Suppressed Breast Cancer Cell Proliferation Research Articles

Identification and validation of an m6A-related gene signature to predict prognosis and evaluate immune features of breast cancer.

Breast cancer is the most prevalent cancer, and it is accompanied by high heterogeneity. N6-methyladenosine (m6A) modification significantly contributes to breast cancer tumorigenesis and progression. However, how m6A-related genes affect the clinical outcomes and tumor immune microenvironment (TIME) of breast cancer is largely unknown. Our study developed an m6A-related gene signature on the basis of The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases. The m6A-related gene signature was constructed using univariate, least absolute shrinkage and selection operator (LASSO), and multivariate Cox regression analyses. Breast cancer patients were classified into low- and high-risk groups depending on the median risk score. The reliability and efficiency of the signature were validated using Kaplan-Meier analysis, receiver operating characteristic (ROC) curves, and principal component analysis (PCA). The risk score was validated as an independent indicator associated with overall survival, and a nomogram model was created to estimate the overall survival of patients with breast cancer. Functional annotation suggested that the risk score had a strong relationship with immune-related pathways. Different proportions of immune cell infiltration between the two groups were evaluated using various algorithms. The high-risk group had higher immune checkpoint expression levels. We discovered that one of the 6 prognostic genes, TMEM71, was downregulated in breast cancer tissues. In vitro experiments indicated that overexpression of TMEM71 suppressed breast cancer cell proliferation and migration. In conclusion, the m6A-related gene signature may be a sensitive biomarker for overall survival prediction and guide the individualized treatment for breast cancer patients.

Daphnoretin Arrests the Cell Cycle and Induces Apoptosis in Human Breast Cancer Cells.

Emerging evidence has shown that daphnoretin, one of the main active ingredients of Daphne giraldii Nitsche, processes antitumor activities in several tumor cells (e.g., colon cancer, lung cancer, cervical cancer, and osteosarcoma). However, the antitumor effect and its mechanism in breast cancer are unexplored. In this study, our data indicated that daphnoretin obviously suppressed the proliferation of breast cancer MCF-7 and MDA-MB-231 cells. Further studies showed that daphnoretin remarkably increased the p21 level, decreased cyclin E and CDK2 levels, and then arrested the cell cycle at the S phase. Moreover, daphnoretin obviously lowered the BCL-2 level and raised the levels of BAX and cleaved caspase-9 and -3, leading to cell apoptosis. Furthermore, daphnoretin remarkably decreased the ratio of p-PI3K/PI3K and p-AKT/AKT in breast cancer cells. Collectively, these findings demonstrated that daphnoretin could suppress breast cancer cell proliferation through cell cycle arrest and inducing apoptosis, which is related to the PI3K/AKT pathway.

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.

FAM96A and FAM96B function as new tumor suppressor genes in breast cancer through regulation of the Wnt/β-catenin signaling pathway

AimsFamily with sequence similarity 96 member A and B (FAM96A and FAM96B) are two highly conserved homologous proteins belonging to MIP18 family. Some studies have shown that FAM96A and FAM96B are significantly down-regulated in human gastrointestinal stromal tumors, colon cancer, and liver cancer. However, the molecular mechanisms of FAM96A/B in breast cancer are unknown. This work aims to explore the roles of FAM96A/B in breast cancer progression. Main methodsSpecific siRNAs were used to down-regulate FAM96A/B expression, and recombinant plasmids were used to up-regulate FAM96A/B expression in breast cancer cells. Cell proliferation was measured using MTT and colony formation. Cell cycle and apoptosis were detected by flow cytometry. Cell migration and invasion were examined by wound healing and transwell assays. The relationships among FAM96A/B, EMT and Wnt/β-catenin pathway were determined by analyzing expression changes of classical markers. Key findingsWe found that FAM96A/B expression was down-regulated in breast cancer. FAM96A/B overexpression suppressed breast cancer cell proliferation, invasion and migration, induced cell apoptosis and caused cell cycle arrest. Conversely, FAM96A/B knockdown exhibited the opposite effects. Moreover, our data demonstrated that FAM96A/B overexpression suppressed EMT and Wnt/β-catenin pathway, while FAM96A/B knockdown showed the promoting effects on EMT and Wnt/β-catenin pathway. Furthermore, a Wnt pathway inhibitor, XAV-939 reversed the promoting effects of FAM96A/B knockdown on breast cancer progression. SignificanceOur findings suggest that FAM96A/B may function as new tumor suppressor genes and inhibit breast cancer progression via modulating Wnt/β-catenin pathway, which can provide the potential markers for breast cancer diagnosis and therapy.

