M2 Macrophage-Derived Exosomes Regulate miR-199a-3p Promoter Methylation Through the LINC00470-Mediated myc/DNMT3a Axis to Promote Breast Cancer Development.

Breast cancer (BC) is the most common invasive cancer in women. M2 macrophage exosomes promote cancer development and play multiple roles in the tumor microenvironment, but the mechanism of action by which M2 macrophage exosomes promote BC remains unclear. Therefore, the purpose of this study was to investigate the mechanism by which M2 macrophage-derived exosomes promote the development of breast cancer. We collected BC tissues and determined the expression of LINC00470, followed by the establishment of M2 macrophages in culture and the isolation and identification of M2 macrophage exosomes. Next, we investigated the effects of M2 macrophage exosomes on BC cell proliferation, invasion, miR-199a-3p promoter methylation, and the expression of LINC00470, myc, DNMT3A, and miR-199a-3p. Finally, LINC00470 expression was inhibited in M2 macrophage exosomes, while miR-199a-3p expression was inhibited in BC cells, and changes in BC cell proliferation, invasion, miR-199a-3p promoter methylation, and the expression of LINC00470, myc, DNMT3A, and miR-199a-3p were analyzed. We demonstrated that LINC00470 was highly expressed in BC tissues, M2-type macrophages were successfully induced in vitro, and Dil-labeled M2 macrophage exosomes could successfully enter MDA-MB-231 and MCF-7 cells. Coculture of M2 macrophage exosomes with MDA-MB-231 and MCF-7 cells significantly enhanced the proliferation and invasion of MDA-MB-231 and MCF-7 cells, upregulated the expression of LINC00470, myc, and DNMT3A and downregulated the expression of miR-199a-3p. Moreover, the inhibition of LINC00470 expression in M2 macrophage exosomes significantly downregulated the expression of LINC00470, myc, and DNMT3A in MDA-MB-231 and MCF-7 cells, upregulated the expression of miR-199a-3p, and hypomethylated the promoter of the miR-199a-3p locus. Moreover, inhibition of LINC00470 expression in M2 macrophage-derived exosomes significantly attenuated the proliferation and invasive ability of MDA-MB-231 and MCF-7 cells, while miR-199a-3p inhibitor transfection reversed this effect. Collectively, these findings indicated that M2-type macrophage-derived exosomes promote BC proliferation and migration by regulating miR-199a-3p promoter methylation through the LINC00470-mediated myc/DNMT3a axis.

Predicting Pathological Complete Response in Neoadjuvant Dual Blockade With Trastuzumab and Pertuzumab in HER2 Gene Amplified Breast Cancer.

Background: Dual-targeted therapy is the standard treatment for human epidermal growth factor receptor 2 (HER2)-positive breast cancer, and effective biomarkers to predict the response to neoadjuvant trastuzumab and pertuzumab treatment need further investigation. Here, we developed a predictive model to evaluate the dual-targeted neoadjuvant treatment efficacy in HER2 gene-amplified breast cancer. Method: This retrospective study included 159 HER2-amplified patients with locally advanced breast cancer who received neoadjuvant trastuzumab, pertuzumab, and chemotherapy. The correlation between clinicopathological factors and pathological complete response (pCR, in the breast and axilla) was evaluated. Patients were randomly assigned into the training set (n=110) and the testing set (n=49). We used an independent cohort (n=65) for external validation. We constructed our predictive nomogram model with the results of risk variables associated with pCR identified in the multivariate logistic analysis. The area under the curve (AUC) of the receiver operating characteristic (ROC) curve, decision curve analysis, and calibration curves were employed to assess the nomogram's performance. Results: We revealed that the HER2/CEP17 ratio (p=0.001), CD8 levels (p=0.005), and histological grade (p=0.007) were independent indicators for pCR in dual-targeted neoadjuvant treatment after multivariate adjustment. The combined prediction efficacy of the three indicators was significantly higher than that of each single indicator alone. The AUCs were 0.819, 0.773, and 0.744 in the training, testing, and external validation sets, respectively. Conclusions: The HER2/CEP17 ratio, CD8 levels, and histological grade were significantly correlated with pCR in dual-targeted neoadjuvant treatment. The combined model using these three markers provided a better predictive value for pCR than the HER2/CEP17 ratio, CD8 levels, and the histological grade alone, which showed that an immunological effect partially mediates the predictive impact of neoadjuvant treatment.

