Long non‐coding RNA ZEB2‐AS1 promotes the proliferation,metastasis and epithelial mesenchymal transition in triple‐negative breast cancer by epigenetically activating ZEB2

The triple‐negative breast cancer is the most malignant type of breast cancer. Its pathogenesis and prognosis remain poor despite the significant advances in breast cancer diagnosis and therapy. Meanwhile, long noncoding RNAs (LncRNAs) play a pivotal role in the progression of malignant tumors. In this study, we found that LncRNA‐ZEB2‐AS1 was dramatically up‐regulated in our breast cancer specimens and cells (MDA231), especially in metastatic tumor specimens and highly invasive cells, and high lncRNA‐ZEB2‐AS1 expression is associated with clinicopathologic features and short survival of breast cancer patients. LncRNA‐ZEB2‐AS1 promotes the proliferation and metastasis of MDA231 cells in SCID mice. Thus, it is regarded as an oncogene in triple‐negative breast cancer. It is mainly endo‐nuclear and situated near ZEB2, positively regulating ZEB2 expression and activating the epithelial mesenchymal transition via the PI3K/Akt/GSK3β/Zeb2 signaling pathway. Meanwhile, EGF‐induced F‐actin polymerization in MDA231 cells can be suppressed by reducing lncRNA‐ZEB2‐AS1 expression. The migration and invasion of triple‐negative breast cancer can be altered through cytoskeleton rearrangement. In summary, we demonstrated that lncRNA‐ZEB2‐AS1 is an important factor affecting the development of triple‐negative breast cancer and thus a potential oncogene target.     

Aromatic‐turmerone attenuates invasion and expression of MMP‐9 and COX‐2 through inhibition of NF‐κB activation in TPA‐induced breast cancer cells

Recent evidence suggests that breast cancer is one of the most common forms of malignancy in females, and metastasis from the primary cancer site is the main cause of death. Aromatic (ar)‐turmerone is present in Curcuma longa and is a common remedy and food. In the present study, we investigated the inhibitory effects of ar‐turmerone on expression and enzymatic activity levels of 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA)‐induced matrix metalloproteinase (MMP)‐9 and cyclooxygenaase‐2 (COX‐2) in breast cancer cells. Our data indicated that ar‐turmerone treatment significantly inhibited enzymatic activity and expression of MMP‐9 and COX‐2 at non‐cytotoxic concentrations. However, the expression of tissue inhibitor of metalloproteinase (TIMP)‐1, TIMP‐2, MMP‐2, and COX‐1 did not change upon ar‐turmerone treatment. We found that ar‐turmerone inhibited the activation of NF‐κB, whereas it did not affect AP‐1 activation. Moreover, The ChIP assay revealed that in vivo binding activities of NF‐κB to the MMP‐9 and COX‐2 promoter were significantly inhibited by ar‐turmerone. Our data showed that ar‐turmerone reduced the phosphorylation of PI3K/Akt and ERK1/2 signaling, whereas it did not affect phosphorylation of JNK or p38 MAPK. Thus, transfection of breast cancer cells with PI3K/Akt and ERK1/2 siRNAs significantly decreased TPA‐induced MMP‐9 and COX‐2 expression. These results suggest that ar‐turmerone suppressed the TPA‐induced up‐regulation of MMP‐9 and COX‐2 expression by blocking NF‐κB, PI3K/Akt, and ERK1/2 signaling in human breast cancer cells. Furthermore, ar‐turmerone significantly inhibited TPA‐induced invasion, migration, and colony formation in human breast cancer cells. J. Cell. Biochem. 113: 3653–3662, 2012. © 2012 Wiley Periodicals, Inc.     

High-density array analysis of DNA methylation in Tamoxifen-resistant breast cancer cell lines

Roughly two-thirds of all breast cancers are ERα-positive and can be treated with the antiestrogen, Tamoxifen, however resistance occurs in 33% of women who take the drug for more than 5 y. Aberrant DNA methylation, an epigenetic mechanism that alters gene expression in cancer, is thought to play a role in this resistance. To develop an understanding of Tamoxifen-resistance and identify novel pathways and targets of aberrant methylation, DNA from MCF-7 breast cancer cells and Tamoxifen-resistant derivatives, TMX2–11 and TMX2–28, were analyzed using the Illumina HumanMethylation450 BeadChip. Normalizing against MCF-7 values, ERα-positive TMX2–11 had 4000 hypermethylated sites and ERα-negative TMX2–28 had over 33?000. Analysis of CpG sites altered in both TMX2–11 and TMX2–28 revealed that the Tamoxifen-resistant cell lines share 3000 hypermethylated and 200 hypomethylated CpGs. ZNF350 and MAGED1, two genes hypermethylated in both cell lines, were examined in greater detail. Treatment with 5-aza-2′deoxycitidine caused a significant reduction in promoter methylation of both ZNF350 and MAGED1 and a corresponding increase in expression in TMX2–28. A similar relationship between methylation and expression was not detected in TMX2–11. Our findings are indicative of the variable responses to methylation-targeted breast cancer therapy and highlight the need for biomarkers that accurately predict treatment outcome.     

