Cyanidin-3-O-glucoside alleviates ethanol-induced liver injury by promoting mitophagy in a Gao-binge mouse model of alcohol-associated liver disease.

BACKGROUND: Alcohol-associated liver disease (ALD) is a leading cause of liver disease-related deaths worldwide. Unfortunately, approved medications for the treatment of this condition are quite limited. One promising candidate is the anthocyanin, Cyanidin-3-O-glucoside (C3G), which has been reported to protect mice against hepatic lipid accumulation, as well as fibrosis in different animal models. However, the specific effects and mechanisms of C3G on ALD remain to be investigated. EXPERIMENTAL APPROACH: In this report, a Gao-binge mouse model of ALD was used to investigate the effects of C3G on ethanol-induced liver injury. The mechanisms of these C3G effects were assessed using AML12 hepatocytes. RESULTS: C3G administration ameliorated ethanol-induced liver injury by suppressing hepatic oxidative stress, as well as through reducing hepatic lipid accumulation and inflammation. Mechanistically, C3G activated the AMPK pathway and enhanced mitophagy to eliminate damaged mitochondria, thus reducing mitochondria-derived reactive oxidative species in ethanol-challenged hepatocytes. CONCLUSIONS: The results of this study indicate that mitophagy plays a potentially important role underlying the hepatoprotective action of C3G, as demonstrated in a Gao-binge mouse model of ALD. Accordingly, C3G may serve as a promising, new therapeutic drug candidate for use in ALD.

LINC00173 silence and estrone supply suppress ER+ breast cancer by estrogen receptor α degradation and LITAF activation.

Persistent activation of estrogen receptor alpha (ERα)-mediated estrogen signaling plays a pivotal role in driving the progression of estrogen receptor positive (ER+) breast cancer (BC). In the current study, LINC00173, a long non-coding RNA, was found to bind both ERα and lipopolysaccharide (LPS)-induced tumor necrosis factor alpha (TNFα) factor (LITAF), then cooperatively to inhibit ERα protein degradation by impeding the nuclear export of ERα. Concurrently, LITAF was found to attenuate TNFα transcription after binding to LINC00173, and this attenuating transcriptional effect was quite significant under lipopolysaccharide stimulation. Distinct functional disparities between estrogen subtypes emerge, with estradiol synergistically promoting ER+ BC cell growth with LINC00173, while estrone (E1) facilitated LITAF-transcriptional activation. In terms of therapeutic significance, silencing LINC00173 alongside moderate addition of E1 heightened TNFα and induced apoptosis, effectively inhibiting ER+ BC progression.

EGR3 and estrone are involved in the tamoxifen resistance and progression of breast cancer.

BACKGROUND: Tamoxifen (Tam) is an effective treatment for estrogen receptor (ER) positive breast cancer. However, a significant proportion of patients develop resistance under treatment, presenting a therapeutic challenge. The study aims to determine the role of early growth response protein (EGR) 3 in tamoxifen resistance (TamR) and elucidate its molecular mechanism. METHODS: TamR cell models were established and NGS was used to screening signaling alternation. Western blot and qRT-PCR were used to analysis the expression of ERα, EGR3, MCL1 and factors associated with apoptosis. CCK8, colony formation and apoptosis assay were used to analysis resistance to Tam. Immunofluorescence, chromatin immunoprecipitation, and dual luciferase assays were used to investigate mechanism of regulation. RESULTS: We observed that EGR3, a deeply rooted ERα response factor, showed increased upregulation in response to both estrone (E1) and Tam in TamR cells with elevated level of E1 and ERα expression, indicating a potential connection between EGR3 and TamR. Mechanically, manipulating EGR3 expression revealed that it imparted resistance to Tam through increased expression of the downstream molecule MCL1 (apoptosis suppressor gene) that it regulated. Mechanismly, EGR3 directly binds to the promoter of the anti-apoptotic factor MCL1 gene, facilitating its transcription. Furthermore, apoptosis assays revealed that E1 reduces Tam induced apoptosis by upregulating EGR3 expression. Importantly, clinical public database confirmed the high expression of EGR3 in breast cancer tissue and in Tam-treated patients. CONCLUSIONS: These findings shed light on the novel estrogen/EGR3/MCL1 axis and its role in inducing TamR in ER positive breast cancer. EGR3 emerges as a promising target to overcome TamR. The elucidation of this mechanism holds potential for the development of new therapeutic modalities to overcome endocrine therapy resistance in clinical settings.

