Axis In Breast Cancer Cells Research Articles

USP7 deubiquitinates epigenetic reader ZMYND8 to promote breast cancer cell migration and invasion

The ubiquitin-proteasome system (UPS), which involves E3 ligases and deubiquitinases (DUBs), is critical for protein homeostasis. The epigenetic reader ZMYND8 (zinc finger MYND-type containing 8) has emerged as an oncoprotein, and its protein levels are elevated in various types of cancer, including breast cancer. However, the mechanism by which ZMYND8 protein levels are increased in cancer remains elusive. Although ZMYND8 has been reported to be regulated by the E3 ligase FBXW7, it is still unknown whether ZMYND8 could be modulated by DUBs. Here, we identified USP7 (ubiquitin carboxyl-terminal hydrolase 7) as a bona fide DUB for ZMYND8. Mechanically, USP7 directly binds to the PBP (PHD-BRD-PWWP) domain of ZMYND8 via its TRAF (tumor necrosis factor receptor-associated factor) domain and UBL (ubiquitin-like) domain and removes F-box and WD repeat domain containing 7 (FBXW7)-catalyzed poly-ubiquitin chains on lysine residue 1034 (K1034) within ZMYND8, thereby stabilizing ZMYND8 and stimulating the transcription of ZMYND8 target genes ZEB1 (zinc finger E-box binding homeobox 1) and VEGFA (Vascular Endothelial Growth Factor A). Consequently, USP7 enhances the capacity of breast cancer cells for migration and invasion through antagonizing FBXW7-mediated ZMYND8 degradation. Importantly, the protein levels of USP7 positively correlates with those of ZMYND8 in breast cancer tissues. These findings delineate an important layer of migration and invasion regulation by the USP7-ZMYND8 axis in breast cancer cells.

Integrating bioinformatics and experimental models to investigate the mechanism of the chelidonine-induced mitotic catastrophe via the AKT/FOXO3/FOXM1 axis in breast cancer cells.

Breast cancer (BC) is currently the most frequent and lethal cancer among women, and therefore, identification of novel biomarkers and potential anticancer agents for BC is crucial. Chelidonine is one of the main active ingredients of Chelidonium majus, which has been applied in Chinese medicine prescriptions to treat cancer. This paper aimed to evaluate the ability of chelidonine to trigger mitotic catastrophe in BC cells and to clarify its mechanism through the AKT/FOXO3/FOXM1 pathway. Bioinformatics analysis revealed that forkhead box O3 (FOXO3) was downregulated in different subtypes of BC. Factors such as age, stage, Scarff-Bloom-Richardson (SBR) grade, diverse BC subclasses, and triple-negative status were inversely correlated to FOXO3 levels in BC patients compared with healthy controls. Notably, patients exhibiting higher FOXO3 expression levels demonstrated better overall survival (OS) and relapse-free survival (RFS). Moreover, FOXM1 levels were negatively correlated with both OS and RFS in BC patients. These results revealed that FOXO3 might be considered a predictive biomarker for the prognosis of BC. By utilizing Gene Set Enrichment Analysis (GSEA), we delved into the main Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment pathways of FOXO3, and the results suggested that FOXO3 was mainly involved in cancer-related pathways and the cell cycle. Thereafter, MTT and flow cytometry (FCM) analysis indicated that chelidonine inhibited BC cell line proliferation and induced M phase arrest. It was found that chelidonine treatment induced MCF-7 cell apoptosis, significantly reduced the expression of survivin and promoted the expression of p53 and caspase-9. Further morphological observation illustrated depolymerization of the actin skeleton and shortening of actin filaments in BC cells, leading to the typical characteristics of mitotic catastrophe, such as abnormal mitosis and multinucleated cells. Western blot analysis demonstrated that chelidonine inhibited the expression of p-AKT to promote the expression of FOXO3 protein and weaken the expression levels of FOXM1 and polo-like kinase 1 (PLK1). Taken together, our present work proved that FOXO3 might be considered a potential therapeutic target for BC. Chelidonine emerges as a promising agent to treat BC by inducing M phase arrest of BC cells and hindering the AKT/FOXO3/FOXM1 axis, thereby inducing mitotic catastrophe in BC.

CENPA knockdown restrains cell progression and tumor growth in breast cancer by reducing PLA2R1 promoter methylation and modulating PLA2R1/HHEX axis.

