Tumorigenicity Of Breast Cancer Cells Research Articles

The Tumorigenicity of Breast Cancer Cells Is Reduced upon Treatment with Small Extracellular Vesicles Isolated from Heparin Treated Cell Cultures.

As a member of the HPSG family, heparin is often used as a specific probe of their role in cell physiology; indeed, we have previously shown a reduction in the tumorigenicity of breast cancer cells when cultured in its presence. However, a partial reversal of the anti-tumorigenic effect occurred when the treated cells were cultured in fresh medium without heparin, which led us to consider whether a more persistent effect could be achieved by treatment of the cells with small extracellular vesicles (sEV) from heparin-treated cells. The tumorigenicity was analyzed using sEV isolated from the culture medium of heparin-treated MCF-7 and MDA-MB231 breast cancer cells (sEV-HT) or from conditioned medium following the termination of treatment (heparin discontinued, sEV-HD). Tumorigenicity was reduced in cells cultured in the presence of sEV-HT compared to that of cells cultured in the presence of sEV from untreated cells (sEV-Ctrl). sEV-HD were also observed to exert an anti-tumorigenic effect on the expression of pro-tumorigenic and cell cycle regulatory proteins, as well as signaling activities when added to fresh cultures of MCF-7 and MDA-MB231 cells. The anti-tumorigenic activity of the heparin-derived sEV may arise from observed changes in the miRNA content or from heparin, which was observed to be bound to the sEV. sEV may constitute a relatively stable reservoir of circulating heparin, allowing heparin activity to persist in the circulation even after therapy has been discontinued. These findings can be considered as a special additional pharmacological characteristic of heparin clinical therapy.

MicroRNA-944 inhibits breast cancer cell proliferation and promotes cell apoptosis by reducing SPP1 through inactivating the PI3K/Akt pathway.

Breast cancer is a common malignancy in women with poor prognosis. This study aimed to investigate the molecular mechanism of microRNA-944 (miR-944) mediated secreted phosphoprotein-1 (SPP1) in breast cancer progression and its regulatory effect on the phosphoinositide 3-kinase (PI3K)/Akt signaling pathway. Differential gene analysis was performed to identify key genes associated with breast cancer development by screening breast cancer-related microarray data. The expression of miR-944 and SPP1 and their relationship were determined in clinical samples and cells. sh-SPP1, oe-SPP1, LY294002 or miR-944 mimic were transfected into MCF-7 cells to investigate the role of miR-944 mediated SPP1 in breast cancer development and its regulatory effect on the PI3K/Akt pathway. Finally, the tumorigenicity of breast cancer cells was observed in nude mice. Through bioinformatics analysis, we identified SPP1 as a key gene in breast cancer, and miR-944 as an upstream miRNA of SPP1. In breast cancer tissues and cells, the expression of miR-944 was decreased while that of SPP1 was increased. miR-944 negatively regulated the expression of SPP1. In breast cancer cells, SPP1 activated the PI3K/Akt pathway to promote cell proliferation and inhibit apoptosis. In vitro cell experiments showed that the downregulation of miR-944 promoted the high expression of SPP1, which then activated the PI3K/Akt signaling pathway, promoting breast cancer cell proliferation. In vivo experiments further confirmed the anti-cancer role of miR-944 mediated SPP1 in breast cancer. Our study highlights the role of miR-944 mediated SPP1 in inhibiting breast cancer progression by blocking the PI3K/Akt pathway.