RUNX regulated immune-associated genes predicts prognosis in breast cancer.

Background: Breast cancer is the most common malignant tumor in women. RUNX family has been involved in the regulation of different carcinogenic processes and signaling pathways with cancer, which is closely related to immunity and prognosis of various tumors, and also plays an important role in the development and prognosis of breast cancer. Methods: We discovered the expression of RUNX family through GEPIA Dataset and then evaluated the relationship between RUNX family and immune-related genes and the prognosis of breast cancer through analyzing TCGA database. A prognostic model was established and verified via cox proportional hazards regression model using R packages. We evaluated the accuracy of the prognostic model by Kaplan-Meier curves and receiver operating characteristic (ROC) curves. Additionally, we obtained the relationship between the RUNX family and immune infiltration by TIMER database. Finally, the dual luciferase reporter assay was used to verify the regulation of RUNX3 on potential target genes ULBP2 and TRDV1, and the effects of ULBP2 and TRDV1 on the growth of breast cancer cells were explored by CCK-8, colony formation and wound healing assays. Results: We screened out RUNX family-regulated immune-related genes associated with the prognosis of breast cancer. These predictors included PSME2, ULBP2, IL-18, TSLP, NPR3, TRDV1. Then a prognosis-related risk score model was built using the independent risk factors to provide a clinically appropriate method predicting the overall survival (OS) probability of the patients with breast cancer. In addition, a further research was made on the functions of high risk immune gene ULBP2 and low risk immune gene TRDV1 which regulated by RUNX3, the results showed that down-regulation of ULBP2 suppressed breast cancer cell proliferation and TRDV1 had the opposite functions. The prognostic model we constructed could promote the development of prognostic, and was associated with lower immune infiltration. Conclusion: The expression of RUNX family was closely related to the prognosis of breast cancer. At the same time, RUNX family could modulate the functions of immune-related genes, and affect the development and prognosis of breast cancer. These immune-related genes regulated by RUNX family could be promising prognostic biomarkers and therapeutic targets in breast cancer.

Circ_0008717 Sponges miR-326 to Elevate GATA6 Expression to Promote Breast Cancer Tumorigenicity.

Circular RNAs (circRNAs) have been reported to paly roles in the progression and management of breast cancers (BC). This work aimed to detect the role and mechanism of circ_0008717 in BC tumorigenesis. Expression levels of genes and proteins were evaluated by quantitative real-time polymerase chain reaction and western blot. In vitro assays were conducted using cell counting kit-8, colony formation, transwell, tube formation, and flow cytometry assays, respectively. The interaction between miR-326 and circ_0008717 or GATA6 (GATA Binding Protein six) was confirmed by bioinformatics analysis, and dual-luciferase reporter assay and RNA immunoprecipitation assay. The murine xenograft models were established to perform in vivo assay. Circ_0008717 and GATA6 were highly expressed, while miR-326 was lowly expressed in BC tissues and cells. Functionally, knockdown of circ_0008717 not only suppressed breast cancer cell proliferation, angiogenesis, migration, invasion and epithelial-mesenchymal transition (EMT) in vitro, but also hindered tumor growth and EMT process in vivo. Mechanistically, Circ_0008717 directly bound to miR-326, which targeted GATA6. Rescue experiments showed that miR-326 reversed the anticancer action of circ_0008717 knockdown on BC cells. Moreover, miR-326 restoration repressed BC cell growth and metastasis, which were attenuated by GATA6 overexpression. In addition, we also observed that circ_0008717 could regulate GATA6 expression by sponging miR-326. Circ_0008717 promoted breast cancer growth and metastasis through miR-326/GATA6 axis, revealing a potential therapeutic target for breast cancer treatment.

