IGF2BP2 Drives Cell Cycle Progression in Triple-Negative Breast Cancer by Recruiting EIF4A1 to Promote the m6A-Modified CDK6 Translation Initiation Process.

IGF2BP2 is a new identified N6-methyladenosine (m6A) reader and associated with poor prognosis in many tumors. However, its role and related mechanism in breast cancer, especially in triple-negative breast cancer (TNBC), remains unclear. In this study, it is found that IGF2BP2 is highly expressed in TNBC due to the lower methylation level in its promoter region. Functional and mechanical studies displayed that IGF2BP2 could promote TNBC proliferation and the G1/S phase transition of the cell cycle via directly regulating CDK6 in an m6A-dependent manner. Surprising, the regulation of protein levels of CDK6 by IGF2BP2 is related to the changes in translation rate during translation initiation, rather than mRNA stability. Moreover, EIF4A1 is found to be recruited by IGF2BP2 to promote the translation output of CDK6, providing new evidence for a regulatory role of IGF2BP2 between m6A methylation and translation. Downregulation of IGF2BP2 in TNBC cell could enhance the sensitivity to abemaciclib, a CDK4/6 inhibitor. To sum up, our study revealed IGF2BP2 could facilitate the translation output of CDK6 via recruiting EIF4A1 and also provided a potential therapeutic target for the diagnosis and treatment of TNBC, as well as a new strategy for broadening the drug indications for CDK4/6 inhibitors.

Histocompatibility Minor 13 (HM13), targeted by miR-760, exerts oncogenic role in breast cancer by suppressing autophagy and activating PI3K-AKT-mTOR pathway.

Histocompatibility Minor 13 (HM13) is reported to participate in regulating multiple cancers. In the present study, we uncovered that HM13 was highly expressed in breast cancer and correlated with worse prognosis. Downregulation of HM13 could suppress breast cancer cell proliferation and metastasis abilities. Tumorigenicity mediated by HM13 was also observed in the xenograft model. Knockdown of HM13 could activate autophagy by inducing endoplasmic reticulum (ER) stress. Moreover, further experiments demonstrated that downregulated HM13 could inhibit PI3K-AKT-mTOR pathway. We then verified that HM13 was a direct target of miR-760 functioned as a tumor -suppressor in breast cancer. And the tumor suppressive effects of miR-760 could be partially reversed by HM13. Taken together, these findings elucidated that HM13, targeted by miR-760, could play an oncogenic role in breast cancer by inducing autophagic inhibition and facilitating PI3K-AKT-mTOR pathway. Our findings suggested HM13 could act as a novel therapeutic target candidate for breast cancer and supported the idea that autophagy inducers might represent a new approach to treat breast cancer.

Kinesin family member 23, regulated by FOXM1, promotes triple negative breast cancer progression via activating Wnt/ß-catenin pathway.

BACKGROUND: Triple negative breast cancer (TNBC) is highly malignant and has a worse prognosis, compared with other subtypes of breast cancer due to the absence of therapeutic targets. KIF23 plays a crucial role in the tumorigenesis and cancer progression. However, the role of KIF23 in development of TNBC and the underlying mechanism remain unknown. The study aimed to elucidate the biological function and regulatory mechanism of KIF23 in TNBC. METHODS: Quantitative real-time PCR and Western blot were used to determine the KIF23 expression in breast cancer tissues and cell lines. Then, functional experiments in vitro and in vivo were performed to investigate the effects of KIF23 on tumor growth and metastasis in TNBC. Chromatin immunoprecipitation assay was conducted to illustrate the potential regulatory mechanisms of KIF23 in TNBC. RESULTS: We found that KIF23 was significantly up-regulated and associated with poor prognosis in TNBC. KIF23 could promote TNBC proliferation, migration and invasion in vitro and in vivo. KIF23 could activate Wnt/ß-catenin pathway and promote EMT progression in TNBC. In addition, FOXM1, upregulated by WDR5 via H3K4me3 modification, directly bound to the promoter of KIF23 gene to promote its transcription and accelerated TNBC progression via Wnt/ß-catenin pathway. Both of small inhibitor of FOXM1 and WDR5 could inhibit TNBC progression. CONCLUSIONS: Our findings elucidate WDR5/FOXM1/KIF23/Wnt/ß-catenin axis is associated with TNBC progression and may provide a novel and promising therapeutic target for TNBC treatment.