PDK1 promotes breast cancer progression by enhancing the stability and transcriptional activity of HIF-1α

Pyruvate dehydrogenase kinase 1 (PDK1) phosphorylates the pyruvate dehydrogenase complex, which inhibits its activity. Inhibiting pyruvate dehydrogenase complex inhibits the tricarboxylic acid cycle and the reprogramming of tumor cell metabolism to glycolysis, which plays an important role in tumor progression. This study aims to elucidate how PDK1 promotes breast cancer progression. We found that PDK1 was highly expressed in breast cancer tissues, and PDK1 knockdown reduced the proliferation, migration, and tumorigenicity of breast cancer cells and inhibited the HIF-1α (hypoxia-inducible factor 1α) pathway. Further investigation showed that PDK1 promoted the protein stability of HIF-1α by reducing the level of ubiquitination of HIF-1α. The HIF-1α protein levels were dependent on PDK1 kinase activity. Furthermore, HIF-1α phosphorylation at serine 451 was detected in wild-type breast cancer cells but not in PDK1 knockout breast cancer cells. The phosphorylation of HIF-1α at Ser 451 stabilized its protein levels by inhibiting the interaction of HIF-1α with von Hippel-Lindau and prolyl hydroxylase domain. We also found that PDK1 enhanced HIF-1α transcriptional activity. In summary, PDK1 enhances HIF-1α protein stability by phosphorylating HIF-1α at Ser451 and promotes HIF-1α transcriptional activity by enhancing the binding of HIF-1α to P300. PDK1 and HIF-1α form a positive feedback loop to promote breast cancer progression.

Molecularly Engineered Surfactin Analogues Induce Nonapoptotic-Like Cell Death and Increased Selectivity in Multiple Breast Cancer Cell Types.

Surfactin, a negatively charged amphiphilic lipopeptide biosurfactant, is synthesized by the bacterium Bacillus subtilis. It consists of a cyclic heptapeptide and an 11-15C β-hydroxy fatty acid. To probe how the modification of the molecular skeleton of surfactin influences its selectivity and activity against breast cancer, six synthetic surfactins were generated. Modifications were accomplished by conjugating amine-functionalized molecules to the Glu and Asp carboxyl moieties of the heptapeptide. The resulting synthetic surfactins provided a diverse series of molecules with differences in charge, size, and hydrophilicity. After purification and structural analysis, insights into biological activity and specificity were generated for each compound. Dose-dependent growth inhibition was determined for four tumorigenic breast cancer cell lines in monolayer and spheroid morphologies, as well as nontumorigenic fibroblasts and sheep erythrocytes, which were utilized to determine selectivity indices. Results indicated that two compounds, which have amplified anionic charge, had increased activity on breast cancer, with reduced activity on nontumorigenic fibroblasts and erythrocytes. Cationic derivative surf-ethylenediamine has increased activity on all cell lines tested. Novel correlations between dose-response activities and physicochemical properties of all compounds determined that there is a significant correlation between the critical micelle concentration and activity against multiple cell lines.

Zinc finger and SCAN domain containing 1, ZSCAN1, is a novel stemness-related tumor suppressor and transcriptional repressor in breast cancer targeting TAZ.

Cancer stem cells (CSCs) targeted therapy holds the potential for improving cancer management; identification of stemness-related genes in CSCs is necessary for its development. The Cancer Genome Atlas (TCGA) and the Molecular Taxonomy of Breast Cancer International Consortium (METABRIC) datasets were used for survival analysis. ZSCAN1 correlated genes was identified by Spearman correlation analysis. Breast cancer stem-like cells (BCSLCs) were isolated by sorting CD44+CD24- cells from suspension cultured breast cancer (BC) spheroids. The sphere-forming capacity and sphere- and tumor-initiating capacities were determined by sphere formation and limiting dilution assays. The relative gene expression was determined by qRT-PCR, western blot. Lentivirus system was used for gene manipulation. Nuclear run-on assay was employed to examine the levels of nascent mRNAs. DNA pull-down and Chromatin immunoprecipitation (ChIP) assays were used for determining the interaction between protein and target DNA fragments. Luciferase reporter assay was used for evaluating the activity of the promoter. ZSCAN1 is aberrantly suppressed in BC, and this suppression indicates a bad prognosis. Ectopic expression of ZSCAN1 inhibited the proliferation, clonogenicity, and tumorigenicity of BC cells. ZSCAN1-overexpressing BCSLCs exhibited weakened stemness properties. Normal human mammary epithelial (HMLE) cells with ZSCAN1 depletion exhibited enhanced stemness properties. Mechanistic studies showed that ZSCAN1 directly binds to -951 ~ -925bp region of WWTR1 (encodes TAZ) promoter, inhibits WWTR1 transcription, thereby inhibiting the stemness of BCSCs. Our work thus revealed ZSCAN1 as a novel stemness-related tumor suppressor and transcriptional repressor in BC.