Breast cancer is a lethal malignancy affecting females worldwide. It has been reported that upregulated centromere protein A (CENPA) expression might indicate unfortunate prognosis and can function as a prognostic biomarker in breast cancer. This study aimed to investigate the accurate roles and downstream mechanisms of CENPA in breast cancer progression. CENPA protein levels in breast cancer tissues and cell lines were analyzed by Western blot and immunohistochemistry assays. We used gain/loss-of-function experiments to determine the potential effects of CENPA and phospholipase A2 receptor (PLA2R1) on breast cancer cell proliferation, migration, and apoptosis. Co-IP assay was employed to validate the possible interaction between CENPA and DNA methyltransferase 1 (DNMT1), as well as PLA2R1 and hematopoietically expressed homeobox (HHEX). PLA2R1 promoter methylation was determined using methylation-specific PCR assay. The biological capabilities of CENPA/PLA2R1/HHEX axis in breast cancer cells was determined by rescue experiments. In addition, CENPA-silenced MCF-7 cells were injected into mice, followed by measurement of tumor growth. CENPA level was prominently elevated in breast cancer tissues and cell lines. Interestingly, CENPA knockdown and PLA2R1 overexpression both restrained breast cancer cell proliferation and migration, and enhanced apoptosis. On the contrary, CENPA overexpression displayed the opposite results. Moreover, CENPA reduced PLA2R1 expression through promoting DNMT1-mediated PLA2R1 promoter methylation. PLA2R1 overexpression could effectively abrogate CENPA overexpression-mediated augment of breast cancer cell progression. Furthermore, PLA2R1 interacted with HHEX and promoted HHEX expression. PLA2R1 knockdown increased the rate of breast cancer cell proliferation and migration but restrained apoptosis, which was abrogated by HHEX overexpression. In addition, CENPA silencing suppressed tumor growth in vivo. CENPA knockdown restrained breast cancer cell proliferation and migration and attenuated tumor growth in vivo through reducing PLA2R1 promoter methylation and increasing PLA2R1 and HHEX expression. We may provide a promising prognostic biomarker and novel therapeutic target for breast cancer.

An antioxidant response element regulates the HIF1α axis in breast cancer cells.

The redox sensitive transcription factor NRF2 is a central regulator of the transcriptional response to reactive oxygen species (ROS). NRF2 is widely recognized for its ROS-responsive upregulation of antioxidant genes that are essential for mitigating the damaging effects of oxidative stress. However, multiple genome-wide approaches have suggested that NRF2's regulatory reach extends well beyond the canonical antioxidant genes, with the potential to regulate many noncanonical target genes. Recent work from our lab and others suggests HIF1A, which encodes the hypoxia-responsive transcription factor HIF1α, is one such noncanonical NRF2 target. These studies found that NRF2 activity is associated with high HIF1A expression in multiple cellular contexts, HIF1A expression is partially dependent on NRF2, and there is a putative NRF2 binding site (antioxidant response element, or ARE) approximately 30 kilobases upstream of HIF1A. These findings all support a model in which HIF1A is a direct target of NRF2, but did not confirm the functional importance of the upstream ARE in HIF1A expression. Here we use CRISPR/Cas9 genome editing to mutate this ARE in its genomic context and test the impact on HIF1A expression. We find that mutation of this ARE in a breast cancer cell line (MDA-MB-231) eliminates NRF2 binding and decreases HIF1A expression at the transcript and protein levels, and disrupts HIF1α target genes as well as phenotypes driven by these HIF1α targets. Taken together, these results indicate that this NRF2 targeted ARE plays an important role in the expression of HIF1A and activity of the HIF1α axis in MDA-MB-231cells.

The invariant chain CD74 protein is a cell surface binding partner of TIMP-1 in breast cancer cells.