Inhibitory effect of the carnosine–gallic acid synthetic peptide on MMP‐2 and MMP‐9 in human fibrosarcoma HT1080 cells

Matrix metalloproteinases (MMPs) are a family of zinc‐dependent endopeptidases that degrade extracellular matrix components and play important roles in a variety of biological and pathological processes such as malignant tumor metastasis and invasion. In this study, we constructed carnosine–gallic acid peptide (CGP) to identify a better MMP inhibitor than carnosine. The inhibitory effects of CGP on MMP‐2 and MMP‐9 were investigated in the human fibrosarcoma (HT1080) cell line. As a result, CGP significantly decreased MMP‐2 and MMP‐9 expression levels without a cytotoxic effect. Moreover, CGP may inhibit migration and invasion in HT1080 cells through the urokinase plasminogen activator (uPA)–uPA receptor signaling pathways to inhibit MMP‐2 and MMP‐9. Based on these results, it appears that CGP may play an important role in preventing and treating several MMP‐2 and MMP‐9‐mediated health problems such as metastasis. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.     

Down‐regulation of uPA and uPAR by 3,3′‐diindolylmethane contributes to the inhibition of cell growth and migration of breast cancer cells

3,3′‐Diindolylmethane (DIM) is a known anti‐tumor agent against breast and other cancers; however, its exact mechanism of action remains unclear. The urokinase plasminogen activator (uPA) and its receptor (uPAR) system are involved in the degradation of basement membrane and extracellular matrix, leading to tumor cell invasion and metastasis. Since uPA‐uPAR system is highly activated in aggressive breast cancer, we hypothesized that the biological activity of B‐DIM could be mediated via inactivation of uPA‐uPAR system. We found that B‐DIM treatment as well as silencing of uPA‐uPAR led to the inhibition of cell growth and motility of MDA‐MB‐231 cells, which was in part due to inhibition of VEGF and MMP‐9. Moreover, silencing of uPA‐uPAR led to decreased sensitivity of these cells to B‐DIM indicating an important role of uPA‐uPAR in B‐DIM‐mediated inhibition of cell growth and migration. We also found similar effects of B‐DIM on MCF‐7, cells expressing low levels of uPA‐uPAR, which was due to direct down‐regulation of MMP‐9 and VEGF, independent of uPA‐uPAR system. Interestingly, over‐expression of uPA‐uPAR in MCF‐7 cells attenuated the inhibitory effects of B‐DIM. Our results, therefore, suggest that B‐DIM down‐regulates uPA‐uPAR in aggressive breast cancers but in the absence of uPA‐uPAR, B‐DIM can directly inhibit VEGF and MMP‐9 leading to the inhibition of cell growth and migration of breast cancer cells. J. Cell. Biochem. 108: 916–925, 2009. © 2009 Wiley‐Liss, Inc.     

RhoA/ROCK pathway mediates the effect of oestrogen on regulating epithelial‐mesenchymal transition and proliferation in endometriosis

Endometriosis is a benign gynaecological disease appearing with pelvic pain, rising dysmenorrhoea and infertility seriously impacting on 10% of reproductive‐age females. This research attempts to demonstrate the function and molecular mechanism of RhoA/ROCK pathway on epithelial‐mesenchymal transition (EMT) and proliferation in endometriosis. The expression of Rho family was abnormally changed in endometriotic lesions; in particular, RhoA and ROCK1/2 were significantly elevated. Overexpression of RhoA in human eutopic endometrial epithelial cells (eutopic EECs) enhanced the cell mobility, epithelial‐mesenchymal transition (EMT) and proliferation, and RhoA knockdown exhibited the opposite function. Oestrogen up‐regulated the RhoA activity and expression of RhoA and ROCK1/2. RhoA overexpression reinforced the effect of oestrogen on promoting EMT and proliferation, and RhoA knockdown impaired the effect of oestrogen. oestrogen receptor α (ERα) was involved with the regulation of oestrogen on EMT and proliferation and up‐regulated RhoA activity and expression of RhoA and ROCK1/2. The function of ERα was modulated by the change in RhoA expression. Furthermore, phosphorylated ERK that was enhanced by oestrogen and ERα promoted the protein expression of RhoA/ROCK pathway. Endometriosis mouse model revealed that oestrogen enhanced the size and weight of endometriotic lesions. The expression of RhoA and phosphorylated ERK in mouse endometriotic lesions was significantly elevated by oestrogen. We conclude that abnormal activated RhoA/ROCK pathway in endometriosis is responsible for the function of oestrogen/ERα/ERK signalling, which promoted EMT and proliferation and resulted in the development of endometriosis.     