Upregulation of NUAK2: A novel prognostic marker in breast cancer.

Breast cancer is the most commonly diagnosed neoplasm in women worldwide. New molecular biomarkers and effective prognostic models are being developed. This study aimed to investigate the clinical and prognostic significance of NUAK2 expression in patients with breast cancer. The expression of NUAK 2 was examined in breast cancer cells and tissues by real-time PCR, western blotting, and immunohistochemical staining. CCK-8 and colony formation assays were performed to verify the effect of NUAK2 on the proliferation and tumor progression of breast cancer cells. A tumor formation assay in nude mice was performed to analyze the effect of NUAK2 on the tumorigenicity of breast cancer cells. The expression of NUAK2 in breast cancer tissues was higher than that in paracarcinoma and normal breast tissues. The overall survival of patients with high NUAK2 expression was significantly lower than that of patients with low NUAK2 expression. Multivariate analyses indicated that NUAK2 was an independent prognostic indicator of survival in breast cancer. In vitro experiments demonstrated that knocking down NUAK2 in breast cancer cells inhibited cell proliferation and tumor-forming ability, and overexpression of NUAK2 showed the opposite effects. NUAK2 overexpression promoted the tumorigenicity of breast cancer cells in vivo. These findings suggest that NUAK2 is involved in breast cancer development and progression. NUAK2 may be a valuable prognostic indicator in patients with breast cancer.

Non-tumorigenic epithelial breast cells and ionizing radiation cooperate in the enhancement of mesenchymal traits in tumorigenic breast cancer cells

AimsRadioresistance and recurrences are crucial hindrances in cancer radiotherapy. Fractionated irradiation can elicit a mesenchymal phenotype in irradiated surviving cells and a deep connection exists between epithelial mesenchymal transition, radioresistance, and metastasis. The aim of this study was to analyze the effect of the secretoma of irradiated non-tumorigenic mammary epithelial cells on surviving irradiated breast tumor cells regarding the gain of mesenchymal traits and migratory ability. Main methodsMDA-MB-231 and MCF-7 breast cancer cells, irradiated or not, were incubated with conditioned media from MCF-10A non-tumorigenic epithelial breast cells, irradiated or not. After five days, we evaluated the expression and localization of epithelial and mesenchymal markers (by western blot and indirect immunofluorescence), cell migration (using transwells) and metalloproteinases activity (by zymography). We also assessed TGF-β1 content in conditioned media by immunoblot, and the effect of A83-01 (a selective inhibitor of TGF-β receptor I) and PP2 (a Src-family tyrosine kinase inhibitor) on nuclear Slug and cell migration. Key findingsConditioned media from MCF-10A cells caused phenotypic changes in breast tumor cells with attainment or enhancement of mesenchymal traits mediated at least in part by the activation of the TGF-β type I receptor and a signaling pathway involving Src activation/phosphorylation. The effects were more pronounced mostly in irradiated tumor cells treated with conditioned media from irradiated MCF-10A. SignificanceOur results suggest that non-tumorigenic epithelial mammary cells included in the irradiation field could affect the response to irradiation of post-surgery residual cancer cells enhancing EMT progression and thus modifying radiotherapy efficacy.

Endotoxin Triggers Tumor Initiation Events in Nontumorigenic Breast Epithelial Cells and Enhances Invasion-Related Phenotype in Pretumorigenic and Tumorigenic Breast Epithelial Cells.