N6-methyladenosine-mediated upregulation of LINC00520 accelerates breast cancer progression via regulating miR-577/POSTN axis and downstream ILK/AKT/mTOR signaling pathway

Various lncRNAs have been reported to be closely associated with cancer initiation and progression in breast cancer (BC), including LINC00520. However, the role and underlying mechanisms by which LINC00520 affects BC aggressiveness have not been fully delineated, and this study aimed to explore this issue. Through performing qRT-PCR analysis, we proved that LINC00520 was significantly upregulated in BC tissues and cells, compared with normal tissues and cells. Higher expression of LINC00520 was closely related to higher tumor grade, poor differentiation and shorter survival in BC patients. Next, the loss-of-function experiments evidenced that silencing LINC00520 suppressed BC cell proliferation, migration and epithelial-mesenchymal transition (EMT) in vitro, and inhibited tumorigenesis in vivo. Interestingly, we found that LINC00520 expression was positively regulated by METTL3-mediated N6-methyladenosine(m6A) modification in BC. Furthermore, we identified the tumor-suppressor miR-577 as the binding target of LINC00520 in BC. Mechanistically, LINC00520 elevated POSTN level via sponging miR-577, resulting in the activation of the downstream tumor-promoting ILK/Akt/mTOR pathway. Finally, the rescuing experiments evidenced that both POSTN knockdown and ILK/Akt/mTOR pathway inhibitor OSU-T315 abrogated the promoting effects of miR-577 ablation on the malignant phenotypes in BC. Collectively, this study firstly verified that LINC00520 acted as a ceRNA of miR-577 to advance BC aggressiveness in a m6A-dependent manner, providing novel biomarkers for BC diagnosis and therapy.

Integrating UPLC-HR-MS/MS, Network Pharmacology, and Experimental Validation to Uncover the Mechanisms of Jin'gan Capsules against Breast Cancer.

In the theory of traditional Chinese medicine (TCM), “liver-qi” stagnation and heat-induced toxicity represent the main etiologies of breast cancer. Recently, several TCMs with heat-clearing and detoxification efficacy have shown inhibitory effects on breast cancer. Jin’gan capsules (JGCs), initially approved to treat colds in China, are a heat-clearing and detoxification TCM formula. However, the anticancer activity of JGCs against breast cancer and its underlying mechanisms remain unclear. First, we assessed the antiproliferative activity of JGCs in breast cancer cell lines and evaluated their effects on cell apoptosis and the cell cycle by flow cytometry. Furthermore, we identified the potential bioactive components of JGCs and their corresponding target genes and constructed a bioactive compound–target interaction network by ultra-performance liquid chromatography–high-resolution tandem mass spectrometry (UPLC-HR-MS/MS) and network pharmacology analysis. Finally, the underlying mechanism was investigated through gene function enrichment analysis and experimental validation. We found that JGCs significantly inhibited breast cancer cell growth with IC50 values of 0.56 ± 0.03, 0.16 ± 0.03, and 0.94 ± 0.09 mg/mL for MDA-MB-231, MDA-MB-468, and MCF-7, respectively. In addition, JGC treatment dramatically induced apoptosis and S phase cell cycle arrest in breast cancer cells. Western blot analysis confirmed that JGCs could regulate the protein levels of apoptosis- and cell cycle-related genes. Utilizing UPLC-HR-MS/MS analysis and network pharmacology, we identified 7 potential bioactive ingredients in JGCs and 116 antibreast cancer targets. Functional enrichment analysis indicated that the antitumor effects of JGCs were strongly associated with apoptosis and the Janus kinase (JAK)-signal transducer and activator of transcription (STAT) signaling pathway. Western blot analysis validated that JGC treatment markedly decreased the expression levels of p-JAK2, p-STAT3, and STAT3. Our findings suggest that JGCs suppress breast cancer cell proliferation and induce cell cycle arrest and apoptosis partly by inhibiting the JAK2/STAT3 signaling pathway, highlighting JGCs as a potential therapeutic candidate against breast cancer.

Flavonoid extract Kushenol a exhibits anti-proliferative activity in breast cancer cells via suppression of PI3K/AKT/mTOR pathway.