SMC1A regulated by KIAA1429 in m6A-independent manner promotes EMT progress in breast cancer.

As a component of N6-methyladenosine (m6A) "writers," KIAA1429 was reported to promote breast cancer proliferation and growth in m6A-independent manners. However, the related mechanism of KIAA1429 in breast cancer metastasis has not been reported. In the present study, we found KIAA1429 could significantly promote the migration and invasion of breast cancer cells. Then we demonstrated that knockdown of KIAA1429 could impede breast cancer metastasis in nude mice in vivo. The level of SNAIL expression and epithelial-mesenchymal transition (EMT) progress was positively related with KIAA1429. Furthermore, we confirmed that the suppression of cell migration, invasion, and EMT progress by knockdown of KIAA1429 could be reversed by the upregulation of SNAIL. However, structural maintenance of chromosomes 1A (SMC1A), not KIAA1429, bound with the SNAIL promoter region directly and promoted the transcription of SNAIL. Then we confirmed that KIAA1429 could bind to the motif in the 3' UTR of SMC1A mRNA directly and enhance SMC1A mRNA stability. In conclusion, our study revealed a novel mechanism of the KIAA1429/SMC1A/SNAIL axis in the regulation of metastasis of breast cancer. Moreover, it first provided detailed investigation of how KIAA1429 regulated the targeted gene expression at posttranscriptional levels as an RNA binding protein unrelated to its m6A modification.

RNA 5-methylcytosine regulates YBX2-dependent liquid-liquid phase separation.

5-Methylcytosine (m5C) is one of the most prevalent internal modifications of messenger RNA (mRNA) in higher eukaryotes. Here we report that Y box protein 2 (YBX2) serves as a novel mammalian m5C binding protein to undergo liquid-liquid phase separation (LLPS) both in vivo and in vitro, and this YBX2-dependent LLPS is enhanced by m5C marked RNA. Furthermore, the crystal structure assay revealed that W100, as a distinct m5C binding site of YBX2, is critical in mediating YBX2 phase separation. Our study resolved the relationship between RNA m5C and phase separation, providing a clue for a new regulatory layer of epigenetics.

CircMETTL3, upregulated in a m6A-dependent manner, promotes breast cancer progression.

Growing evidence indicates N6-methyladenosine (m6A) has biological function in oncogenesis. METTL3, the catalytic component, is the most important part of methyltransferase complex and plays a crucial role in cancers. However, the biological function of circRNAs derived from METTL3 in breast cancer and the underlying molecular mechanism remains unclear. Herein, we report circMETTL3, which has not been explored in breast cancer, and it is markedly upregulated in breast cancer. Moreover, we uncovered that circMETTL3 could facilitate cell proliferation, migration and invasion in breast cancer. Mechanism investigation showed that circMETTL3 might act as a competing endogenous RNA (ceRNA) of miR-31-5p and upregulate its target cyclin-dependent kinases (CDK1). Moreover, m6A modification of circMETTL3 might affect its expression. Taken together, our results elucidate that circMETTL3 promotes breast cancer progression through circMETTL3/miR-31-5p/CDK1 axis. Moreover, METTL3, the host gene of circMETTL3, may regulate circMETTL3 expression in an m6A-dependent manner, while circMETTL3 has no effect on METTL3 expression, providing a new relationship between the circRNA and the corresponding host gene. Thus, it may serve as a new therapeutic target for breast cancer.

Main N6-Methyladenosine Readers: YTH Family Proteins in Cancers.

Among the over 150 RNA modifications, N6-methyladenosine (m6A) is the most abundant internal modification in eukaryotic RNAs, not only in messenger RNAs, but also in microRNAs and long non-coding RNAs. It is a dynamic and reversible process in mammalian cells, which is installed by "writers," consisting of METTL3, METTL14, WTAP, RBM15/15B, and KIAA1429 and removed by "erasers," including FTO and ALKBH5. Moreover, m6A modification is recognized by "readers," which play the key role in executing m6A functions. IYT521-B homology (YTH) family proteins are the first identified m6A reader proteins. They were reported to participate in cancer tumorigenesis and development through regulating the metabolism of targeted RNAs, including RNA splicing, RNA export, translation, and degradation. There are many reviews about function of m6A and its role in various diseases. However, reviews only focusing on m6A readers, especially YTH family proteins are few. In this review, we systematically summarize the recent advances in structure and biological function of YTH family proteins, and their roles in human cancer and potential application in cancer therapy.