Inflammation is associated with the development of several cancers, including breast cancer. However, the molecular mechanisms driving breast cancer initiation or enhancement by inflammation are yet to be deciphered. Hence, we opted to investigate the role of inflammation in initiating and enhancing tumor-like phenotypes in nontumorigenic, pretumorigenic, and tumorigenic breast epithelial cells. Noncytotoxic endotoxin (ET) concentrations capable of inducing an inflammatory phenotype were determined for the different cell lines. Results showed that short-term ET exposure upregulated matrix metalloproteinase-9 (MMP-9) activity in nontumorigenic mammary epithelial cells of mouse (SCp2) and human origins (HMT-3522 S1; S1) and upregulated inflammatory mediators including nitric oxide (NO) and interleukin 1-β in tumorigenic human breast cells (MDA-MB-231), all in a dose-dependent manner. Long-term ET treatment, but not short-term, triggered the migration of SCp2 cells, and proliferation and migration of tumorigenic human breast cells MCF-7 and MDA-MB-231. Both short- and long-term ET exposures preferentially enhanced the invasion of pretumorigenic S1-connexin 43 knockout (Cx43-KO S1) cells compared to their nontumorigenic S1 counterparts. Moreover, both ET exposures disrupted lumen formation and apicolateral distribution of β-catenin in 3D cultures of S1 cells. In conclusion, ET treatment at concentrations that elicited inflammatory phenotype triggered tumor initiation events in nontumorigenic and pretumorigenic breast cells, and increased tumorigenicity of breast cancer cells. Our findings highlight the role of inflammation in enhancing migration, invasion, and loss of normal 3D morphology and suggest that such inflammatory insults can “add injury” to pretumorigenic and tumorigenic breast epithelial cells.

The Interleukin-6/Signal Transducer and Activator of Transcription-3/Cystathionine γ-Lyase Axis Deciphers the Transformation Between the Sensitive and Resistant Phenotypes of Breast Cancer Cells.

Drug resistance of cancer cells is associated with redox homeostasis. The mechanism of acquired resistance of cancer cells to antitumor drugs is not well understood. Our previous studies revealed that drug resistance and highly expressed P-glycoprotein (P-gp) of MCF-7 breast cancer cells was dependent on intracellular redox homeostasis and declined capacity for scavenging reactive oxygen species (ROS). Recently, we observed that, unlike nontumorigenic cells MCF-10A, three tumorigenic breast cancer cells (MCF-7S, BT474, MDA-MB-231) reprogrammed their metabolism, highly expressed cystathionine-γ-lyase (CTH), and acquired a particular specialty to use methionine (Met) to synthesize glutathione (GSH) through the transsulfuration pathway. Interestingly, doxorubicin (adriamycin) further reprogrammed metabolism of MCF-7 cells sensitive to adriamycin (MCF-7S) and induced them to be another MCF-7 cell line resistant to adriamycin (MCF-7R) with dramatically downregulated CTH. The two MCF-7 cell lines showed distinctly different phenotypes in terms of intracellular GSH, ROS levels, expression and activity of P-gp and CTH, and drug resistance. We showed that CTH modulation or the methionine supply brought about the interconversion between MCF-7S and MCF-7R. Methionine deprivation or CTH silencing induced a resistant MCF-7R and lowered paclitaxel activity, yet methionine supplementation or CTH overexpression reversed the above effects, induced a sensitive phenotype of MCF-7S, and significantly increased the cytotoxicity of paclitaxel both in vitro and in vivo. Interleukin-6 (IL-6)/signal transducer and activator of transcription-3 (STAT3) initiated CTH expression and activity, and the effect on the resistant phenotype was exclusively dependent on CTH and ROS. This study suggests that the IL-6/STAT3/CTH axis plays a key role in the transformation between sensitive and resistant MCF-7 cells. SIGNIFICANCE STATEMENT: Cystathionine γ-lyase (CTH) plays a key role in transformation between the sensitive and resistant phenotypes of MCF-7 cells and is dependent on the interleukin-6 (IL-6)/signal transducer and activator of transcription-3 (STAT3) signaling axis. Modulation of the transsulfuration pathway on CTH or IL-6/STAT3 or methionine supplementation is beneficial for reversing the resistance of MCF-7 cells, which indicates a clinical translation potential.