Role of GPER on proliferation,migration and invasion in ligand‐independent manner in human ovarian cancer cell line SKOV3

G protein‐coupled estrogen receptor (GPER) is identified as a critical estrogen receptor, in addition to the classical estrogen receptors ERα and ERβ. In ERα‐negative ovarian cancer cells, our previous studies have found that estrogen stimulated cell proliferation and metastasis via GPER. However, the ligand‐independent function of GPER in ovarian cancer cells is still not clear. Herein, we describe that GPER has a co‐expression with ERα and ERβ, which are first determined in SKOV3 ovarian cancer cell line. In the absence of estrogen, GPER depletion by specific siRNA inhibits the proliferation, migration and invasion of SKOV3 cells. Whereas abrogation of ERα or ERβ by specific antagonist MPP and PHTPP has the opposite effects for stimulation of cell growth. Markedly, GPER knockdown attenuates MPP or PHTPP‐induced cell proliferation, migration and invasion. Furthermore, GPER modulates protein expression of the cell cycle critical components, c‐fos and cyclin D1 and factors for cancer cell invasion and metastasis, matrix metalloproteinase 2 (MMP‐2) and MMP‐9. These findings establish that GPER ligand‐independently stimulates the proliferation, migration and invasion of SKOV3 cells. Knockdown of GPER attenuates the progression of ovarian cancer that caused by functional loss of ERα or ERβ. Targeting GPER provides new aspect as a potential therapeutic strategy in ovarian cancer. Copyright © 2015 John Wiley & Sons, Ltd.     

ODAM inhibits RhoA‐dependent invasion in breast cancer

Odontogenic ameloblast‐associated protein (ODAM) contributes to cell adhesion. In human cancer, ODAM is down‐regulated, and the overexpression of ODAM results in a favourable prognosis; however, the molecular mechanisms underlying ODAM‐mediated inhibition of cancer invasion and metastasis remain unclear. Here, we identify a critical role for ODAM in inducing cancer cell adhesion. ODAM induced RhoA activity and the expression of downstream factors, including Rho‐associated kinase (ROCK). ODAM‐mediated RhoA signalling resulted in actin filament rearrangement by activating PTEN and inhibiting the phosphorylation of AKT. When ODAM is overexpressed in MCF7 breast cancer cells and AGS gastric cancer cells that activate RhoA at high levels, it decreases motility, increases adhesion and inhibits the metastasis of MCF7 cells. Conversely, depletion of ODAM in cancer cells inhibits Rho GTPase activation, resulting in increased cancer migration and invasion. These results suggest that ODAM expression in cells maintains their adhesion, resulting in the prevention of their metastasis via the regulation of RhoA signalling in breast cancer cells. Copyright © 2015 John Wiley & Sons, Ltd. SIGNIFICANCE Breast cancer represents the first most frequent cancer, and the ratio of mortality is high in women. Of utmost importance for reducing risk by breast cancer are their anti‐invasion mechanisms, particularly in the non‐invasive cancer cells because metastasis is the principal cause of death among cancer patients. ODAM induced RhoA activity. ODAM‐mediated RhoA signalling resulted in actin filament rearrangement, increased cell adhesion and inhibited the migration/invasion of MCF7 cells. These results suggest that ODAM expression maintains their adhesion, resulting in the prevention of their metastasis via the regulation of RhoA signalling in breast cancer cells.     