Tissue inhibitor of metalloproteinases-1 (TIMP-1) regulates the proteolytic activity of matrix metalloproteinases (MMPs), playing an important role in the homeostasis of the extracellular matrix. Beyond its well-known role in tissue maintenance, TIMP-1 has been associated with multiple MMP-independent cytokine-like functions. The protein structure of TIMP-1, with two distinct domains, one interacting with MMPs and another able to bind multiple partners, provides a rationale for this multi-functionality. The identification of CD63 as a cell surface receptor for TIMP-1, able to mediate intracellular signaling through the Erk/MAPK axis, provided a molecular basis for the role of TIMP-1 in cellular signaling. However, several lines of evidence suggest that TIMP-1 may be able to associate with many interaction partners, thus attaining multiple functions. To enable the identification of previously unknown interaction partners that may underpin the core cellular functions of TIMP-1, known as well as unknown, we performed a yeast two-hybrid screening using a mammary gland complementary DNA (cDNA) library. We report here the identification of multiple interactors, including MHC class II-associated invariant chain γ (CD74). We verified that CD74 interacts with TIMP-1 in breast cancer cells and that this interaction contributes to cellular internalization of TIMP-1 and mediates intracellular signaling through the Akt signaling axis in breast cancer cells. These data provide new insights into the complex nature of the functions of TIMP-1 and their potential mechanistic basis.

In vitro Anti-malignant Property of PCMT1 Silencing and Identification of the SNHG16/miR-195/PCMT1 Regulatory Axis in Breast Cancer Cells

BackgroundProtein L-isoaspartate (D-aspartate) O-methyltransferase (PCMT1) is a highly conserved protein repair enzyme that participates in regulating the progression of human cancers. We therefore studied the function and the related mechanisms of PCMT1 in breast cancer cells. MethodsExpression profile and prognostic analysis of PCMT1 in breast cancer patients were analyzed using online databases. PCMT1 expression in breast cancer cells was detected by western blot analysis. Cell proliferation was determined by CCK-8 and colony formation assays. Apoptosis was evaluated using flow cytometry analysis and caspase-3/7 activity assay. Cell invasion was assessed by Transwell invasion assay. The small nucleolar RNA host gene 16 (SNHG16)/miR-195/PCMT1 regulatory axis was identified using bioinformatics analysis. ResultsPCMT1 expression was increased in breast cancer tissues and cells. High PCMT1 expression was correlated with poor prognosis in breast cancer patients. PCMT1 knockdown suppressed cell proliferation and colony formation ability in breast cancer cells. Moreover, PCMT1 knockdown induced apoptosis and restrained the invasive ability in breast cancer cells. PCMT1 overexpression increased the proliferative and invasive abilities of breast cancer cells. miR-195 was identified as the unique upstream miRNA of PCMT1. SNHG16 was identified as the unique upstream lncRNA of miR-195. SNHG16 knockdown downregulated PCMT1 by increasing miR-195 expression. Breast cancer cell proliferation was regulated by the SNHG16/miR-195/PCMT1 axis. ConclusionPCMT1 silencing inhibited cell proliferation and invasion and induced apoptosis in breast cancer cells and the SNHG16/miR-195/PCMT1 regulatory axis might serve as a potential therapeutic target for breast cancer.

HULC targets the IGF1R–PI3K-AKT axis in trans to promote breast cancer metastasis and cisplatin resistance

Insulin-like growth factor I receptor (IGF1R) is frequently upregulated in breast cancer. Due to its intrinsic tyrosine kinase activity, aberrant activation of the IGF1R signaling axis may enhance tumor cell proliferation and cancer stemness, causing tumor relapse, metastasis and resistance to chemotherapy. We utilized a chromatin RNA in situ reverse transcription (CRIST) approach to characterize molecular factors that regulate the IGF1R network. We identified lncRNA HULC (Highly Upregulated in Liver Cancer) as a key trans-regulator of IGF1R in breast cancer cells. Loss of HULC suppressed the expression of IGF1R and the activation of its downstream PI3K/AKT pathway, while HULC overexpression activated the axis in breast cancer cells. Using a transcription-associated trap (RAT) assay, we demonstrated that HULC functioned as a nuclear lncRNA and epigenetically activated IGF1R by directly binding to the intragenic regulatory elements of the gene, orchestrating intrachromosomal interactions, and promoting histone H3K9 acetylation. The activated HULC-IGF1R/PI3K/AKT pathway mediated tumor resistance to cisplatin through the increased expression of cancer stemness markers, including NANOG, SOX2, OCT4, CD44 and ALDH1A1. In immunodeficient mice, stimulation of the HULC-IGF1R pathway promoted tumor metastasis. These data suggest that HULC may be a new epigenetic target for IGF1R axis-targeted therapeutic intervention.