Adaptor SH3BGRL promotes breast cancer metastasis through PFN1 degradation by translational STUB1 upregulation.

Metastatic recurrence is still a major challenge in breast cancer treatment, but the underlying mechanisms remain unclear. Here, we report that a small adaptor protein, SH3BGRL, is upregulated in the majority of breast cancer patients, especially elevated in those with metastatic relapse, indicating it as a marker for the poor prognosis of breast cancer. Physiologically, SH3BGRL can multifunctionally promote breast cancer cell tumorigenicity, migration, invasiveness, and efficient lung colonization in nude mice. Mechanistically, SH3BGRL downregulates the acting-binding protein profilin 1 (PFN1) by accelerating the translation of the PFN1 E3 ligase, STUB1 via SH3BGRL interaction with ribosomal proteins, or/and enhancing the interaction of PFN1 with STUB1 to accelerate PFN1 degradation. Loss of PFN1 consequently contributes to downstream multiple activations of AKT, NF-kB, and WNT signaling pathways. In contrast, the forced expression of compensatory PFN1 in SH3BGRL-high cells efficiently neutralizes SH3BGRL-induced metastasis and tumorigenesis with PTEN upregulation and PI3K-AKT signaling inactivation. Clinical analysis validates that SH3BGRL expression is negatively correlated with PFN1 and PTEN levels, but positively to the activations of AKT, NF-kB, and WNT signaling pathways in breast patient tissues. Our results thus suggest that SH3BGRL is a valuable prognostic factor and a potential therapeutic target for preventing breast cancer progression and metastasis.

The association between Annexin A2 and epithelial cell adhesion molecule in breast cancer cells.

BackgroundThe epithelial cell adhesion molecule (EpCAM) is a type I transmembrane and glycosylated protein, which is overexpressed in many neoplasms. However, EpCAM has no known ligand partners and the mechanisms by which it functions are not fully understood.AimThis study was performed to discover novel partners of EpCAM, which may provide a better understanding of its functions.MethodsThe membrane fraction of the ERα+ noninvasive breast cancer cell line ZR‐75‐1 and MCF‐7 was extracted and followed by co‐immunoprecipitation of EpCAM using C‐10, a mouse monoclonal antibody raised against amino acids 24–93 of the EpCAM molecule. As a negative control, MDA‐MB‐231 and Hs578T were used since they express a negligible amount of EpCAM and are known as EpCAM−/low ERα−/low invasive and tumorigenic breast cancer cell lines.ResultsAnnexin A2 (ANXA2) was found to be selectively and differentially co‐immunoprecipitated with EpCAM in the ERα+ breast cancer cells MCF‐7 and ZR‐75‐1. ANXA2 is a multifunctional protein and known to act as a co‐receptor for tissue plasminogen activator (tPA) on the surface of endothelial and cancer cells, thereby affecting fibrinolytic activity and neoangiogenesis as well as invasive and metastatic properties. In this study, the association between EpCAM and ANXA2 was found to affect the activity of tPA.ConclusionThis study concludes that ANXA2 co‐localizes with EpCAM at the plasma membrane, and the co‐localization may have functional implications. Data suggest that EpCAM supports ANXA2 to function as a co‐receptor for the tPA, and that EpCAM has a regulatory function on the expression and subcellular localization of ANXA2.

LncRNA MALAT1/miR‑26a/26b/ST8SIA4 axis mediates cell invasion and migration in breast cancer cell lines.

Metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) is a long non-coding RNA that is overexpressed in various human cancers, including breast cancer. Evidence has associated the function of the α-2,8-sialyltransferase (ST8SIA) family with breast cancer. The present study aimed to investigate the potential roles of MALAT1 in breast cancer development and progression using analyses of both breast cancer tissues and cell lines. The mRNA levels of MALAT1, microRNA (miR)-26a/26b and ST8SIA4 were detected by reverse transcription-quantitative PCR (RT-qPCR) and the protein level of ST8SIA4 was assessed by western blot analysis. Cell proliferation, invasion and migration were detected by CCK-8, wound healing and Transwell assays, respectively. Interactions between MALAT1 and miR-26a/26b were assessed using fluorescence in situ hybridization, RNA immunoprecipitation and luciferase reporter assays. Herein, different levels of MALAT1 were primarily observed in human breast cancer samples and cells. Upregulated MALAT1 was a crucial predictor of poor breast cancer prognosis. Altered MALAT1 modulated cell progression in breast cancer. Moreover, miR-26a/26b was confirmed as a direct regulator of MALAT1, and ST8SIA4 was predicted as a target of miR-26a/26b. Functional analysis in human breast cancer cell lines demonstrated that MALAT1 modulated breast cancer cell tumorigenicity by acting as a competing endogenous lncRNA (ceRNA) to regulate ST8SIA4 levels by sponging miR-26a/26b. The identification of the MALAT1/miR-26a/26b/ST8SIA4 axis which contributes to breast cancer progression may constitute a potential new therapeutic target.

Silencing of KIF18B restricts proliferation and invasion and enhances the chemosensitivity of breast cancer via modulating Akt/GSK-3β/β-catenin pathway.

Kinesin family member 18B (KIF18B) is a new tumor-associated protein that contributes to the carcinogenesis of multiple malignancies. However, the detailed relevance of KIF18B in breast cancer has not been fully elucidated. This work aimed was to evaluate a possible relationship between KIF18B and breast cancer progression. Our findings show KIF18B is increased in breast cancer and demonstrate that high KIF18B level predicts a reduced survival rate. Cellular functional studies revealed that knockdown of KIF18B markedly reduces the proliferation, invasion, and epithelial-mesenchymal transition of breast cancer cells and enhances their chemosensitivity toward doxorubicin. Further studies showed that KIF18B modulates the level of phospho-Akt, phospho-glycogen synthase kinase-3β, and β-catenin. Notably, suppression of Akt abolished KIF18B-overexpression-induced increases in activation of Wnt/β-catenin pathway. In addition, re-expression of β-catenin reversed KIF18B-silencing-induced cancer-promoting effect. In vivo animal experiments elucidated that knockdown of KIF18B significantly weakened the tumorigenicity of breast cancer cells. Taken together, data of this study illustrate that KIF18B exerts a potential cancer-promoting function in breast cancer via enhancement of Wnt/β-catenin pathway through modulation of the Akt/GSK-3β axis.

Isolation and Establishment of a Highly Proliferative, Cancer Stem Cell-Like, and Naturally Immortalized Triple-Negative Breast Cancer Cell Line, KAIMRC2.

In vitro studies of a disease are key to any in vivo investigation in understanding the disease and developing new therapy regimens. Immortalized cancer cell lines are the best and easiest model for studying cancer in vitro. Here, we report the establishment of a naturally immortalized highly tumorigenic and triple-negative breast cancer cell line, KAIMRC2. This cell line is derived from a Saudi Arabian female breast cancer patient with invasive ductal carcinoma. Immunocytochemistry showed a significant ratio of the KAIMRC2 cells’ expressing key breast epithelial and cancer stem cells (CSCs) markers, including CD47, CD133, CD49f, CD44, and ALDH-1A1. Gene and protein expression analysis showed overexpression of ABC transporter and AKT-PI3Kinase as well as JAK/STAT signaling pathways. In contrast, the absence of the tumor suppressor genes p53 and p73 may explain their high proliferative index. The mice model also confirmed the tumorigenic potential of the KAIMRC2 cell line, and drug tolerance studies revealed few very potent candidates. Our results confirmed an aggressive phenotype with metastatic potential and cancer stem cell-like characteristics of the KAIMR2 cell line. Furthermore, we have also presented potent small molecule inhibitors, especially Ryuvidine, that can be further developed, alone or in synergy with other potent inhibitors, to target multiple cancer-related pathways.