MCT4/Lactate Promotes PD-L1 Glycosylation in Triple-Negative Breast Cancer Cells.

Triple-negative breast cancer (TNBC) has the highest percentage of lymphocytic infiltration among breast cancer subtypes, and TNBC patients may benefit from anti-PD-1/PD-L1 immunotherapy. However, some cases whether the immune checkpoint blockade (ICB) shows low targeting efficiency have occurred and effective synergistic targets need to be found, which inspired our exploration of the co-expression analysis of MCT4 (SLC16A3) and PD-L1 (CD274) and their potential regulatory mechanisms. After bioinformatic analysis of the relationship between MCT4 and PD-L1, we validated their positive co-expression relationship in triple-negative breast cancer through multiple immunohistochemical staining (mIHC), CRISPR/Cas9, and lentiviral transduction for MCT4 knockout (sgMCT4/231 KO) or overexpression (pEGFP-N1-MCT4/231). We examined the effect of lactate treatment on PD-L1 expression in triple-negative breast cancer cells by qRT-PCR and Western blot. Combined with our results, we found that MCT4 positively regulated PD-L1 expression through discharging lactate and stabilized PD-L1 through promoting its glycosylation by the classic WNT pathway in MDA-MB-231 cells. More importantly, the high co-expression of MCT4 and PD-L1 appears to predict more effective targets for treating TNBC, which would improve immune checkpoint therapy for TNBC.

Glucagon-like peptide-1 receptor activation by liraglutide promotes breast cancer through NOX4/ROS/VEGF pathway.

AIMS: Scientific evidence imply the strong correlation between diabetes and breast cancer. Glucagon-like peptide-1 (GLP-1) and its analogue liraglutide, have been widely used for diabetes treatment. However, the role of GLP-1 receptor (GLP-1R) in breast cancer requires further elucidation. This study aimed to investigate the risk and the molecular mechanisms of liraglutide using in breast cancer. MATERIALS AND METHODS: Quantitative real-time polymerase chain reaction, western blot or immunohistochemistry were used to detect the expressions of GLP-1R, NADPH oxidase 4 (NOX4) and vascular endothelial growth factor (VEGF) in human triple negative breast cancer (TNBC) cells (MDA-MB-231 and MDA-MB-468) and tissues derived from BALB/cfC3H mouse bearing 4T1 cells inoculation. Cell proliferation and migration was detected using the Cell Counting Kit-8, adenosine triphosphate assay, and transwell assay, respectively. Flow cytometry was used to measure the level of reactive oxygen species (ROS). KEY FINDINGS: We found that the expression of GLP-1R increased after liraglutide treatment in breast cancer cells and the transplanted tumors. Liraglutide, at a slightly higher concentration, accelerated breast cancer progress in vitro (100 nM) and in vivo (400µg/kg) through the NOX4/ROS/VEGF signal pathway after activating GLP-1R. The GLP-1R inhibitor, Exendin (9-39), significantly inhibited the effect of liraglutide, inducing a reversed function of GLP-1R activation. SIGNIFICANCE: Our study illustrated that in an approximately toxicology context, liraglutide may promote the malignant progression of TNBC. The dosage and the phenotype of the breast cancer should be considered as important factors for the rational administration of antidiabetic drugs, especially that of liraglutide in breast cancer patients.

ZEB1 induces ROS generation through directly promoting MCT4 transcription to facilitate breast cancer.

