Retracted: Glabridin attenuates the migratory and invasive capacity of breast cancer cells by activating microRNA‐200c

Current treatments for breast cancer, a common malignancy in human females, are less than satisfactory because of high rates of metastasis. Glabridin (GLA), which acts through the FAK/ROS signaling pathway, has been used as an antioxidant and anti‐metastatic agent. However, little is known regarding the effect of microRNA (miRNA) on GLA's anti‐metastatic activity. The miRNA‐200 family, which is frequently expressed at low levels in triple negative breast cancers, inhibits metastasis by blocking the epithelial–mesenchymal transition. Here, we found that GLA attenuated the migratory and invasive capacity of breast cancer cells by activating miR‐200c. GLA induced the mesenchymal–epithelial transition in vitro and in vivo, as determined by increased expression of the epithelial marker, E‐cadherin, and decreased expression of the mesenchymal marker, vimentin. Overexpression of miR‐200c enhanced the expression of E‐cadherin and decreased the expression of vimentin. Furthermore, in MDA‐MB‐231 and BT‐549 breast cancer cells exposed to GLA, knockdown of miR‐200c blocked the GLA‐induced mesenchymal–epithelial transition and alleviated the GLA‐induced inhibition of migration and invasion. Thus, elevation of miR‐200c by GLA has considerable therapeutic potential for anti‐metastatic therapy for breast cancer patients.     

MiR‐200c inhibits autophagy and enhances radiosensitivity in breast cancer cells by targeting UBQLN1

Radioresistance is a major challenge during the treatment of breast cancer. A further understanding of the mechanisms of radioresistance could provide strategies to address this challenge. In our study, we compared the expression of miR‐200c in four distinct breast cancer cell lines: two representative basal cancer cells (MDA‐MB‐231 and BT549) vs. two representative luminal cancer cells (MCF‐7 and BT474). The results revealed practically lower expression of miR‐200c in the two basal cancer cell lines and higher expression of miR‐200c in luminal cancer cells compared to the normal breast epithelial cell line MCF‐10A. Ectopic expression of miR‐200c in MDA‐MB‐231 cells inhibited irradiation‐induced autophagy and sensitized the breast cancer cells to irradiation. We also identified UBQLN1 as a direct functional target of miR‐200c involved in irradiation‐induced autophagy and radioresistance. In 35 human breast cancer tissue samples, we detected an inverse correlation between the expression of miR‐200c vs. UBQLN1 and LC3. These results indicate that the identified miR‐200c/UBQLN1‐mediated autophagy pathway may help to elucidate radioresistance in human breast cancer and might represent a therapeutic strategy.     

The tumor suppressor microRNA‐29c is downregulated and restored by celecoxib in human gastric cancer cells

MicroRNAs (miRNAs) are small noncoding RNAs that function as endogenous silencers of target genes and play critical roles during carcinogenesis. The selective cyclooxygenase‐2 (COX‐2) inhibitor celecoxib has been highlighted as a potential drug for treatment of gastrointestinal tumors. The aim of this study was to investigate the role of miRNAs in gastric carcinogenesis and the feasibility of a new therapeutic approach for gastric cancer. miRNA expression profiles were examined in 53 gastric tumors including gastric adenomas (atypical epithelia), early gastric cancers and advanced gastric cancers and in gastric cancer cells treated with celecoxib. miRNA microarray analysis revealed that miR‐29c was significantly downregulated in gastric cancer tissues relative to nontumor gastric mucosae. miR‐29c was significantly activated by celecoxib in gastric cancer cells. Downregulation of miR‐29c was associated with progression of gastric cancer and was more prominent in advanced gastric cancers than in gastric adenomas and early gastric cancer. In addition, expression of the oncogene Mcl‐1, a target of miR‐29c, was significantly increased in gastric cancer tissues relative to nontumor gastric mucosae. Activation of miR‐29c by celecoxib induced suppression of Mcl‐1 and apoptosis in gastric cancer cells. These results suggest that downregulation of the tumor suppressor miR‐29c plays critical roles in the progression of gastric cancer. Selective COX‐2 inhibitors may have clinical promise for the treatment of gastric cancer via restoration of miR‐29c.     

miR‐212 is downregulated and suppresses methyl‐CpG‐binding protein MeCP2 in human gastric cancer

To clarify the role of micro (mi) RNAs in gastric carcinogenesis, we studied the expression and function of miRNAs in gastric carcinoma (GC) cells. Initially, we performed microarray analysis using total RNA from 3 human GC cell lines and noncancerous gastric tissue. Among the downregulated miRNAs in GC cells, miR‐212 expression was decreased in all 8 GC cell lines examined and a significant decrease of miR‐212 expression in human primary GC tissues was also observed in 6 of 11 cases. Transfection of the precursor miR‐212 molecule induced decreased growth of 3 GC cell lines. Using 3 different databases, methyl‐CpG‐binding protein MeCP2 was postulated to be a target of miR‐212. As seen on reporter assaying, miR‐212 repressed the construct with the MECP2 3′‐UTR. Ectopic expression of miR‐212 repressed expression of the MeCP2 protein but not the MECP2 mRNA level. These data suggest that downregulation of miR‐212 may be related to gastric carcinogenesis through its target genes, such as MECP2.     