MiR-4709-3p Inhibits Cell Proliferation by Downregulating TSP50 Expression in Breast Cancer Cells.

Breast cancer is a serious threat to the physical and mental health of women all over the world. Our previous results have shown that Serine protease 50 (TSP50), an oncogene overexpressed in breast cancer, can promote proliferation, migration, and invasion of breast cancer cells. Mechanistic studies have revealed that TSP50 promoted tumorigenesis mainly by activating NF-kappa B (NF-κB) and inhibiting activin signaling pathway, indicating that TSP50 played a critical role in the occurrence and development of breast cancer. However, there are few reports on the regulation of TSP50 expression in breast cancer. MicroRNAs (miRNAs) have emerged as an essential posttranscriptional regulator in gene expression and they played a significant role in breast cancer regulation. In the present study, bioinformatics software miRBase and TargetScan were first used to predict and analyze miRNAs that could target TSP50 mRNA 3'UTR and six miRNAs were found. Results from quantitative real-time PCR (qRT-PCR) and western blot suggested that miR-4709-3p could bind to TSP50 mRNA 3'UTR and significantly inhibit the expression of TSP50 protein. Moreover, the effects of miR-4709-3p on the proliferation of breast cancer cells and mammary epithelial cells were detected in vitro. Our data suggested that overexpression of miR-4709-3p mimic greatly inhibited the proliferation of breast cancer cells, whereas overexpression of miR-4709-3p inhibitors significantly promoted the proliferation of breast epithelial cells. Furthermore, the effect of miR-4709-3p on the tumorigenicity of breast cancer cells in vivo was tested, and the results showed that miR-4709-3p significantly reduced the volume and weight of tumor in nude mice. All these results suggested that miR-4709-3p could inhibit the tumorigenesis of breast cancer cells by targeting TSP50. Finally, the underlying molecular mechanisms were investigated and we found that both NF-κB and activin signaling were involved in miR-4709-3p-related tumor inhibitory 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.

Spindle and kinetochore‑associated complex subunit 3 accelerates breast cancer cell proliferation and invasion through the regulation of Akt/Wnt/β-catenin signaling.

Spindle and kinetochore‑associated complex subunit 3 (SKA3) has recently been identified as a novel regulator of carcinogenesis in multiple types of cancers. However, the function and potential regulatory mechanisms of SKA3 in breast cancer remain poorly understood. The present study was designed to gain a detailed relevance of SKA3 in breast cancer. Expression of SKA3 in breast cancer was examined via real-time quantitative PCR, western blotting and immunohistochemistry analysis. Malignant behaviors of breast cancer cells were investigated via cell counting kit-8, cell apoptosis, and transwell invasion assays. The activity of Wnt/β-catenin signaling was monitored via luciferase reporter assay. The tumorigenicity of breast cancer cells in vivo was assessed via xenograft tumor assay. SKA3 expression was elevated in breast cancer tissue and was correlated with shorter survival rates in breast cancer patients. Knockdown of SKA3 caused marked reductions in cellular proliferation and invasion in breast cancer cells, whereas SKA3 overexpression accelerated proliferation and invasion. Knockdown of SKA3 resulted in decreased Akt and glycogen synthase kinase-3β phosphorylation, and decreased expression of active β-catenin, which lead to the inactivation of Wnt/β-catenin signaling. Inhibition of Akt significantly reversed the SKA3 overexpression-induced activation of Wnt/β-catenin signaling. Inhibition of Wnt/β-catenin signaling markedly abrogated SKA3 overexpression-induced tumor-promotion effects, while re-activation of Wnt/β-catenin signaling significantly reversed SKA3 knockdown-mediated tumor-inhibition effects. Knockdown of SKA3 resulted in a significant decrease in breast cancer tumor formation in vivo. SKA3 accelerates proliferation and invasion in breast cancer through the modulation of Akt/Wnt/β-catenin signaling.