TRIM32 promotes radioresistance by disrupting TC45-STAT3 interaction in triple-negative breast cancer.

Radioresistance is common in the treatment of triple-negative breast cancer (TNBC), but the molecular mechanisms involved remain unclear. Herein, we reveal that tripartite motif-containing protein 32 (TRIM32) is upregulated in TNBC and is negatively associated with survival of TNBC patients. Radiotherapy resulted in enhanced expression of TRIM32, whereas TRIM32 depletion reduced TNBC radioresistance in vitro and in vivo. Mechanistically, radiotherapy promoted the association between TRIM32 and nuclear STAT3, which suppressed TC45-induced dephosphorylation of STAT3, resulting in increased STAT3 transcriptional activation and TNBC radioresistance. Finally, we demonstrated that TRIM32 and STAT3 phosphorylation are co-expressed in TNBC tissues. Moreover, high expression of TRIM32 and STAT3 phosphorylation is positively linked to poor prognosis of TNBC patients. Our study demonstrates that TRIM32 is a novel target for predicting radioresistance in TNBC patients.

lncRNA HEIH, an indicator of high malignancy and poor prognosis, functions as an oncogene in breast cancer.

Long non­coding RNA high expression in hepatocellular carcinoma (lncRNA HEIH) acts as an oncogene in multiple tumors, including hepatocellular carcinoma, colorectal cancer, melanoma and non­small cell lung cancer. However, the role of HEIH in breast cancer remains unknown. The present study focused on the clinical significance and biological function of HEIH in breast cancer. Specifically, the expression levels of HEIH in breast cancer tissues and breast cancer cell lines were investigated. The results indicated high expression levels of HEIH in human breast cancer tissues, and its expression was positively associated with malignancy status and poor disease prognosis. High expression levels of HEIH were detected in the breast cancer cell lines, including MCF­7, SK­BR­3, MDA­MB­231 and MDA­MB­468. These data were consistent with those derived from the in vivo study. Therefore, small interfering RNA was used to knockdown HEIH expression in order to explore whether HEIH exhibits an oncogenic function in breast cancer. Following HEIH knockdown, the proliferative and metastatic activity of MDA­MB­231 cells was decreased, whereas the induction of cell apoptosis was increased. These results suggested the oncogenic role of HEIH in breast cancer and the potential application of HEIH as an index of malignancy and poor prognosis in breast cancer.

Up-regulated lncRNA AFAP1-AS1 indicates a poor prognosis and promotes carcinogenesis of breast cancer.

BACKGROUND: Long noncoding RNAs (lncRNAs) have been reported to play crucial roles in breast cancer. This study aimed to determine the clinical significance and biological functions of lncRNA AFAP1-AS1 in breast cancer. METHODS: The expression of AFAP1-AS1 in breast cancer tissue and adjacent normal tissue from 160 patients and breast cancer cell lines were determined by qRT-PCR. The clinical characteristics of patients were collected to analyse the correlation between AFAP1-AS1 expression and malignancy status. Kaplan-Meier and Cox proportional hazards model were used to analyze whether AFAP1-AS1 expression impacted prognosis. To assess the effect of AFAP1-AS1 on MCF-7 cells proliferation, cell viability, EdU incorporation and colony formation assays were conducted after AFAP1-AS1 knockdown by siRNA. The apoptosis was detected by Caspase-3 activity, cell cycle analysis, Bcl-2 and Bax protein expression. Wound scratch assay and EMT-related protein expression (E-cadherin, N-cadherin and Vimentin) were conducted to evaluate the metastasis ability. To further determine the effect of AFAP1-AS1 on AFAP1, the mRNA and protein expression of AFAP1 and subsequent actin filament integrity were measured after AFAP1-AS1 knockdown. RESULTS: The expression of AFAP1-AS1 was up-regulated in human breast cancer tissue and associated with malignancy status, high expression of AFAP1-AS1 had a poor prognosis in breast cancer patients. AFAP1-AS1 expression was up-regulated in 4 breast cancer cell lines (MCF-7, SK-RB-3, MDA-MB-231and MDA-MB-468) compared with normal breast cell line HBL-100. MCF-7, the most up-regulation cancer cell, was used for following studies. AFAP1-AS1 knockdown can inhibit the proliferation, metastasis and promote apoptosis of MCF-7. However, the AFAP1 expression and actin filament integrity was not affected after AFAP1-AS1 knockdown. CONCLUSION: Up-regulated lncRNA AFAP1-AS1 indicates a poor prognosis in breast cancer patients and regulated the breast cancer cells proliferation, apoptosis and metastasis.