PURPOSE: Transcription factor zinc finger E-box binding homeobox 1(ZEB1) was well-known as a transcription factor in epithelial-mesenchymal transition (EMT) process of cancer, but little is known about its role in cancer metabolism. We found metabolism-related gene monocarboxylate transporter 4 (MCT4) contained E-box motifs in its promoter region, which is the potential binding site of ZEB1. Thus, the correlation between ZEB1 and MCT4 was also investigated in this study. METHODS: Two human breast cancer cell lines MDA-MB-231 and MCF7 were used for ZEB1 and MCT4 expression by double fluorescence staining, ChIP as well as luciferase reporter. ROS levels were revealed by DCFDA and DHE fluorescence probes. The role of ZEB1/MCT4/ROS/autophagy was also determined in xenograft tumor mice model. RESULTS: MCT4 and ZEB1 were synchronously expressed in two types of breast cancer cells. Moreover, ZEB1 positively regulated the expression and the function of MCT4 through binding to the E-box motifs in MCT4' promoter. In addition, the in vitro and in vivo experiments showed that ZEB1/MCT4 in synergy promoted the growth of breast cancer through ROS generation and autophagy, which can be reversed by a MCT4 inhibitor, 7ACC1. CONCLUSION: ZEB1 directly binds to E-box elements of MCT4 promoter and enhance MCT4 expression, inducing ROS accumulation, which cooperatively resulting in breast cancer growth and shorten survival. Our findings provide a theoretical basis for interfering the metabolism in breast cancer therapeutics.

Blocking MCT4 SUMOylation inhibits the growth of breast cancer cells.

Monocarboxylate transporter 4 (MCT4) is highly expressed in various types of solid neoplasms including breast cancer (BC); however, the pro-tumor functions underlying its increased expression have not been explained. Here, we examined the roles of posttranslational modifications to MCT4 in BC, particularly SUMOylation. Our findings revealed that SUMOylation of MCT4 inhibited its degradation and stabilized MCT4 protein levels, while ubiquitination facilitated MCT4 degradation. The E3 ubiquitin ligases ß-TRCP and FBW7 interacted with MCT4 at the DSG-box and TPETS sequences, respectively, and Lys448 (K448) of MCT4 could be modified by SUMO chains. Our key finding was that K448 was crucial for MCT4 SUMOylation. Moreover, mutations of K448 abolished MCT4 expression, delaying the growth of BC. This study suggested that SUMOylation of K448 increased MCT4 levels, and mutations of K448 in MCT4 could have therapeutic significance in BC.

ZEB1 directly inhibits GPX4 transcription contributing to ROS accumulation in breast cancer cells.

PURPOSE: Prior studies have noted that zinc finger E-box binding homeobox 1 (ZEB1) is a master transcription regulator, affecting the expression of nearly 2000 genes in breast cancer cells, especially in the epithelial-mesenchymal transition (EMT) process. We now tested the role of ZEB1 on the oxidative stress of cancer cells and explored its possible mechanisms. METHODS: Two human breast cancer cell lines MDA-MB-231 and MCF7 were selected for the ROS test, PCR, immunofluorescence, Western blot, chromatin immunoprecipitation assay, luciferase assay, and enzyme assay. Mouse models experiments and bioinformatics analysis were conducted to test the indicated molecules. RESULTS: We observed ZEB1 could inhibit GPX4 transcription by binding to the E-box motifs and promote breast cancer progression by accumulating intracellular ROS. From the perspective of ROS clearance, Vitamin E enhanced GPX4 function to consume L-glutathione and eliminated excess intracellular ROS. CONCLUSIONS: ZEB1 could not only regulate EMT, but also inhibit GPX4 transcription by binding to the E-box motif. It was important to note that the ZEB1/GPX4 axis had a therapeutic effect on breast cancer metabolism.

[Design of Detection Module in Coagulometers Based on Dual-magnetic Circuit Beads Method].

Coagulometer, known as blood coagulation analyzer, is a product that can provide accurate test results for medical diagnosis and treatment analysis by detecting a series of items closely related to thrombosis and hemostasis in coagulation reaction. On the basis of previous traditional methods, and with our deep understanding about the principles of hemagglutination detection, we propose a hemagglutination detection method by using the dual-magnetic circuit beads method. Then, the corresponding hemagglutination detection module is designed. The coagulation time of plasma can be measured by detecting the movement of the magnetic beads when the magnetic field intensity is appropriate. The activated partial thromboplastin time(APTT) of plasma is tested when the most suitable magnetic field intensity is found. The results preliminarily show that this blood coagulation test method is valid and the corresponding test module has a potential value in business.

Downregulation of caveolin-1 promotes murine breast cancer cell line progression by highly glycosylated CD147.