Down‐regulating Myoferlin inhibits the vasculogenic mimicry of melanoma via decreasing MMP‐2 and inducing mesenchymal‐to‐epithelial transition

Vasculogenic mimicry (VM) constitutes a novel approach for tumour blood supply and contributes to tumour metastasis and poor prognosis in patients with melanoma. Myoferlin (MYOF), a type II membrane protein involved in membrane regeneration and repair, is elevated in several malignant tumours, especially in advanced melanomas. This study aims to investigate the role and mechanism of MYOF in the regulation of VM. VM structures were found in 14 of 52 tested melanoma samples, and high MYOF expression correlated with VM structures. According to Kaplan–Meier survival curves, VM channels and elevated MYOF expression both correlated with poor prognosis in melanoma patients. Down‐regulation of MYOF by siRNA severely impaired the capability of A375 cells to form VM structures in vitro. Further studies demonstrated MYOF knockdown inhibited cell migration and invasion, which is required for VM formation, via decreasing MMP‐2 expression as evidenced by Western blotting, RT‐RCP and ELISA results. SB‐3CT, a specific inhibitor of MMP‐2, showed similar inhibiting effects with siMYOF, further supporting that MYOF down‐regulation inhibits MMP‐2 expression to affect VM formation. Moreover, MYOF knockdown suppress VM formation by A375 cells by inducing mesenchymal‐to‐epithelial transition (MET). After down‐regulating MYOF, focal adhesions were enlarged and A375 cells developed into a clear epithelial morphology. Such cells acquired the expression of E‐cadherin at adherens junctions along with a loss of mesenchymal markers, such as Vimentin and Twist1. In conclusion, MYOF plays an important role in VM and knockdown of MYOF suppresses VM formation via decreasing MMP‐2 and inducing MET in A375 melanoma cells.     

miR‐655 suppresses epithelial‐to‐mesenchymal transition by targeting Prrx1 in triple‐negative breast cancer

Triple‐negative breast cancer (TNBC) is a highly aggressive breast cancer subtype that lacks effective targeted therapies. The epithelial‐to‐mesenchymal transition (EMT) is a key contributor in the metastatic process. In this study, we found that miR‐655 was down‐regulated in TNBC, and its expression levels were associated with molecular‐based classification and lymph node metastasis in breast cancer. These findings led us to hypothesize that miR‐655 overexpression may inhibit EMT and its associated traits of TNBC. Ectopic expression of miR‐655 not only induced the up‐regulation of cytokeratin and decreased vimentin expression but also suppressed migration and invasion of mesenchymal‐like cancer cells accompanied by a morphological shift towards the epithelial phenotype. In addition, we found that miR‐655 was negatively correlated with Prrx1 in cell lines and clinical samples. Overexpression of miR‐655 significantly suppressed Prrx1, as demonstrated by Prrx1 3′‐untranslated region luciferase report assay. Our study demonstrated that miR‐655 inhibits the acquisition of the EMT phenotype in TNBC by down‐regulating Prrx1, thereby inhibiting cell migration and invasion during cancer progression.     

CD147 regulates melanoma metastasis via the NFAT1‐MMP‐9 pathway

Although accumulating evidence had revealed that NFAT1 has oncogenic characteristics, the role of this molecule in melanoma cells remains unclear. Previous studies proved that CD147 plays a crucial function in melanoma cell metastasis and invasion through matrix metalloproteinase 9 (MMP‐9) expression; however, the details of how CD147 regulates MMP‐9 expression remain elusive. In this study, we demonstrated that CD147 and NFAT1 are overexpressed in the tissues of patients with primary and metastatic melanoma, which has shown a positive correlation. Further, we observed that CD147 regulates NFAT1 activation through the [Ca²⁺]i‐calcineurin pathway. Knockdown of NFAT1 significantly suppresses melanoma metastasis, and we demonstrated that CD147 affects melanoma metastasis in an NFAT1‐dependent manner. Moreover, we verified that NFAT1 directly binds to MMP‐9 promoter. Inhibition of CD147 expression significantly abrogates MMP‐9 promoter luciferase gene reporter activity as well as NFAT1 association with MMP‐9 promoter. Taken together, this study demonstrated that CD147 affects MMP‐9 expression through regulating NFAT1 activity and provided a novel mechanism by which NFAT1 contributes to melanoma metastasis through the regulation of MMP‐9.     