Comprehensive circular RNA profiling reveals the regulatory role of the circRNA-000911/miR-449a pathway in breast carcinogenesis.

Circular RNAs (circRNAs) are key regulators in the development and progression of human cancers; however their roles in breast tumorigenesis are not yet well understood. Thus, the present study aimed to investigate the expression profiles and potential modulatory effects of circRNAs on breast carcinogenesis. A human circRNA microarray analysis was performed to screen for abnormally expressed circRNAs in breast cancer tissue and circRNA-000911 was identified as a circRNA which was significantly downregulated in breast cancer cells. Mechanistic investigations suggested that the enhanced expression of circRNA-000911 suppressed cell proliferation, migration and invasion, and promoted the apoptosis of breast cancer cells. By using a biotin-labeled circRNA-000911 probe to perform RNA precipitation in breast cancer cells, we identified miR­449a as the circRNA­000911-associated microRNA. Gain- and loss-of-function assays indicated that miR­449a antagonized circRNA-000911 to regulate breast cancer progression. Subsequently, Notch1 was identified as the functional target of miR­449a, and the overexpression of circRNA-000911 in breast cancer elevated Notch1 expression. Furthermore, Cignal Signal Transduction Reporter Array and western blot analysis identified nuclear factor-κB (NF-κB) signaling as a functional target of the circRNA-000911/miR­449a pathway. On the whole, our findings indicate that circRNA-000911 plays an anti-oncogenic role in breast cancer and may thus serve as a promising therapeutic target for patients with breast cancer. Therefore, the overexpression of circRNA-000911 may provide a future direction which may aid in the development of a novel treatment strategy for breast cancer.

Matrine Suppresses the ER-positive MCF Cells by Regulating Energy Metabolism and Endoplasmic Reticulum Stress Signaling Pathway.

Matrine (C15 H24 N2 O), an alkaloid that is one of the main active components from Sophora flavescens. Matrine has been demonstrated to have therapeutic effects on various solid tumors, including breast cancer, but the mechanism still needs further study. Endoplasmic reticulum (ER)-positive Michigan Cancer Foundation cells were cultured, and matrine was added in various amounts to measure the dose-dependent and time-dependent cytotoxicity. Hoechst 33258 staining was used to observed nuclear morphological changes. Apoptosis was measured by AnnexinV/PI double staining assay kit. Intracellular adenosine triphosphate and glycometabolism were detected by assay kit. The protein levels GRP78, p-eIF2α, CHOP, cytochrome c, and HexokinaseII were analyzed. Mechanistic investigations revealed that matrine treatment causes ER dilation and up-regulated the expression of ER stress markers GRP78, eIF2α, and CHOP, increases the levels of apoptotic in Michigan Cancer Foundation cells, subsequently, blocking the ER stress-mediated apoptosis pathway, significantly decreased matrine-induced apoptotic but still has significant difference between control group. In addition, matrine not only promoted the occurrence of ER stress but also inhibited the expression of hexokinase II, down-regulated energy metabolism. In summary, the present study suggests that the induction of ER stress-mediated apoptosis by matrine and down-regulated energy metabolism may account for its cytotoxic effects in human breast cancer cells. Copyright © 2017 John Wiley & Sons, Ltd.