Kushenol A is natural flavonoid extract discovered in recent years, with potential anti-tumor activity. Its role in breast cancer is poorly understood. To investigate biological function of Kushenol A in breast cancer (BC), Cell Counting Kit-8 assay, colony formation assay, flow cytometry, western blotting, qPCR analysis, and xenograft mouse model were performed. We found that Kushenol A treatment reduced proliferative capability and induced G0/G1 phase cell cycle arrest and apoptosis of BC cells in a concentration-dependent manner. Besides, Kushenol A treatment contributed to the upregulation of apoptosis-related and cell cycle-associated genes. In nude mice, Kushenol A administration repressed BC xenograft tumor growth. Mechanistically, phosphorylation levels of AKT and mTOR were markedly attenuated in Kushenol A-treated BC cells; however, there were no significant differences in total AKT and mTOR expressions. Moreover, PI3K inhibitor combined with Kushenol A exhibited synergistic inhibitory activity on cell proliferation. Taken together, our findings suggested that Kushenol A suppressed BC cell proliferation by modulating PI3K/AKT/mTOR signaling pathway. Kushenol A may be a promising therapeutic drug for treating BC.

A photochemical-responsive nanoparticle boosts doxorubicin uptake to suppress breast cancer cell proliferation by apoptosis

In the course of chemotherapy for breast cancer, doxorubicin (DOX) is one of the most commonly prescribed agents. However, it has been recognized as clinically circumscribed on account of its poor selectivity and toxic reactions to normal tissues. Fortunately, the distinct merit of photochemical-responsive nanoparticle delivery systems to enhance cellular drugs uptake through localized concentration, adequate selective and minimizing systemic toxicity has aroused substantial interest recently. In this study, we synthesized photochemical-responsive nanoparticle by incorporating DOX, curcumin (CUR), and perfluorooctyl bromide (PFOB) into poly(lactic-co-glycolic acid) (PLGA) via double emulsification (DOX–CUR–PFOB–PLGA). The synthesized composite nanoparticles, which featured good ultrasound imaging, engendered photochemical activation for drug release when given laser irradiation. Cumulative release rates for DOX were 76.34%, and for CUR were 83.64%, respectively. Also, MCF-7 cells displayed significant intracellular DOX uptake and reactive oxygen species (ROS) levels, degraded cytoskeleton, and decreased cell growth and migration capacity. At the molecular level, cellular pAKT levels decreased, which resulted in downregulated HIF-1α and BAX/BCl-2 levels, leading to Caspase-3 activation and thus induction of apoptosis. Therefore, the photochemical-responsive nanoparticles possess the potential to elicit apoptosis in MCF-7 cells via enhanced DOX uptake.

NR2F1, a Tumor Dormancy Marker, Is Expressed Predominantly in Cancer-Associated Fibroblasts and Is Associated with Suppressed Breast Cancer Cell Proliferation.

Simple SummaryTumor dormancy is a state in which some cancer cells are dormant by less cell proliferation, and are resistant to treatments, thus leading to late recurrence. As nuclear receptor subfamily 2 group F member 1 (NR2F1) is a biomarker of dormancy shown in experimental settings, we studied its clinical relevance by analyzing comprehensive gene expression profiles of approximately 7000 breast cancer patients using computational biological approaches. NR2F1 gene expression of bulk tumors correlated with the suppression of cell proliferation and metastasis-related pathways, but not with metastasis or survival. Using single-cell sequence cohorts, we found that NR2F1 was predominantly expressed in cancer-associated fibroblasts, particularly in inflammatory type, rather than in cancer cells. Further, NR2F1 expression in breast cancer cells did not consistently correlate with stem cell-like traits. Our findings suggest that the NR2F1 gene expression signal from a patient’s bulk tumor is not from the cancer cells.Background: Tumor dormancy is a crucial mechanism responsible for the late recurrence of breast cancer. Thus, we investigated the clinical relevance of the expression of NR2F1, a known dormancy biomarker. Methods: A total of 6758 transcriptomes of bulk tumors from multiple breast cancer patient cohorts and two single-cell sequence cohorts were analyzed. Results: Breast cancer (BC) with high NR2F1 expression enriched TGFβ signaling, multiple metastases, and stem cell-related pathways. Cell proliferation-related gene sets were suppressed, and MKi67 expression was lower in high NR2F1 BC. In tumors with high Nottingham grade, NR2F1 expression was found to be lower. There was no consistent relationship between NR2F1 expression and metastasis or survival. Cancer mutation rates, immune responses, and immune cell infiltrations were lower in high NR2F1 tumors, whereas the infiltration of stromal cells including cancer-associated fibroblasts (CAFs) was higher. NR2F1 was predominantly expressed in CAFs, particularly inflammatory CAFs, rather than in cancer cells, consistently in the two single-cell sequence cohorts. Conclusions: NR2F1 expression in breast cancer is associated with tumor dormancy traits, and it is predominantly expressed in CAFs in the tumor microenvironment.

