Mammary serine protease inhibitor inhibits epithelial growth factor‐induced epithelial‐mesenchymal transition of esophageal carcinoma cells

BACKGROUND:

By using proteomic technology, the authors previously observed the substantial down‐regulation of mammary serine protease inhibitor (maspin) in esophageal squamous cell carcinoma and metastases. In the current study, they examined the effects of maspin re‐expression in a maspin‐null esophageal cancer cell line EC109 and also investigated the underlying mechanism.

METHODS:

A cell line with stable maspin expression was established. An epithelial growth factor (EGF)‐induced epithelial‐mesenchymal transition (EMT) model was used to mimic some aspects of the metastatic process in vitro. The effects of maspin reintroduction on EGF‐induced EMT and cell growth characteristics were evaluated. Comparative proteomic analysis of transfected cells versus parental cells was then performed to explore the potential mechanism.

RESULTS:

The introduction of maspin into EC109 cells was able to inhibit EGF‐induced EMT and altered cell growth characteristics, including the serum dependence, proliferative response to EGF stimulation, and colony formation ability in soft agar, indicating a conversion from a malignant phenotype to a benign phenotype. Proteomic analysis revealed a significant down‐regulation of a group of glycolytic enzymes in maspin‐transfected cells. In addition, maspin‐transfected cells expressed much lower levels of hypoxia‐inducible factor 1α than parental cells or empty vector transfected cells.

CONCLUSIONS:

Maspin exhibited a metastasis‐suppressive effect, which may be a consequence of the reversal of the malignant phenotype of EC109 cells. The switch of cellular metabolic phenotype to low glycolysis by the gain of maspin function may play a key role in the process. This finding provides additional evidence of the tumor metastasis‐suppressive activity of maspin and may indicate a new direction for future studies of the mechanism of maspin. Cancer 2009. © 2008 American Cancer Society.     

Interaction of transforming growth factor‐β‐Smads/microRNA‐362‐3p/CD82 mediated by M2 macrophages promotes the process of epithelial‐mesenchymal transition in hepatocellular carcinoma cells

Abnormal tumor microenvironment and the epithelial‐mesenchymal transition (EMT) are important features of tumor metastasis. However, it remains unknown how signals can form complicated networks to regulate the sustainability of the EMT process. The aim of our study is to explore the possible interaction between tumor‐associated macrophages and tumor cells in the EMT process mediated by microRNA (miR)‐362‐3p. In this study, we found that by releasing TGF‐β, M2 macrophages mediate binding of Smad2/3 to miR‐362‐3p promoter, leading to overexpression of miR‐362‐3p. MicroRNA‐362‐3p maintains EMT by regulating CD82, one of the most important members of the family of tetraspanins. Our finding suggests that miR‐362‐3p can serve as a core factor mediating cross‐talk between the TGF‐β pathway in tumor‐associated macrophages and tetraspanins in tumor cells, and thus facilitates the EMT process.     

Extracellular ATP promotes breast cancer invasion and epithelial‐mesenchymal transition via hypoxia‐inducible factor 2α signaling

Extracellular ATP has been shown to play an important role in invasion and the epithelial‐mesenchymal transition (EMT) process in breast cancer; however, the mechanism is unclear. Here, by using a cDNA microarray, we demonstrated that extracellular ATP could stimulate hypoxia‐inducible factor (HIF) signaling and upregulate hypoxia‐inducible factor 1/2α (HIF‐1/2α) expression. After knocking down HIF‐1/2α using siRNA, we found that ATP‐driven invasion and EMT were significantly attenuated via HIF2A‐siRNA in breast cancer cells. By using ChIP assays, we revealed that the biological function of extracellular ATP in invasion and EMT process depended on HIF‐2α direct targets, among which lysyl oxidase‐like 2 (LOXL2) and matrix metalloproteinase‐9 (MMP‐9) mediated ATP‐driven invasion, and E‐cadherin and Snail mediated ATP‐driven EMT, respectively. In addition, using silver staining and mass spectrometry, we found that phosphoglycerate kinase 1 (PGK1) could interact with HIF‐2α and mediate ATP‐driven HIF‐2α upregulation. Furthermore, we demonstrated that expressions of HIF‐2α and its target proteins could be regulated via ATP by AKT‐PGK1 pathway. Using a Balb/c mice model, we illustrated the function of HIF‐2α in promoting tumor growth and metastasis in vivo. Moreover, by exploring online databases, we found that molecules involved in ATP‐HIF‐2α signaling were highly expressed in human breast carcinoma tissues and were associated with poor prognosis. Altogether, these findings suggest that extracellular ATP could promote breast carcinoma invasion and EMT via HIF‐2α signaling, which may be a potential target for future anti–metastasis therapy.     