Metformin exhibits antiproliferation activity in breast cancer via miR-483-3p/METTL3/m6A/p21 pathway.

Evidence suggests that metformin might be a potential candidate for breast cancer treatment. Yet, its relevant molecular mechanisms remain to be fully investigated. We found that metformin could suppress the N6-methyladenosine (m6A) level in breast cancer cells significantly. The latter has an essential role in breast cancer progression and is newly considered as a therapeutic target. In this study, we measured the m6A level by m6A colorimetric analysis and dot blot assay. We then performed qRT-PCR, western blot, MeRIP, dual-luciferase reporter assay, and others to explore the m6A-dependent pathway associated with metformin. In vivo effect of metformin was investigated using a mouse tumorigenicity model. In addition, breast cancer and normal tissues were used to determine the role of METTL3 in breast cancer. Metformin could reduce the m6A level via decreasing METTL3 expression mediated by miR-483-3p in breast cancer. METTL3 is known to be able to promote breast cancer cell proliferation by regulating the p21 expression by an m6A-dependent manner. Metformin can take p21 as the main target to inhibit such effect. To specify, this study exhibited that metformin can inhibit breast cancer cell proliferation through the pathway miR-483-3p/METTL3/m6A/p21. Our findings suggest that METTL3 may be considered as a potential therapeutic target of metformin for breast cancer.

Oncogenic action of the exosome cofactor RBM7 by stabilization of CDK1 mRNA in breast cancer.

RNA exosome can target the specific RNAs for their processing/degradation by distinct exosome cofactors. As a key component in exosome cofactors, RNA binding motif protein 7 (RBM7) shows the binding specificity for uridine-rich sequences in mRNAs via its RNA recognition motifs. However, the specific function of RBM7 in human breast cancer remains unclear. In vitro, experiments revealed that knockdown of RBM7 dramatically inhibited breast cancer cell proliferation, while inducing G1 cell cycle arrest; the opposite was true when RBM7 was overexpressed. Meanwhile, experiments in vivo confirmed the oncogenic function of RBM7 in breast cancer. RNA sequencing and the following pathway analysis found that cyclin-dependent kinase1 (CDK1) was one of the main gene regulated by RBM7. Overexpression of RBM7 increased CDK1 expression, while RBM7 knockdown decreased it. RIP assays additionally found that RBM7 bound directly to CDK1 mRNA. It was also showed that RBM7 could directly bind to the AU-rich elements (AREs) in 3'-UTR of CDK1 mRNA, which contributed to the stability of CDK1 mRNA by lengthening its half-life. More importantly, the oncogenic activity reduced by knockdown of RBM7 could be rescued by overexpression of CDK1 both in vitro and in vivo, but mutant CDK1 failed. All the evidences implied RBM7 promoted breast cancer cell proliferation by stabilizing CDK1 mRNA via binding to AREs in its 3'-UTR. As we knew, it was the first attempt to connect the RNA exosome to the tumor development, providing new insights into the mechanisms of RNA exosome-linked diseases.

Cell division cycle proteinising prognostic biomarker of breast cancer.

