4+ T cells, CD8+ T cells, neutrophils, macrophages, and dendritic cells, and negative correlation with Treg cells in breast cancer. CONCLUSIONS: This study demonstrated that down-regulation of METTL14 and ZC3H13 which act as two tumor suppressor genes was found in breast cancer and predicted poor prognosis. Their abnormal expression promoted breast cancer invasion by affecting pathways related to tumor progression and mediating immunosuppression.

Corrigendum: Hypoxia-Associated Prognostic Markers and Competing Endogenous RNA Co-Expression Networks in Breast Cancer.

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Prognostic value of DKK2 from the Dickkopf family in human breast cancer.

Breast cancer is one of the most frequently diagnosed types of cancer with a high mortality and malignancy rate in women worldwide. The Dickkopf (DKK) protein family, as a canonical Wnt/ß-catenin pathway antagonist, has been implicated in both physiological and pathological processes. This study aimed to comprehensively characterize the prognostic value and elucidate the mechanisms of DKKs in breast cancer and its subtypes. Firstly, DKK mRNA expression and corresponding outcome were analyzed by means of the Gene Expression-Based Outcome for Breast Cancer Online (GOBO) platform based on PAM50 intrinsic breast cancer subtypes. Subsequently, we extracted breast cancer datasets from the Cancer Genome Atlas (TCGA) and Molecular Taxonomy of Breast Cancer International Consortium (METABRIC) to validate the expression profile and prognostic values from the GOBO platform. Moreover, a protein-protein network was created and functional enrichment was conducted to explore the underlying mechanisms of action of the DKKs. In addition, we uncovered the genetic and epigenetic alterations of DKK2 in breast cancer. The main finding of this study was the differential roles of DKKs in the PAM50 subtypes of breast cancer analyzed. The overall trend was that a high level of DKK2 was associated with a good survival in breast cancer, although it played an opposite role in the Normal-like subtype. We also found that DKK2 carried out its functions through multiple signaling pathways, not limited to the Wnt/ß-catenin cascade in breast cancer. Finally, we used our own data to validate the bioinformatics analysis data for DKK2 by RT-qPCR. Taken together, our findings suggest that DKK2 may be a potential prognostic biomarker for the Normal-like subtype of breast cancer. However, the prognostic role of DKKs in the subtypes of breast cancer still requires validation by larger sample studies in the future.

The role of Dickkopf family in cancers: from Bench to Bedside.

Numerous epidemiological studies indicate that cancer will be responsible for millions of deaths in one year. Although multiple therapeutic strategies exist, and vast research efforts are being focused on developing newer and better regimens, cancer-related morbidity and mortality remain high. Metastasis and recurrence are prominent causes of treatment failure in cancers. Moreover, early diagnosis and treatment initiation are difficult to achieve in clinical practice. Fortunately, targeted therapy, which exerts its function at the molecular level, has proved to be greatly beneficial in several human diseases including cancers. The Wnt signaling pathway is a crucial regulator of embryogenesis and development in humans, and its dysfunction has been implicated in the incidence and development of cancers and other diseases. The Dickkopf family (Dkks) is a widely studied Wnt signaling pathway antagonist and plays multiple roles in human physiological and pathological process through both Wnt pathway-dependent and -independent manners. However, the precise roles of Dkks in tumorigenesis and the causal mechanisms have not been clearly elucidated. We discuss the pleiotropic roles of Dkks, with a specific focus on the underlying mechanisms, in cancer biology. We review recent literature to explore the potential use of Dkks as a tumor diagnosis biomarker and therapeutic target.

Bleomycin Suppresses the Proliferation and the Mobility of Human Gastric Cancer Cells Through the Smad Signaling Pathway.

BACKGROUND/AIMS: Extensive studies have demonstrated that Bleomycin (BLM) is a glycopeptide antibiotic that has been used as an anticancer chemotherapeutic reagent. It can induce both single- and double-strand DNA damage, inhibit synthesis of DNA, suppress proliferation, and induce apoptosis in cancer cells. Smad signaling transducers are considered as important molecules in tumor development and progression, and may closely be related to the biological behaviors of some malignant carcinomas, including gastric cancer. METHODS: The effects of different concentrations of BLM on the proliferation, cell cycle, apoptosis, migration, and invasion on gastric cancer cell lines MKN45 and AGS were assayed by using CCK-8 assay, Annexin V/PI double staining, PI staining, and transwell assay. Western blot and Immunohistochemistry were applied to analyze the potential mechanism(s). RESULTS: BLM treatment resulted in a low proliferation, high apoptosis, low migration and invasion in MKN45 and AGS cells. Furthermore, the possible mechanisms underlying that Smad3 activity could be changed after binding with BLM, and subsequently the Smad signaling pathway had a cascade response. CONCLUSION: These results highlight BLM as an exciting theme for gastric cancer treatment, which may represent an effective clinical therapeutic reagent for gastric cancer patients.