E‐cadherin‐Fc chimera protein matrix enhances cancer stem‐like properties and induces mesenchymal features in colon cancer cells

Cancer stem cells (CSC) are a subpopulation of tumor cells with properties of high tumorigenicity and drug resistance, which lead to recurrence and poor prognosis. Although a better understanding of CSC is essential for developing cancer therapies, scarcity of the CSC population has hindered such analyses. The aim of the present study was to elucidate whether the E‐cadherin‐Fc chimera protein (E‐cad‐Fc) enhances cancer stem‐like properties because studies show that soluble E‐cadherin stimulates human epithelial growth factor receptor (EGFR) and downstream signaling pathways that are reported to play a crucial role in CSC. For this purpose, we used ornithine decarboxylase (ODC)‐degron–transduced (Degron(+)) KM12SM cells as a CSC model that retains relatively low CSC properties. Compared to cultures without E‐cad‐Fc treatment, we found that E‐cad‐Fc treatment further suppressed proteasome activity and largely enhanced cancer stem‐like properties of ODC‐degron–transduced KM12SM cells. These results include increased expression of stem cell markers Lgr5, Bmi‐1, SOX9, CD44, and CD44v9, aldehyde dehydrogenase (ALDH), and enhancement of robust spheroid formation, and chemoresistance to 5‐fluorouracil (5‐FU) and oxaliplatin (L‐OHP). These effects could be attributed to activation of the EGFR pathway as identified by extensive phosphorylation of EGFR, ERK, PI3K, AKT, and mTOR. In SW480 cells, E‐cad‐Fc matrix induced some CSC markers such as CD44v9 and ALDH. We also found that E‐cad‐Fc matrix showed high efficiency of inducing mesenchymal changes in colon cancer cells. Our data suggest that the E‐cad‐Fc matrix may enhance CSC properties such as enhancement of chemoresistance and sphere formation.     

Epidermal growth factor down‐regulates the expression of neutrophil gelatinase‐associated lipocalin (NGAL) through E‐cadherin in pancreatic cancer cells

BACKGROUND:

The authors previously reported that neutrophil gelatinase‐associated lipocalin (NGAL) overexpression significantly blocked invasion and angiogenesis of pancreatic ductal adenocarcinoma (PDAC). They also demonstrated a loss of NGAL expression in the advanced stages of PDAC. However, little is known regarding the mechanisms of NGAL regulation in PDAC. Because the epidermal growth factor (EGF)‐EGF receptor (EGFR) axis is up‐regulated significantly in PDAC, they examined EGF‐mediated NGAL regulation in these cells.

METHODS:

The NGAL‐positive cell lines AsPC‐1 and BxPC‐3 were used as a model system. Quantitative real‐time polymerase chain reaction (RT‐PCR), Western blot analysis, and immunofluorescence studies were used to investigate EGF‐mediated effects on NGAL expression. E‐cadherin expression was manipulated using lentiviral overexpression or small hairpin RNA constructs. NGAL promoter activity was assessed by luciferase‐reporter assay and electrophoretic mobility shift assay.

RESULTS:

NGAL expression was positively associated with tumor differentiation and was down‐regulated significantly after EGF treatment along with a concomitant reduction of E‐cadherin expression in PDAC cells. E‐cadherin down‐regulation was partly through the EGFR‐dependent mitogen‐activated protein kinase (MEK)/extracellular signal‐regulated kinase (ERK) (MEK‐ERK) signaling pathway. In addition, E‐cadherin down‐regulation reduced NGAL expression in PDAC cells, whereas overexpression of E‐cadherin led to increased NGAL expression and partly rescued the inhibition of NGAL expression by EGF. Furthermore, EGF, in part through E‐cadherin, reduced NGAL promoter activity by blocking nuclear factor κB (NF‐κB) activation.

CONCLUSIONS:

The current study demonstrated for the first time that EGF potently blocked NGAL expression in PDAC cells. This effect was mediated in part through activation of the EGFR‐MEK‐ERK signaling pathway, which, in turn, down‐regulated E‐cadherin with a subsequent reduction in NF‐κB activation. These findings illustrate a novel mechanism by which EGF regulates NGAL expression in PDAC. Cancer 2011;. © 2010 American Cancer Society.     