Caveolin-1 (CAV-1) can extensively regulate lipid transportation, cell growth and cell death. In the present study, we revealed a novel function of CAV-1 in inhibiting glycosylation of other molecules in murine breast cancer cell line. After the silencing of CAV-1, we found that the mRNA and protein expressions of cluster of differentiation 147 (CD147) and its related molecules (MCT4, matrix metalloproteinase MMP2 and MMP9) increased in the breast cancer cells. Meanwhile, the migration and invasion of the breast cancer cells were significantly enhanced assessed by cell wound healing experiment and transwell assays. Further, the gelatin zymography and lactate assay in the cells also showed the strengthened enzyme activity of MMP9 and the increased extracellular lactate concentration, respectively, after the silencing of CAV-1. Notably, the glycosylation level of CD147 overtly increased after the inhibition of CAV-1 detected by Western Blot analysis, whereas upregulation of CAV-1 did the opposite. Therefore, the findings suggest that the downregulation of CAV-1 can promote breast cancer cell progression probably by highly glycosylated CD147.

Interleukin-23 derived from CD16+ monocytes drives IL-17 secretion by TLR4 pathway in children with mycoplasma pneumoniae pneumonia.

AIMS: The study aimed to investigate whether IL-23 is amplified in monocyte subsets of MP pneumonia and to determine its relevant pathway. MATERIALS AND METHODS: We firstly analyze the IL-23p19 expression in monocyte subgroups in MP pneumonia patients and healthy controls subjects by using flow cytometry. Then, we also analyzed the percentage of IL-17+γδT cells and Th17 cells in patients with MP pneumonia and controls subjects. At the same time, the relation between IL-23 and IL-17 were also assessed. Furthermore, we constructed the recombinant community-acquired respiratory distress syndrome (CARDS) toxin and intend to stimulate peripheral blood mononuclear cells and RAW264.7 cells in vitro. IL-23p19 was detected by flow cytometry and the mRNA levels were measured by real-time PCR. Finally, TLR4 pathway was also investigated by TAK242 inhibitor. KEY FINDINGS: It turned out that the expression of IL-23p19 was increased in CD14brightCD16+ monocyte of MP pneumonia patients than controls subjects. The patients with MP pneumonia had significantly higher the percentage of IL-17+γδT cells and Th17 cells than controls subjects. Interestingly, the levels of IL-23 were positively related to IL-17 in MP pneumonia patients. CD16+ monocytes and RAW264.7 cells, respectively can be induced by CARDS toxin to secrete IL-23 by TLR4 pathway in vitro. SIGNIFICANCE: These results indicated that IL-23-IL-17+γδT/Th17 axis may play a role in the pathogenesis of MP pneumonia, whereas IL-23 derived from CD16+ monocytes was expanded in MP pneumonia by TLR4 pathway.

Activation of ß2-Adrenergic Receptor Promotes Growth and Angiogenesis in Breast Cancer by Down-regulating PPARγ.

PURPOSE: Chronic stress and related hormones are key in cancer progression. Peroxisome proliferator-activated receptor γ (PPARγ) and its agonists was reported that inducing anti-tumor effect. However, the function of PPARγ in pro-tumorigenic effects induced by chronic stress in breast cancer remains unknown. Herein, we have characterized a novel role of PPARγ and vascular endothelial growth factor (VEGF)/fibroblast growth factor 2 (FGF2) signals in breast cancer promoted by chronic stress. MATERIALS AND METHODS: We performed experiments in vivo and in vitro and used bioinformatics data to evaluate the therapeutic potential of PPARγ in breast cancer promoted by stress. RESULTS: Chronic stress significantly inhibited the PPARγ expression and promoted breast cancer in vivo. VEGF/FGF2-mediated angiogenesis increased in the chronic stress group compared to the control group. PPARγ agonist pioglitazone (PioG) injection offset the pro-tumorigenic effect of chronic stress. Moreover, specific ß2-adrenergic receptor (ß2R) antagonist ICI11-8551 inhibited the effect of chronic stress. In vitro, norepinephrine (NE) treatment had a similar tendency to chronic stress. The effect of NE was mediated by the ß2R/adenylate cyclase signaling pathway and suppressed by PioG. PPARγ suppressed VEGF/FGF2 through reactive oxygen species inhibition. Bioinformatics data confirmed that therewas a lowPPARγ expression in breast invasive carcinoma. Lower PPARγ was associated with a significantly worse survival. CONCLUSION: ß2R activation induced by chronic stress and related hormones promotes growth and VEGF/FGF2-mediated angiogenesis of breast cancer by down-regulating PPARγ. Our findings hint that ß receptor and PPARγ as two target molecules and the novel role for their agonists or antagonists as clinical medicine in breast cancer therapy.