Retraction Statement.

Retraction Statement.

Novel O-acylated (E)-3-aryl-6,7-dihydrobenzisoxazol-4(5H)-one oximes targeting HSP90-HER2 axis in breast cancer cells

Novel O-acylated (E)-3-aryl-6,7-dihydrobenzisoxazol-4(5H)-one oximes were designed as potential HSP90 inhibitors. A series of the compounds was synthesized by oximation of (E)-3-aryl-6,7-dihydrobenzisoxazol-4(5H)-ones followed by O-acylation with acylamidobenzoic acids. The obtained compounds showed an antiproliferative effect on three breast cancer cell lines (MCF7, MDA-MB-231 and HCC1954). Compound 16s exhibited high antiproliferative potency against HCC1954 breast cancer cells with the IC50 value of 6 µM was selected for in-depth evaluation. Compound 16s did not inhibit the growth of normal epithelial cells. We have demonstrated that the compound 16s can induce apoptosis in cancer cells via inhibition of HSP90 “client” proteins including a key oncogenic receptor, HER2/neu. Described here compounds can be considered for further basic and preclinical investigation as a part of HSP90/HER2-targeted therapies.

Docosahexaenoic acid supresses breast cancer cell proliferation and migration by promoting the expression of miR-99a and targeting mTOR signaling

Docosahexaenoic acid (DHA) shows different anti-cancer effects on breast cancer (BC) cell proliferation and progression; however, the underlying molecular mechanism yet still is blanketed in mystery. Herein, we aimed to reveal whether the inhibitory effects of DHA on BC proliferation and migration are exerted, at least in part, through promoting the expression of miR-99a and targeting mTOR signaling. DHA lessened the BC cell viability in a time- and concentration-dependent manner. Besides, DHA-treatment significantly suppressed the proliferation and migration, while promoted BC cell apoptosis by regulating Bax and Bcl-2. We also demonstrated that DHA activated caspase-3/7 in MDA-MB-231 BCE cells. Also, we determined that miR-99a was upregulated in DHA-treated cells and mTOR was a direct and functional target of this miRNA, verified by the ability of anti-miR-99a to rescue the suppressive effects of DHA on mTOR expression in BC cells. Furthermore, DHA was shown to inhibit the mTOR-HIF-1α-VEGF signaling via regulating miR-99a in BC cells. DHA treatment caused a significant dose-dependent reduction of VEGF secretion from BC cells. When miR-99a was knocked down, DHA did not inhibit the BC proliferation and migration. We concluded that the anti-cancer effects of DHA can be attributed to the up-regulation of miR-99a that ipso facto inhibit the mTOR-HIF-1α-VEGF axis in BC cells. It is thought that DHA treatment might be considered as a promising supplement for BC therapy.

Discovery and Characterization of a Novel MASTL Inhibitor MKI-2 Targeting MASTL-PP2A in Breast Cancer Cells and Oocytes.

Although microtubule-associated serine/threonine kinase-like (MASTL) is a promising target for selective anticancer treatment, MASTL inhibitors with nano range potency and antitumor efficacy have not been reported. Here, we report a novel potent and selective MASTL inhibitor MASTL kinase inhibitor-2 (MKI-2) identified in silico through a drug discovery program. Our data showed that MKI-2 inhibited recombinant MASTL activity and cellular MASTL activity with IC50 values of 37.44 nM and 142.7 nM, respectively, in breast cancer cells. In addition, MKI-2 inhibited MASTL kinase rather than other AGC kinases, such as ROCK1, AKT1, PKACα, and p70S6K. Furthermore, MKI-2 exerted various antitumor activities by inducing mitotic catastrophe resulting from the modulation of the MASTL-PP2A axis in breast cancer cells. The MKI-2 treatment showed phenocopies with MASTL-null oocyte in mouse oocytes, which were used as a model to validate MKI-2 activity. Therefore, our study provided a new potent and selective MASTL inhibitor MKI-2 targeting the oncogenic MAST-PP2A axis in breast cancer cells.