Abstract 424: Development of HJC0152-based proteolysis-targeting chimera degraders for breast cancer therapy

Abstract Signal transducer and activator of transcription 3 (STAT3), a member of the STAT family, is constitutively activated in various human cancers, and has been recognized as an attractive therapeutic target in cancer. STAT3 protein dimerizes and translocates to the nucleus to eventually transactivate downstream target genes. Despite some progress has been made in the past two decades, to date there is still lack of FDA-approved STAT3 inhibitors available for clinical use. Recently, proteolysis-targeting chimera (PROTAC) technology has dramatically advanced the development of therapeutics by introducing targeted protein degradation. Previously, our team has developed HJC0152, a promising STAT3 inhibitor, via structure- and fragment-based drug design strategies and in silico molecular modeling and docking. HJC0152 has demonstrated an attractive cancer therapeutic and preventive efficacy by targeting the non- estrogen receptor (ER)-based STAT3 signaling pathway. To achieve better potency and inhibition of STAT3 signaling, we therefore developed a series of HJC0152-based PROTAC protein degraders. These potential anticancer agents have been initially evaluated and several showed favorable potency in inhibiting the proliferation in a panel of breast cancer cell lines. We found that compounds JMX1122, JMX1123, and JMX1124 significantly inhibited proliferation and colony formation of a highly aggressive triple-negative breast cancer (TNBC) cell line, MDA-MB-231, and the ER-positive breast cancer cell lines. Different from the lead compound HJC0152, which showed an early inhibition on the phosphorylation of STAT3 at the Tyr705 and Ser727 residues, the newly developed HJC0152-PROTACs showed a significantly late differential inhibition of STAT3 phosphorylation at the Tyr705 and Ser727 residues, and reduced the total STAT3 protein level at 72 hours of treatment. Our current results support the notion that HJC0152-based PROTAC degraders potently suppress breast cancer cell proliferation and induce apoptosis, via direct targeting of STAT3 and its protein degradation. These compounds are currently being investigated for breast cancer cell migration and invasion, and will be evaluated for the in vivo inhibition of tumor growth and metastasis in different mouse models. This project is supported by NIH/NCI Awards R01CA226001 and R01CA231150 to Q.S. and J.Z. Citation Format: Junhai Xu, Jimin Xu, Ruixia Ma, Mingxiang Zhou, Doerte R. Fricke, Haiying Chen, Hyejin Kim, Xi Liu, Jia Zhou, Qiang Shen. Development of HJC0152-based proteolysis-targeting chimera degraders for breast cancer therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 424.

MiR-143-5p suppresses breast cancer progression by targeting the HIF-1α-related GLUT1 pathway.