Macrophage migration inhibitory factor promotes the invasion and metastasis of oral squamous cell carcinoma through matrix metalloprotein‐2/9

Macrophage migration inhibitory factor (MIF) has been shown to closely associate with the malignant progression of a variety of human carcinomas. However, the role and its underlying molecular mechanisms of MIF in the invasion and metastasis of oral squamous cell carcinoma (OSCC) still remains unclear. Here, we found that MIF silencing reduced the cell proliferation, migration, and invasion, as well as matrix metalloprotein‐2 (MMP‐2) and MMP‐9 in OSCC cells. Overexpression of MMP‐2 or MMP‐9 restored the migration and invasion of MIF‐knockdown cells, indicating that MMP‐2 and MMP‐9 are downstream targets of MIF. In the xenograft model, MIF silencing inhibited tumor growth and in lymph metastasis model, MIF silencing reduced tumor metastasis. More importantly, immunohistochemistry staining in a tissue microarray (TMA) demonstrated that MIF expression was positively correlated with clinic stage, recurrence, metastasis, and poor prognosis of patients with OSCC as well as with the levels of MMP‐2 or MMP‐9 in TMA. Therefore, our findings suggest that MIF may promote the invasion and metastasis of OSCC through the activation of MMP‐2 and MMP‐9 and prompt further investigation into the therapeutic value of MIF for OSCC treatment.     

Epigenetics of epithelial‐to‐mesenchymal transition in pancreatic carcinoma

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal malignant diseases with late diagnosis, rapid progression, high invasiveness, and early metastasis. Epithelial‐to‐mesenchymal transition (EMT) is a crucial step in metastasis that enables polarized immotile epithelial cells to gain fibroblast‐like mesenchymal abilities such as enhanced motility. The dynamic process of EMT in PDAC with its powerful influence on disease progression and especially metastasis is of vigorous interest in biomedical research to elucidate its signaling pathways and regulation mechanisms. It is evident that epigenetics such as histone and DNA modification or noncoding RNAs such as microRNAs and long noncoding RNAs are of high importance in initiation and progress of EMT in PDAC. This review analyzes the latest research dealing with EMT and its epigenetic regulation in PDAC and summarizes its potentials in diagnostic, prognostic, and therapeutic management.     

Persistent activation of STAT3 by PIM2‐driven positive feedback loop for epithelial‐mesenchymal transition in breast cancer

Metastasis of breast cancer is promoted by epithelial–mesenchymal transition (EMT). Emerging evidence suggests that STAT3 is a critical signaling node in EMT and is constitutively activated in many carcinomas, including breast cancer. However, its signaling mechanisms underlying persistent activation of STAT3 associated with EMT remain obscure. Here, we report that PIM2 promotes activation of STAT3 through induction of cytokines. Activation of STAT3 caused an increase in PIM2 expression, implicating a positive feedback loop between PIM2 and STAT3. In agreement, targeting of either PIM2, STAT3 or PIM2‐dependent cytokines suppressed EMT‐associated migratory and invasive properties through inhibition of ZEB1. Taken together, our findings identify the signaling mechanisms underlying the persistent activation of STAT3 and the oncogenic role of PIM2 in EMT in breast cancer.     