Chemotherapy-elicited exosomal miR-378a-3p and miR-378d to promote breast cancer stemness and chemoresistance via the activation of EZH2/STAT3 signaling.

e12615 Background: Not all breast cancer (BC) patients who receive neoadjuvant chemotherapy achieve a pathologic complete response (pCR), but the reasons for this are unknown. Previous studies have shown that exosomes produced in the tumor microenvironment in response to chemotherapy promote a chemotherapy-resistant phenotype in tumors. However, the role of BC chemo-elicited exosomes in regulating chemoresistance is poorly understood. Methods: Using commercial kits, serum exosomes were extracted from patients before neoadjuvant chemotherapy, after one cycle of chemotherapy and after four cycles of chemotherapy consisting of doxorubicin (DOX) and paclitaxel (PTX). Their miRNAs were sequenced, and the correlation between the sequencing results and chemotherapy effects was further verified by RT-qPCR using patient serum exosomes. Cell Counting Kit-8 (CCK-8) was used to detect chemosensitivity. Stemness was assessed by CD44+/CD24- population analysis and mammosphere formation assays. Chromatin immunoprecipitation (ChIP) experiments were performed to verify the binding of signal transducer and activator of transcription 3 (STAT3) to the promoter of miRNAs. Results: Here, we provide clinical evidence that chemotherapy-elicited exosomal miR-378a-3p and miR-378d are closely related to the chemotherapy response and that exosomes produced by BC cells after stimulation with DOX or PTX deliver miR-378a-3p and miR-378d to neighboring cells to activate WNT and NOTCH stemness pathways and induce drug resistance by targeting Dickkopf 3 (DKK3) and NUMB. In addition, STAT3, which is enhanced by zeste homolog 2 (EZH2), bound to the promoter regions of miR-378a-3p and miR-378d, thereby increasing their expression in exosomes. More importantly, chemotherapeutic agents combined with the EZH2 inhibitor tazemetostat reversed chemotherapy-elicited exosome induced drug resistance in a nude mouse tumor xenograft model. Conclusions: This study revealed a novel mechanism of acquired chemoresistance whereby chemotherapy activates the EZH2/STAT3 axis in BC cells, which then secrete chemotherapy-elicited exosomes enriched in miR-378a-3p and miR-378d. These exosomes are absorbed by chemotherapy-surviving BC cells, leading to activation of WNT and NOTCH stem cell pathways via the targeting of DKK3 and NUMB and subsequently resulting in drug resistance. Therefore, blocking this adaptive mechanism during chemotherapy may reduce the development of chemotherapy resistance and maximize the therapeutic effect.

LncRNA MAFG-AS1 affects the tumorigenesis of breast cancer cells via the miR-574-5p/SOD2 axis

Amounting evidence suggested that long non coding RNAs (lncRNAs) played vital roles in the progression of various cancers. The aim of this study is to examine the biological roles and underlying mechanisms of lncRNA MAFG-AS1 in the tumorigenesis of breast cancer (BC) cells. Here we showed that downregulation of MAFG-AS1 inhibited the viability, migration, and invasion of BC cells. Mechanism investigation showed that inhibition of MAFG-AS1 induced apoptosis via the intrinsic apoptotic pathway and overexpression of Bcl-2 could inhibited it. Further, MAFG-AS1 acts as a sponge of miR-574-5p which directly binds to SOD2 mRNA. Re-expression of SOD2 using a 3′-UTR mutant SOD2 reversed the effects of silencing of MAFG-AS1 on BC cells. Finally, downregulation of MAFG-AS1 inhibited the growth of tumour in vivo. Together, MAFG-AS1 acts as an oncogene via regulation of miR-574-5p/SOD2 axis in BC cells.

Chemotherapy-elicited exosomal miR-378a-3p and miR-378d promote breast cancer stemness and chemoresistance via the activation of EZH2/STAT3 signaling