Breast cancer (BC) is a commonly identified life-threatening type of cancer and a major cause of death among women worldwide. Several microRNAs (miRs), including miR-143-5p, have been reported to be vital for regulating hallmarks of cancer; however, the effect of miR-143-5p on BC requires further exploration. The present study performed bioinformatics analysis on GSE42072 and GSE41922 datasets from the National Center for Biotechnology Information Gene Expression Omnibus (GEO) database to identify miR-143-5p expression patterns. Furthermore, miR-143-5p expression was detected in BC cell lines and tissues via reverse transcription-quantitative PCR. Post-transfection with miR-143-5p mimics, Cell Counting Kit-8, colony formation and Transwell assays were performed to explore the effects of miR-143-5p on BC cell proliferation, colony formation, and migration. The association of miR-143-5p with the hypoxia-inducible factor-1α (HIF-1α)-associated glucose transporter 1 (GLUT1) pathway was explored via western blotting, immunofluorescence and dual-luciferase reporter assay. The present study detected high expression of miR-143-5p in BC tissue of the GSE42072 and serum of the GSE41922 datasets by GEO chip analysis. Additionally, the expression levels of miR-143-5p were decreased in BC tissues compared with those in adjacent healthy tissues, and low miR-143-5p expression was associated with a poorer prognosis and shorter survival time in patients with BC. In vitro, miR-143-5p expression levels were decreased in BC cells, and transfection with miR-143-5p mimics suppressed BC cell proliferation, colony formation, migration. Furthermore, miR-143-5p targeted the HIF-1α-related GLUT1 pathway, and inhibited HIF-1α and GLUT1 expression. Additionally, HIF-1α agonists reversed the miR-143-5p-induced inhibition during tumorigenesis. In conclusion, miR-143-5p exhibited low expression in BC tissues, and suppressed BC cell proliferation, colony formation, migration. Moreover, the antitumor effects of miR-143-5p targeted the HIF-1α-related GLUT1 pathway.

Knockdown of circ_0102273 inhibits the proliferation, metastasis and glycolysis of breast cancer through miR-1236-3p/PFKFB3 axis.

The key regulatory roles of circular RNAs (circRNAs) in human diseases have been demonstrated, including breast cancer (BC). The purpose of this study is to explore the role of circ_0102273, a newly discovered circRNA, in BC progression. The expression levels of circ_0102273, microRNA (miR)-1236-3p and 6-phosphofructo-2-kinase/fructose-2, 6-biphosphatase 3 (PFKFB3) were determined by quantitative real-time PCR. Cell proliferation, migration and invasion were measured using colony formation assay, EdU staining, wound healing assay and transwell assay. Glucose consumption, lactate production and ATP level were detected to evaluate cell glycolysis. The interaction between miR-1236-3p and circ_0102273 or PFKFB3 was confirmed by dual-luciferase reporter assay and RIP assay. Additionally, western blot analysis was utilized for measuring PFKFB3 protein expression. In-vivo experiments were performed to further explore the function of circ_0102273 in BC tumorigenesis. Our data showed that circ_0102273 was highly expressed in BC tumor tissues and cells, and its downregulation could inhibit BC cell proliferation, metastasis and glycolysis. MiR-1236-3p was confirmed to be sponged by circ_0102273, and its inhibitor could reverse the negative regulation of sh-circ_0102273 on BC cell proliferation, metastasis and glycolysis. PFKFB3 could be targeted by miR-1236-3p, and its expression could be positively regulated by circ_0102273. In addition, miR-1236-3p could suppress BC cell proliferation, metastasis and glycolysis, while this effect could be abolished by PFKFB3. Furthermore, circ_0102273 knockdown also had been discovered to reduce BC tumorigenesis in vivo. In summary, our research proposed that circ_0102273 might be a potential target for BC treatment, which could inhibit BC proliferation, metastasis and glycolysis through the miR-1236-3p/PFKFB3 axis.

Midkine promotes breast cancer cell proliferation and migration by upregulating NR3C1 expression and activating the NF-κB pathway

Breast cancer (BC) is the most common malignancy in females and is the second leading cause of cancer-related death among women worldwide. Midkine (MDK) is a heparin-binding growth factor that is abnormally expressed at high levels in various human malignancies. We aimed to uncover the biological function and molecular mechanism of MDK in BC cells. MDA-MB-231-shMDK and T47D-shMDK BC cells were established. The in vitro biological functions of MDK were demonstrated by CCK-8 assays, Transwell assays and Western blotting, whereas qPCR pathway arrays were implemented to explore the mechanism of MDK in BC cells. Functionally, we verified that silencing MDK significantly suppressed BC cell proliferation and migration by inhibiting the activation of the nuclear factor kappa B (NF-κB) pathway and the nuclear distribution of NF-κB. Meanwhile, Ingenuity Pathway Analysis (IPA) and a qPCR pathway array revealed that silencing MDK decreased the expression of NR3C1, a potential downstream target of the NF-κB pathway. We also confirmed that treatment with an NF-κB inhibitor suppressed NR3C1 expression in BC cells. Finally, we demonstrated that silencing NR3C1 repressed BC cell proliferation and migration. Our findings highlight a novel mechanism by which MDK influences BC progression via regulation of the NF-κB-NR3C1 pathway.