The PI3K/Akt/GSK-3ß/ROS/eIF2B pathway promotes breast cancer growth and metastasis via suppression of NK cell cytotoxicity and tumor cell susceptibility.

OBJECTIVE: To examine the effect of pSer9-GSK-3ß on breast cancer and to determine whether the underlying metabolic and immunological mechanism is associated with ROS/eIF2B and natural killer (NK) cells. METHODS: We employed TWS119 to inactivate GSK-3ß by phosphorylating Ser9 and explored its effect on breast cancer and NK cells. The expression of GSK-3ß, natural killer group 2 member D (NKG2D) ligands, eIF2B was quantified by PCR and Western blot. We measured intracellular reactive oxygen species (ROS) and mitochondrial ROS using DCFH-DA and MitoSOXTM probe, respectively, and conducted quantitative analysis of cellular respiration on 4T1 cells with mitochondrial respiratory chain complex I/III kits. RESULTS: Our investigation revealed that TWS119 downregulated NKG2D ligands (H60a and Rae1), suppressed the cytotoxicity of NK cells, and promoted the migration of 4T1 murine breast cancer cells. Nevertheless, LY290042, which attenuates p-GSK-3ß formation by inhibiting the PI3K/Akt pathway, reversed these effects. We also found that higher expression of pSer9-GSK-3ß induced higher levels of ROS, and observed that abnormality of mitochondrial respiratory chain complex I/III function induced the dysfunction of GSK-3ß-induced electron transport chain, naturally disturbing the ROS level. In addition, the expression of NOX3 and NOX4 was significantly up-regulated, which affected the generation of ROS and associated with the metastasis of breast cancer. Furthermore, we found that the expression of pSer535-eIF2B promoted the expression of NKG2D ligands (Mult-1 and Rae1) following by expression of pSer9-GSK-3ß and generation of ROS. CONCLUSIONS: The PI3K/Akt/GSK-3ß/ROS/eIF2B pathway could regulate NK cell activity and sensitivity of tumor cells to NK cells, which resulted in breast cancer growth and lung metastasis. Thus, GSK-3ß is a promising target of anti-tumor therapy.

IDO1 impairs NK cell cytotoxicity by decreasing NKG2D/NKG2DLs via promoting miR-18a.

BACKGROUND: Indoleamine-2,3-dioxygenase 1 (IDO1) is an important enzyme for altering the tumour microenvironment and assisting tumour cells to escape the immune system. RESULTS: In this study, a significant reduction in NK cell cytotoxicity that was associated with a high expression of IDO1 in a reconstructed tumour microenvironment was observed. In a co-culture system of tumour cell culture supernatant (TSN) and murine NK cell, IDO1 was substantially increased, while NKG2D was markedly downregulated in NK cells. Based on computational predictions, miR-18a, which has two definite binding sites consisting of the 3'UTR of NKG2D and the 3'UTR of NKG2D ligand (Mult-1), was suspected to be a negative regulator of which its conjoined. As expected, the IDO1 could promote the expression of miR-18a and promote the downregulation effect of miR-18a on NKG2D and NKG2DL, and INCB024360 (INCB) could reverse the result. For digging the mechanism deeper, we authenticated IDO1 promoted the combination of miR-18a and AGO2 after argonaute 2 (AGO2) co-immunoprecipitation, which then degraded Mult-1 mRNA and inhibited the translation of it, further destructing NK cell cytotoxicity. CONCLUSION: Our findings revealed a new regulatory axis, IDO1/miR-18a/NKG2D/NKG2DL, in the regulation of NK cell function. This may provide insight into the mechanism of the priming effect of IDO1 inhibitors and miR-18a interference, then elicit possible new methods of cancer treatment.