[Effects of miR-449a on proliferation and migration of human breast cancer cell line MCF-7].

OBJECTIVE: To study the effects of knockdown of miR-449a on the proliferation and migration of human breast cancer cell line Michigan Cancer Foundation-7 (MCF-7). METHODS: Using miRNA chip screening of the differential expressions of miRNA in human breast cancer cell MCF-7 and normal breast cells MCF-10A. The inhibitor of miR-449a was synthesized by chemical and detected by real-time PCR after transfection aimed to verify the expression. Cell Counting Kit-8 (CCK-8) assay was used to detect the ability of cell proliferation after transfection with miR-449a inhibitor. Scratch assay was used to detect cell migration of MCF-7, and cell invasion ability was showed by transwell assay; The MCF-7 cell proliferation and migration related proteins, ß-catenin and E-cadherin, were detected by Western blot. The potential target gene of miR-449a was predicted by bioinformatics software, and Notch homolog 1 (Notch 1) was proved to be the target gene of miR-449a by luciferase assay. RESULTS: MCF-7 and MCF-10a cells were collected separately, and miRNA chip results showed that the level of miR-449a in MCF-7 cells was significantly higher than that of MCF-10A. In this study, the cells were divided into mock group, negative control group (NC group) and treatment group, the MCF-7 cells were collected before and after treatment and CCK-8 results showed that knockdown of miR-449a decreased MCF-7 cell proliferation ability significantly. Scratch assay results showed that downregulated miR-449a was related to the decreased metastasis of MCF-7 cells. Transwell results showed that knockdown of miR-449a inhibited the invasion of MCF-7 cells. Western blot showed the expression of ß-catenin was decreased and the expression of E-cadherin was increased after knockdown of miR-449a. Luciferase assay showed that miR-449a could significantly decrease the luciferase activity of Notch homolog 1-untranslated region (Notch 1-3'-UTR) plasmid (P<0.01). CONCLUSIONS: Inhibition of miR-449a in breast cancer cell line MCF-7 can significantly inhibit the proliferation and migration of cancer cells, which may be achieved by decreasing the expression of Notch 1 protein.

Activation of BK(Ca) channels in zoledronic acid-induced apoptosis of MDA-MB-231 breast cancer cells.

BACKGROUND: Zoledronic acid, one of the most potent nitrogen-containing biphosphonates, has been demonstrated to have direct anti-tumor and anti-metastatic properties in breast cancer in vitro and in vivo. In particular, tumor-cell apoptosis has been recognized to play an important role in the treatment of metastatic breast cancer with zoledronic acid. However, the precise mechanisms remain less clear. In the present study, we investigated the specific role of large conductance Ca(2+)-activated potassium (BK(Ca)) channel in zoledronic acid-induced apoptosis of estrogen receptor (ER)-negative MDA-MB-231 breast cancer cells. METHODOLOGY/PRINCIPAL FINDINGS: The action of zoledronic acid on BK(Ca) channel was investigated by whole-cell and cell-attached patch clamp techniques. Cell apoptosis was assessed with immunocytochemistry, analysis of fragmented DNA by agarose gel electrophoresis, and flow cytometry assays. Cell proliferation was investigated by MTT test and immunocytochemistry. In addition, such findings were further confirmed with human embryonic kidney 293 (HEK293) cells which were transfected with functional BK(Ca) α-subunit (hSloα). Our results clearly indicated that zoledronic acid directly increased the activities of BK(Ca) channels, and then activation of BK(Ca) channel by zoledronic acid contributed to induce apoptosis in MDA-MB-231 cells. The possible mechanisms were associated with the elevated level of intracellular Ca(2+) and a concomitant depolarization of mitochondrial membrane potential (Δψm) in MDA-MB-231 cells. CONCLUSIONS: Activation of BK(Ca) channel was here shown to be a novel molecular pathway involved in zoledronic acid-induced apoptosis of MDA-MB-231 cells in vitro.