MiR-135a-5p suppresses breast cancer cell proliferation, migration, and invasion by regulating BAG3

BackgroundMicroRNAs (miRNAs) are involved in the progression of diverse human cancers. This work aimed to delve into how microRNA-135a-5p (miR-135a-5p) affects the biological behaviors of Breast Cancer (BC) cells. MethodsGene Expression Omnibus (GEO) datasets were used to analyze the expression differences of miR-135a-5p in cancer tissues of BC patients. Quantitative real-time PCR and western blot were conducted to detect miR-135a-5p and Bcl-2 Associated Athanogene (BAG3) expression levels in BC tissues and cells, respectively. The proliferation, migration, invasion, and cell cycle of BC cells were detected by cell counting kit-8 assay, BrdU assay, wound healing assay, transwell assay, and flow cytometry. The targeted relationship between miR-135a-5p and BAG3 mRNA 3′UTR predicted by bioinformatics was further testified by a dual-luciferase reporter gene assay. Pearson's correlation analysis was adopted to analyze the correlation between miR-135a-5p expression and BAG3 expression. The downstream pathways of BAG3 were analyzed by the LinkedOmics database. ResultsMiR-135a-5p was significantly down-regulated and BAG3 expression was significantly raised in BC tissues. MiR-135a-5p overexpression repressed the viability, migration and invasion of BC cells, and blocked cell cycle progression in G0/G1 phase while inhibiting miR-135a-5p worked oppositely. BAG3 was verified as a target of miR-135a-5p. Overexpression of BAG3 reversed the impacts of miR-135a-5p on the malignant biological behaviors of BC cells. The high expression of BAG3 was associated with the activation of the cell cycle, mTOR and TGF-β signaling pathways. ConclusionMiR-135a-5p regulates BAG3 to repress the growth, migration, invasion, and cell cycle progression of BC cells.

N6-Methyladenosine-regulated LINC00675 suppress the proliferation, migration and invasion of breast cancer cells via inhibiting miR-513b-5p

ABSTRACT Breast cancer (BC) is the most common cancer among women. LINC00675 and miR-513b-5p has been reported to be abnormally expressed in multiple types of cancers and modulate malignant phenotypes of cancer cells. However, to date, the functional role and underlying regulatory mechanism of LINC00675 and miR-513b-5p in BC remains largely unknown. Here, we found that LINC00675 was significantly downregulated in BC tissues and cell lines. Decrease of LINC00675 expression associated with higher tumor grade, lymphovascular invasion and shorter survival in BC patients. Functional experiments demonstrated that overexpression of LINC00675 suppressed BC cell proliferation, migration and invasion, whereas depletion of LINC00675 exerted opposite effects. Mechanistically, LINC00675 functioned as a competing endogenous RNA (ceRNA) to interact with miR-513b-5p and suppress its expression. Moreover, METTL3 increased the m6A methylation of LINC00675, which enhanced the association between LINC00675 and miR-513b-5p. Collectively, the central findings of our study suggest that LINC00675 represses BC progression through the inhibition of miR-513b-5p in a m6A-dependent manner.

Aberrant promoter hypermethylation inhibits RGMA expression and contributes to tumor progression in breast cancer.

Breast cancer (BC) is the most common cancer in women worldwide, and the exploration of aberrantly expressed genes might clarify tumorigenesis and help uncover new therapeutic strategies for BC. Although RGMA was recently recognized as a tumor suppressor gene, its detailed biological function and regulation in BC remain unclear. Herein, we found that RGMA was downregulated in BC tissues compared with non-tumorous breast tissues, particularly in metastatic BC samples, and that patients with low RGMA expression manifested a poorer prognosis. Furthermore, DNMT1 and DNMT3A were found to be recruited to the RGMA promoter and induced aberrant hypermethylation, resulting in downregulation of RGMA expression in BC. In contrast, RGMA overexpression suppressed BC cell proliferation and colony-formation capabilities and increased BC cell apoptosis. Furthermore, RGMA knockdown accelerated BC cell proliferation and suppressed cellular apoptosis in vitro and in vivo. Reversal of RGMA promoter methylation with 5-Aza-CdR restored RGMA expression and blocked tumor growth. Overall, DNMT1- and DNMT3A-mediated RGMA promoter hypermethylation led to downregulation of RGMA expression, and low RGMA expression contributed to BC growth via activation of the FAK/Src/PI3K/AKT-signaling pathway. Our data thus suggested that RGMA might be a promising therapeutic target in BC.