Downregulation of MCT4 for lactate exchange promotes the cytotoxicity of NK cells in breast carcinoma.

Monocarboxylate transporter-4 (MCT4), a monocarboxylic acid transporter, demonstrates significantly increased expression in the majority of malignancies. We performed an experiment using BALB/C mice, and our results showed that ShMCT4 transfection or the pharmaceutic inhibition of MCT4 with 7acc1 strengthens the activity of NK cells. The results of a calcein assay revealed that the cytotoxicity of NK cells was strengthened via inhibition of MCT4. In addition, ELISA testing showed that the content of perforin and CD107a was increased, and PCR amplification and immunoblotting revealed that the expression of NKG2D and H60 was upregulated after the inhibition of MCT4. Further, we observed an elevated pH value, decreased extracellular lactate flow, and attenuated tumor growth. Therefore, we concluded that the inhibition of MCT4 enhanced the cytotoxicity of NK cells by blocking lactate flux and reversing the acidified tumor microenvironment. In addition to these findings, we also discovered that MCT4 depletion may have a pronounced impact on autophagy, which was surmised by observing that the inhibition of autophagy (3MA) pulled the enhanced cytotoxicity of NK cells downwards. Together, these data suggest that the key effect of MCT4 depletion on NK cells probably utilizes inductive autophagy as a compensatory metabolic mechanism to minimize the acidic extracellular microenvironment associated with lactate export in tumors.

miR-21a negatively modulates tumor suppressor genes PTEN and miR-200c and further promotes the transformation of M2 macrophages.

miR-21a is well-known to inhibit PTEN expression. We have previously shown that PTEN suppressed the transformation of M2 macrophages in the tumor microenvironment. Therefore, we hypothesized that miR-21a could influence M2 macrophage transformation by regulating PTEN expression. In this study, we identified how miR-21a reduced the expression of both PTEN mRNA and protein in murine macrophage cell lines and primary macrophages. Moreover, opposite effects were identified upon the use of a miR-21a inhibitor. Using a cytokine array, we identified the cytokines closely associated with miR-21a-mediated macrophage transformation to the M2 phenotype. miR-21a mimics could also enhance the migratory ability of murine breast cancer 4T1 cells, the growth of breast cancer in vivo and CD206 intratumor expression. In addition, quantitative PCR (qPCR) and methylation-specific PCR analysis showed that miR-21a enhanced miR-200c methylation and then decreased miR-200c and PTEN expression. These effects could be reversed by treatment with 5'-Aza, a DNA-demethylating agent. MiR-200c was predicted to target the PTEN 3'UTR, but qPCR illustrated the miR-200c mimic that increased PTEN expression, and 5'-Aza could enhance its effect. The above results indicate that miR-21a negatively modulates two tumor suppressor genes, miR-200c and PTEN, thereby promoting M2 macrophage transformation. This demonstrates that miR-21a represents a novel target for improving the overall tumor microenvironment.

Autophagy suppresses isoprenaline-induced M2 macrophage polarization via the ROS/ERK and mTOR signaling pathway.

The objective of this study was to examine the effect of autophagy on stress-induced M2 macrophage polarization in the tumor microenvironment of breast cancer and to determine whether the underlying mechanism was related to the reactive oxygen species (ROS)/ERK and mTOR pathway. In vitro, we found that the basal autophagy level in mouse RAW 264.7 macrophages decreased with the incubation of tumor cell culture supernatant. Similarly, the polarization of RAW 264.7 to M2 macrophages was inhibited by the autophagy inducer rapamycin and increased by the autophagy inhibitor 3-MA or by siBeclin1. In addition, we found that not only was M2 molecule expression down-regulated but intracellular ROS generation was also blocked by autophagy induction. In vivo, we observed that mice that received an isoprenaline injection as a stress agent exhibited augmented implanted breast tumor growth, lung metastasis, intratumoral mRNA expression of M2 molecules and serum ROS generation. In contrast, the intratumoral expression of LC3-II and Beclin1 was decreased. In addition, we observed that isoprenaline induced the up-regulation of the intratumoral expression of phosphorylated mTOR, phosphorylated ERK1/2, phosphorylated Tyr705-STAT3 and HIF-1α, whereas rapamycin induced an opposite effect on the same molecules and could abolish the effects of isoprenaline. These results suggest that autophagy might suppress M2 macrophage polarization induced by isoprenaline via the ROS/ERK and mTOR signaling pathway. Our findings provide a theoretical basis for why high levels of stress hormones accelerate the progression of breast cancer, and autophagy may play a role in determining the outcomes of cancer therapy.