Alterations of microRNAs and their targets are associated with acquired resistance of MCF‐7 breast cancer cells to cisplatin

Cancer cells that develop resistance to chemotherapeutic agents are a major clinical obstacle in the successful treatment of breast cancer. Acquired cancer chemoresistance is a multifactorial phenomenon, involving various mechanisms and processes. Recent studies suggest that chemoresistance may be linked to drug‐induced dysregulation of microRNA function. Furthermore, mounting evidence indicates the existence of similarities between drug‐resistant and metastatic cancer cells in terms of resistance to apoptosis and enhanced invasiveness. We studied the role of miRNA alterations in the acquisition of cisplatin‐resistant phenotype in MCF‐7 human breast adenocarcinoma cells. We identified a total of 103 miRNAs that were overexpressed or underexpressed (46 upregulated and 57 downregulated) in MCF‐7 cells resistant to cisplatin. These differentially expressed miRNAs are involved in the control of cell signaling, cell survival, DNA methylation and invasiveness. The most significantly dysregulated miRNAs were miR‐146a, miR‐10a, miR‐221/222, miR‐345, miR‐200b and miR‐200c. Furthermore, we demonstrated that miR‐345 and miR‐7 target the human multidrug resistance‐associated protein 1. These results suggest that dysregulated miRNA expression may underlie the abnormal functioning of critical cellular processes associated with the cisplatin‐resistant phenotype.     

p0071 interacts with E‐cadherin in the cytoplasm so as to promote the invasion and metastasis of non‐small cell lung cancer

As a member of the p120‐catenin (p120ctn) subfamily, the p0071 study in tumor is very limited. We demonstrated the clinicopathological significance of p0071 in non‐small cell lung cancer (NSCLC), as well as E‐cadherin. Co‐immunoprecipitation was used to detect the interaction of p0071 with E‐cadherin in A549 and SPC cells (E‐cadherin is mainly expressed in the cytoplasm of these cells). p0071 cytoplasmic expression was knocked down by siRNA in these cells and this effect on the RhoA activity and cell invasion and migration ability were measured. p0071 overexpression in the cytoplasm of tumor cell was correlated with lymphatic metastase and poor prognosis of NSCLC. The patients with both abnormal expression of p0071 and E‐cadherin (cytoplasmic expression) had a statistically significant shorter survival than the patients without both abnormal expression (P < 0.05). There is a significant correlation between cytoplasmic overexpression of p0071 and E‐cadherin in NSCLC tissues. p0071 interacted with E‐cadherin in the cytoplasm of A549 and SPC cell lines. Treatment with siRNA‐p0071 inhibited the invasion and migration ability of NSCLC cells. Above results confirmed that p0071 interacted with E‐cadherin in the cytoplasm so as to promote the invasion and metastasis of NSCLC.     

Significance of P‐cadherin overexpression and possible mechanism of its regulation in intrahepatic cholangiocarcinoma and pancreatic cancer

It has become evident that P‐cadherin, one of the classical cadherins, contributes to the malignant behavior of several types of cancer. In this study, we analyzed the expression of P‐cadherin and its clinicopathological and prognostic values in intrahepatic cholangiocarcinoma (ICC) and pancreatic cancer. Furthermore, we investigated the functional role of P‐cadherin in these cancer cells by knockdown and overexpression in vitro and by analyzing the correlation between the P‐cadherin expression and its promoter methylation status. Thirty of 59 ICC cases (51%) and 36 of 73 pancreatic cancer cases (49%) stained positive for P‐cadherin with mainly membranous distribution in tumor cells by immunohistochemistry. P‐cadherin expression was significantly correlated with several clinicopathological factors, which reflect tumor behavior, and was identified as an independent adverse prognostic factor for disease‐free survival in patients with ICC (relative risk [RR] 2.93, P = 0.04) and pancreatic cancer (RR 2.68, P = 0.005) via multivariate analyses. P‐cadherin downregulation by siRNA suppressed migration and invasion, and P‐cadherin overexpression induced the opposite effects in both ICC and pancreatic cancer cells, without any effects on cell proliferation. P‐cadherin expression was related to its promoter methylation status in both cell lines and cancer tissues. In summary, P‐cadherin overexpression may serve as a useful biomarker of invasive phenotype and poor prognosis; P‐cadherin expression was found to be regulated by its promoter methylation. These results suggest that P‐cadherin represents a novel therapeutic target for the treatment of ICC and pancreatic cancer.