The role of vulpinic acid as a natural compound in the regulation of breast cancer-associated miRNAs

BackgroundBreast cancer is the most frequently diagnosed cancer, and no effective treatment solution has yet been found. The number of studies based on the research of novel natural compounds in the treatment of breast cancer has been increasing in recent years. The anticancer properties of natural compounds are related to the regulation of microRNA (miRNA) expression. Therefore, changing the profile of miRNAs with the use of natural products is very important in cancer treatment. However, the role of vulpinic acid and related miRNAs in breast cancer progression remains unknown. Vulpinic acid, methyl (as2E)-2-(3-hydroxy-5-oxo-4-phenylfuran-2-ylidene)-2 phenylacetate, is a natural product extracted from the lichen species and shows an anticancer effect on different cancer cells.MethodsThis study examines the effects of vulpinic acid on the miRNA levels of breast cancer (MCF-7) cells and its relationship with cell proliferation and apoptosis levels. The antiproliferative effect of vulpinic acid was screened against MCF-7 breast cancer cells and MCF-12A breast epithelial cells using the xCELLigence real-time cell analysis system. We analyzed the altered miRNA expression profile in MCF-7 breast cancer cells versus MCF-12A cells following their response to vulpinic acid through microarray analysis. The microarray analysis results were confirmed through quantitative real-time PCR and bioinformatics analysis.ResultsThe results of the miRNA array and bioinformatic analyses demonstrated that 12 miRNAs were specifically responsive to vulpinic acid in MCF-7 breast cancer cells. This is the first study to reveal that vulpinic acid inhibits the expression of 12 miRNAs and suppresses breast cancer cell proliferation. The study also revealed that vulpinic acid may downregulate the expression of 12 miRNAs by repressing the FOXO-3 gene. The miRNA targets were mainly found to play a role in the apoptosis, cell cycle and MAPK pathways. Moreover, Bcl-2, Bax, procaspase-3 and procaspase-9 protein levels were assessed by western blot analysis for validation of apoptosis at the protein level.ConclusionThis study revealed the molecular mechanisms of vulpinic acid on breast cancer and showed that vulpinic acid regulates apoptosis signaling pathways by decreasing the expression of miRNAs. The miRNA expression patterns illuminate the underlying effect of vulpinic acid in breast cancer treatment.Graphical

Osteoprotegerin (OPG) mediates the anti-carcinogenic effects of normal breast fibroblasts and targets cancer stem cells through inhibition of the β-catenin pathway.

Normal breast fibroblasts (NBFs) support and maintain the architecture of the organ, and can also suppress tumorigenesis. However, the mechanisms involved are not fully understood. We have shown here that NBFs suppress breast carcinogenesis through secretion of osteoprotegerin (OPG), a soluble decoy receptor for the Receptor Activator of NF-κB ligand (RANKL). Indeed, NBFs and human recombinant OPG (rOPG), suppressed breast cancer cells proliferation and motility through inhibition of the epithelial-to-mesenchymal transition (EMT) process both in vitro and in vivo. Additionally, rOPG inhibited the IL-6/STAT3 and NF-κB pathways as well as the OPG gene, which turned out to be STAT3-regulated. This was confirmed using denosumab, a RANKL-targeted antibody, which also inhibited NF-κB, down-regulated OPG and repressed EMT in breast cancer cells grown in 2D and 3D. Importantly, both rOPG and denosumab targeted cancer stem cells (CSCs). This was mediated through inhibition of the CSC-related pathway β-catenin. Moreover, rOPG reduced tumor growth and inhibited breast CSC biomarkers in orthotopic humanized breast tumors. Therefore, normal mammary fibroblasts can suppress carcinogenesis through OPG, which constitutes great potential as preventive and/or therapeutic molecule for breast carcinomas.