EZH2 overexpression dampens tumor-suppressive signals via an EGR1 silencer to drive breast tumorigenesis.

The mechanism underlying EZH2 overexpression in breast cancer and its involvement in tumorigenesis remain poorly understood. In this study, we developed an approach to systematically identify the trans-acting factors regulating the EZH2 expression, and identified more than 20 such factors. We revealed reciprocal regulation of early growth response 1 (EGR1) and EZH2: EGR1 activates the expression of EZH2, and EZH2 represses EGR1 expression. Using CRISPR-mediated genome/epigenome editing, we demonstrated that EHZ2 represses EGR1 expression through a silencer downstream of the EGR1 gene. Deletion of the EGR1 silencer resulted in reduced cell growth, invasion, tumorigenicity of breast cancer cells, and extensive changes in gene expression, such as upregulation of GADD45, DDIT3, and RND1; and downregulation of genes encoding cholesterol biosynthesis pathway enzymes. We hypothesize that EZH2/PRC2 acts as a "brake" for EGR1 expression by targeting the EGR1 silencer, and EZH2 overexpression dampens tumor-suppressive signals mediated by EGR1 to drive breast tumorigenesis.

Upregulation of CCT-3 Induces Breast Cancer Cell Proliferation Through miR-223 Competition and Wnt/β-Catenin Signaling Pathway Activation.

The clinical significance and the function of chaperonin-containing TCP1 complex 3 (CCT-3) in breast cancer remain unknown. In this study, we found that CCT-3 was markedly overexpressed in breast cancer tissues. Statistical analysis revealed a significant correlation of CCT-3 expression with advanced breast cancer clinical stage and poorer survival. Ablation of CCT-3 knocked down the proliferation and the tumorigenicity of breast cancer cells in vitro and in vivo. CCT-3 may regulate breast cancer cell proliferation through a ceRNA network between miR-223 and β-catenin, thus affecting Wnt/β-catenin signaling pathway activation. We also validated that CCT-3 and β-catenin are novel direct targets of tumor suppressor miR-223. Our results suggest that both mRNA and the protein levels of CCT-3 are potential diagnosis biomarkers and therapeutic targets for breast cancer.

Effect of SALL4 on the Proliferation, Invasion and Apoptosis of Breast Cancer Cells.

Objective:We aimed to identify the expression of Sal-like 4 (SALL4) in breast cancer tissues and to explore the role of this gene in the carcinogenesis of breast cancer cells.Methods:A total of 62 paired breast cancer and noncancerous tissue samples were obtained from patients with breast cancer. SALL4 expression patterns and their association with clinicopathological characteristics were investigated by qRT-PCR, western blotting, and immunochemistry in breast cancer tissues. After the knockdown of SALL4 by short hairpin RNAs (shRNAs), the proliferative, invasive, and apoptotic abilities of MDA-MB-435 and MDA-MB-468 cells (breast cancer cell lines) were measured by colony formation and CCK-8 assays, wound healing and transwell assays, and flow cytometry, respectively.Results:SALL4 expression was higher in breast cancer tissues than that in the paired noncancerous tissues, and increased SALL4 expression in tumor tissues was closely related to tumor size and lymphatic metastasis. Furthermore, functional experiments revealed that SALL4 knockdown inhibited the cell proliferation, induced cell cycle arrest in G0/G1phase and apoptosis, and decreased the ability of migration and invasion in breast cancer cells. Additionally, our study first demonstrated that SALL4 played a critical role in modulating the tumorigenicity of breast cancer cells via the WNT/β-catenin signaling pathway.Conclusions:Our results suggest that the expression of SALL4 is upregulated in breast cancer, and this upregulation is involved in the regulation of cell growth, invasion, and apoptosis. Hence, SALL4 may be a promising target for diagnosis and therapy in patients with breast cancer.