BackgroundNot all breast cancer (BC) patients who receive neoadjuvant chemotherapy achieve a pathologic complete response (pCR), but the reasons for this are unknown. Previous studies have shown that exosomes produced in the tumor microenvironment in response to chemotherapy promote a chemotherapy-resistant phenotype in tumors. However, the role of BC chemotherapy-elicited exosomes in regulating chemoresistance is poorly understood.MethodsUsing commercial kits, serum exosomes were extracted from patients before neoadjuvant chemotherapy, after one cycle of chemotherapy and after four cycles of chemotherapy consisting of doxorubicin (DOX) and paclitaxel (PTX). Their miRNAs were sequenced, and the correlation between the sequencing results and chemotherapy effects was further verified by RT-qPCR using patient serum exosomes. Cell Counting Kit-8 (CCK-8) was used to detect chemosensitivity. Stemness was assessed by CD44+/CD24- population analysis and mammosphere formation assays. Chromatin immunoprecipitation (ChIP) experiments were performed to verify the binding of signal transducer and activator of transcription 3 (STAT3) to the promoter of miRNAs.ResultsHere, we provide clinical evidence that chemotherapy-elicited exosomal miR-378a-3p and miR-378d are closely related to the chemotherapy response and that exosomes produced by BC cells after stimulation with DOX or PTX deliver miR-378a-3p and miR-378d to neighboring cells to activate WNT and NOTCH stemness pathways and induce drug resistance by targeting Dickkopf 3 (DKK3) and NUMB. In addition, STAT3, which is enhanced by zeste homolog 2 (EZH2), bound to the promoter regions of miR-378a-3p and miR-378d, thereby increasing their expression in exosomes. More importantly, chemotherapeutic agents combined with the EZH2 inhibitor tazemetostat reversed chemotherapy-elicited exosome-induced drug resistance in a nude mouse tumor xenograft model.ConclusionThis study revealed a novel mechanism of acquired chemoresistance whereby chemotherapy activates the EZH2/STAT3 axis in BC cells, which then secrete chemotherapy-elicited exosomes enriched in miR-378a-3p and miR-378d. These exosomes are absorbed by chemotherapy-surviving BC cells, leading to activation of the WNT and NOTCH stem cell pathways via the targeting of DKK3 and NUMB and subsequently resulting in drug resistance. Therefore, blocking this adaptive mechanism during chemotherapy may reduce the development of chemotherapy resistance and maximize the therapeutic effect.

Dihydroartemisinin induces pyroptosis by promoting the AIM2/caspase-3/DFNA5 axis in breast cancer cells

BackgroundBreast cancer is a complex disease. Recent research has examined the anticancer effects of dihydroartemisinin (DHA) on breast cancer. However, the molecular mechanism of the antitumour effect of DHA is unclear. MethodsMCF-7 and MDA-MB-231 cell lines were used for in vitro research. BALB/c nude mice were used to establish breast cancer xenografts. The mRNA and protein levels were analysed by qRT-PCR and western blotting, respectively. Flow cytometry was performed to examine cell apoptosis. ELISA kits were used to evaluate the production of interleukin-1β (IL-1β) and IL-18. LDH and ATP release were individually measured with the corresponding kits. A colony formation assay was used to examine the proliferation of breast cancer cells. ResultsDHA inhibited proliferation and induced pyroptosis in breast cancer cells. Mechanistically, DHA activated the expression of absent in melanoma 2 (AIM2), caspase-3 and gasdermin E (DFNA5). In addition, AIM2 promoted DFNA5 expression by activating caspase-3. Knockdown of AIM2 and DFNA5 significantly enhanced breast cancer cell resistance to DHA. In vivo experiments showed that the tumorigenicity of breast cancer cells was significantly suppressed by DHA. Moreover, the AIM2/caspase-3/DFNA5 axis was activated by DHA and then induced pyroptosis. ConclusionsOur findings indicate that DHA inhibits tumorigenesis by inducing pyroptosis in breast cancer cells, highlighting a promising therapeutic strategy for breast cancer.

Long noncoding RNA MEG3 suppresses cell proliferation, migration and invasion, induces apoptosis and paclitaxel-resistance via miR-4513/PBLD axis in breast cancer cells