Cell division cycle protein (CDC20) has been observed to be expressed higher in various kinds of human cancers and was associated with poor prognosis. However, studies on role of CDC20 in breast cancer are seldom reported till now, most of which are not systematic and conclusive. The present study was performed to analyze the expression pattern, potential function, and distinct prognostic effect of CDC20 in breast cancer using several online databases including Oncomine, bc-GenExMiner, PrognoScan, and UCSC Xena. To verify the results from databases, we compared the mRNA CDC20 expression in breast cancer tissues and adjacent normal tissues of patients by real-time PCR. We found that CDC20 was expressed higher in different types of breast cancer, comparing with normal tissues. Moreover, the patients with a more advanced stage of breast cancer tended to express higher level CDC20. CDC20 was expressed higher in breast cancer tissues than normal tissues from patients in our hospital, consistent with the results from databases. Estrogen receptor (ER) and progesterone receptor (PR) status were negatively correlated with CDC20 level. Conversely, Scarff-Bloom-Richardson (SBR) grade, Nottingham prognostic index (NPI), epidermal growth factor receptor-2 (HER-2) status, basal-like status, and triple-negative status were positively related to CDC20 expression in breast cancer patients with respect to normal individuals. Higher CDC20 expression correlated with worse survival. Finally, a positive correlation between CDC20 and Targeting protein for Xenopus kinesin-like protein 2 (TPX2) expression was revealed. CDC20 could be considered as a potential predictive indicator for prognosis of breast cancer with co-expressed TPX2 gene.

TCDD-Inducible Poly-ADP-Ribose Polymerase (TIPARP), A Novel Therapeutic Target Of Breast Cancer.

BACKGROUND: TIPARP (TCDD-inducible poly-ADP-ribose polymerase), a mono-ADP-ribosyltransferase and a transcriptional repressor of aryl hydrocarbon receptor (AHR), was one of the potential therapeutic targets for human cancers identified by CRISPR-Cas9 screens recently. Studies about TIPARP on cancers are scarce till now, most of which just focus on expressions, while the functions have not been widely reported yet. Moreover, the TIPARP prognostic significance and therapeutic value of breast cancer is also uncertain. METHODS: The present study was performed to comprehensively analyze the expression pattern, prognostic effect, potential therapeutic function of TIPARP in breast cancer by pooling all currently available databases online including Oncomine, UALCAN, bc-GenExMiner, Kaplan-Meier Plotter, COSMIC, UCSC Xena, STRING, DAVID and Comparative Toxicogenomics Database. Further, we also performed several cell biology experiments including RT-qPCR, Western blot and CCK-8 in cellular and clinical sample levels to confirm the conclusions from bioinformatics analysis. RESULTS: TIPARP was expressed lower in tumor tissues comparing with normal tissues. Meanwhile, several clinical parameters of breast cancer patients were correlated with TIPARP expression. Further, higher TIPARP expression was related to preferable survival. Moreover, the mutations and DNA methylation of TIPARP might contribute to TIPARP dysregulation in breast cancer. Interactors with TIPARP were significantly enriched in telomere maintenance, telomere organization and mainly participated in pathways in cancer. Finally, several common drugs including metformin were observed to up-regulate the expression of TIPARP. CONCLUSION: TIPARP might act as a preferable prognostic marker of breast cancer through multiple biological processes such as DNA methylation, mutation as well as pathway related to telomere and so on. TIPARP could be considered as a potential therapeutic target for breast cancer. However, large-scale and comprehensive research is needed to clarify our results.

[Preparation and optimization of zhitong micro-emulsion formula].

To prepare Zhitong micro-emulsion in this study, with the empirical formula of Zhitong preparation as the model medicine, the essential oil in the formula as the oil phase, and the water decoction as the water phase. The types of surfactant and co-surfactant were investigated. The changes in micro-emulsion conductivity and construction, the water percentage in the micro-emulsion system, the changing curve of conductivity and the fine pseudo-ternary phase diagram of micro-emulsion were drawn to determine the surfactant-co-surfactant mass ratio (K(m)). Subsequently, the D-mixture design was used to optimize Zhitong Micro-emulsion formula, with particle size and surface tension of micro-emulsion as the indexes. Finally, efforts were made to determine part of physical parameters of Zhitong micro-emulsion and preliminarily detect its stability. The results showed that the micro-emulsion was optimal with the EL-35-tween 20 ratio of 4:1 in surfactant, whereas the absolute ethyl alcohol was recommended as the co-surfactant. The ratio between surfactant and co-surfactant (K(m)) was 1.5. The finalized micro-emulsion formula contains 12% surfactant, 8% co-surfactant, 70% 1 g x mL(-1) water decoction and 8% oil. The results of the preliminary stability experiment showed a better stability of Zhitong micro-emulsion.