The influence of miR-34a expression on stemness and cytotoxic susceptibility of breast cancer stem cells.

In this study, we investigate the effect of miR-34a expression and biological characteristics of breast cancer stem cells (BCSCs). The mammospheres were formed from murine breast cancer cell line 4T1 and regarded as murine BCSCs. Identification of stemness molecules and cloning experiments validate the biological characteristics of BCSCs we have established. We showed that miR-34a, as a tumor suppressor, could separately reduce the stemness of BCSCs and activate the cytotoxic susceptibility of BCSCs to natural killer (NK) cells in vitro via down regulating the expression of Notch1 signaling molecules. Moreover, miR-34a could completely restrain established mice breast tumor xenografts in vivo in the NOD/SCID mice that have functional NK cells at a normal level, whereas it was less effective in NOD/SCID/ CD122/IL-2Rß mice that do not have functional NK cells. We conclude that miR-34a is a crucial, dual tumor suppressor and BCSCs-targeting immunotherapeutic agent and has shown efficacy in the treatment of murine breast cancer. The results also suggest that impaired NK cells could contribute to the resistance to therapies.

Adrenergic receptor ß2 activation by stress promotes breast cancer progression through macrophages M2 polarization in tumor microenvironment.

Stress and its related hormones epinephrine (E) and norepinephrine (NE) play a crucial role in tumor progression. Macrophages in the tumor microenvironment (TME) polarized to M2 is also a vital pathway for tumor deterioration. Here, we explore the underlying role of macrophages in the effect of stress and E promoting breast cancer growth. It was found that the weight and volume of tumor in tumor bearing mice were increased, and dramatically accompanied with the rising E level after chronic stress using social isolation. What is most noteworthy, the number of M2 macrophages inside tumor was up-regulated with it. The effects of E treatment appear to be directly related to the change of M2 phenotype is reproduced in vitro. Moreover, E receptor ADRß2 involved in E promoting M2 polarization was comprehended simultaneously. Our results imply psychological stress is influential on specific immune system, more essential for the comprehensive treatment against tumors.

PTEN inhibits macrophage polarization from M1 to M2 through CCL2 and VEGF-A reduction and NHERF-1 synergism.

PTEN has been studied in several tumor models as a tumor suppressor. In this study, we explored the role of PTEN in the inhibition state of polarized M2 subtype of macrophage in tumor microenvironment (TME) and the underlying mechanisms. To elucidate the potential effect in TME, RAW 264.7 macrophages and 4T1 mouse breast cancer cells were co-cultured to reconstruct tumor microenvironment. After PTEN was down-regulated with shRNA, the expression of CCL2 and VEGF-A, which are definited to promote the formation of M2 macrophages, have a dramatically increase on the level of both gene and protein in co-cultured RAW 264.7 macrophages. And at the same time, NHERF-1 (Na(+)/H(+) exchanger regulating factor-1), another tumor suppressor has a similar tendency to PTEN. Q-PCR and WB results suggested that PTEN and NHERF-1 were consistent with one another no matter at mRNA or protein level when exposed to the same stimulus. Coimmunoprecipitation and immunofluorescence techniques confirmed that PTEN and NHERF-1 were coprecipitated, and NHERF-1 protein expression was properly reduced with rCCL2 effect. In addition, cell immunofluorescence images revealed a profound transferance, in co-cultured RAW 264.7 macrophages, an up-regulation of NHERF-1 could promote the PTEN marked expression on the cell membrane, and this form for the interaction was not negligible. These observations illustrate PTEN with a certain synergy of NHERF-1, as well as down-regulation of CCL2 suppressing M2 macrophage transformation pathway. The results suggest that the activation of PTEN and NHERF-1 may impede the evolution of macrophages beyond the M1 into M2 phenotype in tumor microenvironment.