ABSTRACT Breast cancer remains a general-threat event in the health of women. Currently, increasing records indicate that long non-coding RNA maternally expressed 3 (MEG3) plays a central role in breast cancer. The current research focused on the function of MEG3 in paclitaxel (PTX)-resistance and human breast cancer growth. Levels of MEG3, microRNA (miR)-4513, and phenazine biosynthesis-like domain-containing protein (PBLD) were evaluated using quantitative real-time polymerase chain reaction (qRT-PCR) or western blot assays. 3-(4.5-dimethylghiazol-2-yl)-2,5-diphenyltetrazolium Bromide (MTT) assay was performed to examine the IC50 of PTX and cell proliferation in breast cancer cells. In addition, cell apoptosis was determined utilizing flow cytometry. Transwell was conducted to assay cell migration and invasion in MCF-7 and MDA-MB-231 cells. The interaction between miR-4513 and MEG3 or PBLD was expounded via dual-luciferase reporter assay. Levels of MEG3 and PBLD were decreased, but miR-4513 level was triggered in breast cancer tissues and cell lines. Overexpression of MEG3 could reinforce cell apoptosis, impede proliferation, migration, invasion, and the IC50 of PTX in breast cancer cells. Moreover, the impact of miR-4513 inhibitor on cell progression and PTX-resistance was overturned by MEG3 deficiency. Interestingly, miR-4513 mimic could abolish the role of PBLD upregulation in cell behaviors and PTX-resistance in MCF-7 and MDA-MB-231 cells. Finally, the expression of PBLD was co-modulated by miR-4513 and MEG3 in vitro. MEG3/miR-4513/PBLD axis modulated PTX-resistance and the development of breast cancer cells, which might provide a promising therapeutic strategy for breast cancer.

3-Acetyl-11-keto-β-boswellic acid inhibits cancer cell invasion and induces apoptosis in breast cancer cells by abrogating the EGFR-mediated PI3K/Akt pathway

IntroductionRecent evidence has demonstrated the anticancer potential of 3-acetyl-11-keto-β-boswellic acid (AKBA). The current study was performed to evaluate the anti-invasive and antimotility effect of AKBA against breast cancer (BC) cells.Material and methodsThe antiproliferative assay was used to evaluate cell viability. Wound healing assay and Transwell invasion assay were used for migration and invasion respectively. Annexin V FITC assay and microscopy were used to quantify and detect apoptosis. Zymography and immunoblotting analysis were used for gelatinase activity of matrix metalloproteinases (MMPs) and protein expression of signaling proteins involved in apoptosis, tumor cell invasion and motility of BC cells.ResultsAKBA exhibits significant inhibition of cell proliferation and suppresses BC cell motility. Mechanistically, AKBA suppresses migration and invasion, and attenuates MMP-2 and MMP-9 activity, which plays a key role in metastasis. Furthermore, a dose-dependent manner of AKBA promotes significant induction of apoptosis. Moreover, we observed that a higher concentration of AKBA reduces phosphorylation of EGFR, PI3K, and Akt with a subsequent decrease in MMP-9 and MMP-2 expression, and an increase in TIMP1 and E-cadherin expression. However, pre-treatment with EGF in BC cells restores significant cell motility and invasion even in the presence of AKBA, indicating that inhibition of cell migration by AKBA is operated by the EGFR/PI3K/Akt axis. Additionally, AKBA showed a significant inhibitory and strong synergistic effect on EGFR-mediated PI3K/Akt signaling associated proteins when compared with the known inhibitors gefitinib and erlotinib.ConclusionsAKBA exhibits potential antiproliferative effects by inducing apoptosis and suppresses tumor cell motility and invasion, which is mediated by the EGFR/PI3K/Akt axis in BC cells.

Uev1A promotes breast cancer cell survival and chemoresistance through the AKT-FOXO1-BIM pathway

BackgroundUbiquitin-conjugating enzyme variant UEV1A is required for Ubc13-catalyzed K63-linked poly-ubiquitination that regulates several signaling pathways including NF-κB, MAPK and PI3K/AKT. Previous reports implicate UEV1A as a potential proto-oncogene and have shown that UEV1A promotes breast cancer metastasis through constitutive NF-кB activation. Ubc13-Uev1A along with TARF6 can also ubiquitinate AKT but its downstream events are unclear.MethodsIn this study, we experimentally manipulated UEV1 expression in two typical breast cancer cell lines MDA-MB-231 and MCF7 under serum starvation conditions and monitored AKT activation and its downstream protein levels, as well as cellular sensitivity to chemotherapeutic agents.ResultsWe found that overexpression of UEV1A is sufficient to activate the AKT signaling pathway that in turn inhibits FOXO1 and BIM expression to promote cell survival under serum starvation conditions and enhances cellular resistance to chemotherapy. Consistently, experimental depletion of Uev1 in breast cancer cells inhibits AKT signaling and promotes FOXO1 and BIM expression to reduce cell survival under serum starvation stress and enhance chemosensitivity.ConclusionsUev1A promotes cell survival under serum starvation stress through the AKT-FOXO1-BIM axis in breast cancer cells, which unveals a potential therapeutic target in the treatment of breast cancers.