LIPH promotes metastasis by enriching stem‐like cells in triple‐negative breast cancer

Lipase member H (LIPH), a novel member of the triglyceride lipase family. The clinical implications of its expression in breast cancer are still unclear. Therefore, in this study, we investigated the associations between LIPH and the tumorigenic behaviours of 144 triple‐negative breast cancer (TNBC) patients. The ratio and mammosphere‐forming ability of CD44+/CD24? stem‐like cells were tested. The role of LIPH in breast cancer cell migration and invasion was also evaluated. In addition, the effect of LIPH silencing on mitochondrial respiration was determined using the Seahorse assay. Finally, the effect of LIPH silencing on protein expression was determined via tandem mass tag‐based spectrometry and Western blotting. We found that LIPH expression was associated with metastasis in lymph nodes and distant organs (P = 0.025), resulting in poor survival among breast cancer patients (P = 0.027). LIPH knockdown significantly decreased both the ratio of CD44⁺/CD24^? stem‐like cells and their mammosphere‐forming ability. LIPH silencing promoted apoptosis, arrested cell cycle in the G2/M phase, mitigated the oxidation‐related oxygen consumption rate in the mitochondria, and reduced metabolism. LIPH inhibited adhesion between tumour cells and enhanced the epithelial‐mesenchymal transition. Tandem mass spectrometric analysis presented 68 proteins were differentially expressed in LIPH‐silenced cells and LIPH‐mediated modulation of tumour cell adhesion depended on integrin‐related CAPN2 and paxillin signalling. Overall, our findings provided strong evidence that LIPH up‐regulation promoted metastasis and the stemness of TNBC cells. Therefore, targeting LIPH is a potentially viable strategy for preventing metastasis in TNBC.     

Alpha B‐crystallin promotes the invasion and metastasis of gastric cancer via NF‐κB‐induced epithelial‐mesenchymal transition

Alpha B‐crystallin (CRYAB) is overexpressed in a variety of cancers. However, little is known about its specific function and regulatory mechanism in gastric cancer. Here, we first explore the role of CRYAB in gastric cancer progression and metastasis. The expression of CRYAB was determined by western blot and immunohistochemistry in gastric cancer tissues. Besides, methods including stably transfected against CRYAB into gastric cancer cells, western blot, migration and invasion assays in vitro and metastasis assay in vivo were also conducted. The expression of CRYAB is up‐regulated in gastric cancer tissues compared with matched normal tissues. High expression level of CRYAB is closely correlated with cancer metastasis and shorter survival time in patients with gastric cancer. Additionally, CRYAB silencing significantly suppresses epithelial‐mesenchymal transition (EMT), migration and invasion of gastric cancer cells in vitro and in vivo, whereas CRYAB overexpression dramatically reverses these events. Mechanically, CRYAB facilitates gastric cancer cells invasion and metastasis via nuclear factor‐κ‐gene binding (NF‐κB)‐regulated EMT. These findings suggest that CRYAB expression predicts a poor prognosis in patients with gastric cancer. Besides, CRYAB contributes to gastric cancer cells migration and invasion via EMT, mediated by the NF‐κB signalling pathway, thus possibly providing a novel therapeutic target for gastric cancer.     

Estrogen receptor-alpha 36 mediates the anti-apoptotic effect of estradiol in triple negative breast cancer cells via a membrane-associated mechanism

17β-Estradiol can promote the growth and development of several estrogen receptor (ER)-negative breast cancers. The effects are rapid and non-genomic, suggesting that a membrane-associated ER is involved. ERα36 has been shown to mediate rapid, non-genomic, membrane-associated effects of 17β-estradiol in several cancer cell lines, including triple negative HCC38 breast cancer cells. Moreover, the effect is anti-apoptotic. The aim of this study was to determine if ERα36 mediates this anti-apoptotic effect, and to elucidate the mechanism involved. Taxol was used to induce apoptosis in HCC38 cells, and the effect of 17β-estradiol pre-treatment was determined. Antibodies to ERα36, signal pathway inhibitors, ERα36 deletion mutants, and ERα36-silencing were used prior to these treatments to determine the role of ERα36 in these effects and to determine which signaling molecules were involved. We found that the anti-apoptotic effect of 17β-estradiol in HCC38 breast cancer cells is in fact mediated by membrane-associated ERα36. We also showed that this signaling occurs through a pathway that requires PLD, LPA, and PI3K; Gαs and calcium signaling may also be involved. In addition, dynamic palmitoylation is required for the membrane-associated effect of 17β-estradiol. Exon 9 of ERα36, a unique exon to ERα36 not found in other identified splice variants of ERα with previously unknown function, is necessary for these effects. This study provides a working model for a mechanism by which estradiol promotes anti-apoptosis through membrane-associated ERα36, suggesting that ERα36 may be a potential membrane target for drug design against breast cancer, particularly triple negative breast cancer.