Long non-coding RNA NONHSAT101069 promotes epirubicin resistance, migration, and invasion of breast cancer cells through NONHSAT101069/miR-129-5p/Twist1 axis.

Drug resistance, including epirubicin-based therapeutic resistance, is one of the major reasons responsible for the unfavorable prognosis of patients diagnosed with breast cancer (BC). Acquired chemoresistance and metastatic properties have been identified to be closely associated with the process of epithelial-mesenchymal transition (EMT). Recently, dysregulation of long non-coding RNAs (lncRNAs) have been increasingly reported to perform promotive or suppressive functions in chemoresistance and EMT process in multiple cancers. However, relevant novel lncRNA participating in epirubicin resistance and EMT and its underlying molecular mechanisms remain unknown in BC. Herein, we established the epirubicin-resistant breast cancer cell subline (MCF-7/ADR), which presented mesenchymal phenotype and increased metastatic potential. A panel of differentially expressed lncRNAs, including 268 upregulated and 49 downregulated lncRNAs, were identified by high-flux microarray investigation in MCF-7 and MCF-7/ADR cells. The novel lncRNA NONHSAT101069 was significantly overexpressed in BC specimens, BC cell lines, and epirubicin-resistant cell sublines. The knockdown of NONHSAT101069 significantly repressed, whereas overexpression of NONHSAT101069 promoted the epirubicin resistance, migration, invasion and EMT process of BC cells both in vitro and in vivo. Further mechanism-related researches uncovered that NONHSAT101069 functioned as a ceRNA (competing endogenous RNA) via sponging miR-129-5p. Twist1 was a direct downstream protein of NONHSAT101069/miR-129-5p axis in BC cells. To conclude, NONHSAT101069 was upregulated in BC tissues and promoted epirubicin resistance, migration and invasion of BC cells via regulation of NONHSAT101069/miR-129-5p/Twist1 axis, highlighting its potential as an oncogene and a therapeutic biomarker for BC.

Ursolic Acid Reverses the Chemoresistance of Breast Cancer Cells to Paclitaxel by Targeting MiRNA-149-5p/MyD88.

Paclitaxel (PTX) is widely used as a front-line chemotherapy for breast cancer treatment. However, its clinical applications are limited by the development of chemoresistance. The objective of this study was to investigate the reversal effects of ursolic acid (UA) on PTX resistance and the possible mechanisms in breast cancer. The role of miRNA-149-5p/MyD88 in the regulation of PTX resistance was investigated by the transfection of breast cancer cells with MDA-MB-231 (231) and MDA-MB-231/PTX-resistance (231/PTX) with lentiviruses carrying the MyD88 gene, shRNA specific for MyD88, the miR-149-5p gene, and shRNA specific for miR-149-5p. The PTX sensitivity was assessed by a CCK-8 assay. qRT-PCR and Western blot analyses were used to detect changes in the mRNA and protein levels. Flow cytometry was used to measure the rate of cell apoptosis. A luciferase activity assay was used to detect the binding site of miR-149-5p on the 3′UTR of MyD88. 231/PTX cells were injected into the flanks of female athymic nude mice, and the mice were randomly divided into the five following groups: PBS, PTX (low), PTX (high), UA, and PTX+UA. Our data show that UA reversed the resistance of breast cancer 231/PTX cells to PTX in vitro and in vivo. UA treatment significantly increased the expression of miR-149-5p, which was lower in 231/PTX cells than in 231 cells. Furthermore, the overexpression of miR-149-5p increased the sensitivity of 231/PTX cells to PTX treatment, whereas the knockdown of the miR-149-5p gene attenuated the effects of UA on the regulation of PTX sensitivity. A luciferase assay demonstrated that miR-149-5p could directly regulate the transcriptional activity of MyD88, a known PTX-resistance gene, by targeting the 3′UTR of MyD88. Meanwhile, the downregulation of MyD88 through the overexpression of miR-149-5p or UA treatment inhibited the activation of the Akt signaling pathway in 231/PTX cells. Thus, our data indicate that UA can reverse PTX resistance by targeting the miRNA-149-5p/MyD88 axis in breast cancer cells.