Aberrant methylation of EYA4 promotes epithelial‐mesenchymal transition in esophageal squamous cell carcinoma

EYA4, one of the four members of the EYA gene family, is associated with several human cancers. However, its biological functions and molecular mechanisms in the progression of cancer, particularly in esophageal squamous cell carcinoma (ESCC), remain unknown. In the present study, we found that EYA4 was underexpressed and hypermethylated in most of the ESCC cell lines tested (85.7%, 6/7). Treatment with 5‐aza‐dC and/or trichostatin A (TSA) restored EYA4 expression in ESCC cell lines, which indicates that EYA4 expression was epigenetically regulated. Similarly, EYA4 was aberrantly hypermethylated in ESCC tissues (78%, 39/50) and downregulation of EYA4 occurred in approximately 65% of primary ESCC at protein level where it was associated significantly with TNM stage and lymph node metastases. Knockdown of EYA4 in KYSE30 and KYSE70 ESCC cells using small hairpin RNA increased migration and invasive motility in vitro. Conversely, the overexpression of EYA4 in KYSE180 and KYSE450 promoted an epithelial phenotype, which consisted of decreased migration and invasion abilities and a decrease in TGF‐β1‐induced epithelial‐mesenchymal transition. Mechanistically, EYA4 overexpression reduced the phosphorylation of Akt and glycogen synthase kinase (GSK) 3β, which led to the inactivation of slug. In addition, we found that TGF‐β1 decreased EYA4 expression in both a dose‐dependent and a time‐dependent manner in KYSE30 cells, accompanied by an increase in the expression of DNA methyltransferases, especially DNMT3A. In summary, EYA4 is frequently hypermethylated in ESCC and may function as a tumor suppressor gene in the development of ESCC.     

Lysyl oxidase induces epithelial‐mesenchymal transition and predicts intrahepatic metastasis of hepatocellular carcinoma

Hepatocellular carcinoma (HCC) has high recurrence rates even after curative hepatectomy. Drug therapy for recurrence of HCC is still limited; therefore, identifying new therapeutic targets is urgently needed. We searched for genes that would predict HCC recurrence from intrahepatic metastasis in an exhaustive DNA microarray database by searching genes associated with high early recurrence rate and having higher expression in the tumor area compared to background liver. We detected lysyl oxidase (LOX) and validated the clinical significance of LOX in 358 patients who underwent hepatectomy. Expression of LOX was evaluated by qRT‐ PCR, and immunohistochemical (IHC) staining. High LOX expression group had a significantly higher recurrence rate than the low LOX expression group (2‐year recurrence rate was 64.0% vs 24.2%, P < .0001 for IHC) and poorer survival rate (5‐year rate was 60.1% vs 86.2%, P < .0001 for IHC). Multivariate analysis showed that high LOX expression was an independent risk factor for early recurrence (IHC: HR, 2.52; P < .0001). Bioinformatic analysis showed that LOX expression was associated with hypoxia‐inducible factor‐1α (HIF‐1α) and the hypoxia cascade, suggesting that HIF‐1α or hypoxia regulates LOX expression and induces epithelial‐mesenchymal transition (EMT). In vitro, LOX and HIF‐1α were involved in migration and invasion capability. High LOX expression is associated with EMT markers and predicts early recurrence and poor survival in patients with HCC. These findings indicate that lysyl oxidase could be a potential therapeutic target for early recurrence of HCC.     

Frizzled 2‐induced epithelial‐mesenchymal transition correlates with vasculogenic mimicry,stemness, and Hippo signaling in hepatocellular carcinoma

Prior observation has indicated that Frizzled 2 (FZD2)‐induced epithelial‐mesenchymal transition (EMT) could be a key step in metastasis and early recurrence of hepatocellular carcinoma (HCC). However, the mechanism underlying tumor development and progression due to aberrant FZD2 expression is poorly defined. Here, we provide evidence that FZD2 is a driver for EMT, cancer stem cell properties, and vasculogenic mimicry (VM) in HCC. We found that FZD2 was highly expressed in two cohorts of Chinese hepatitis B virus‐related HCC patients, and that high FZD2 expression was associated with poor prognosis. Concerning the mechanism, gain‐ and loss‐of‐function experiments showed the oncogenic action of FZD2 in HCC cell proliferation, apoptosis, migration, and invasion. Further investigations in vitro and in vivo suggested that FZD2 promotes the EMT process, enhances stem‐like properties, and confers VM capacity to HCC cells. Notably, integrative RNA sequencing analysis of FZD2‐knockdown cells indicated the enrichment of Hippo signaling pathway. Taken together, our data suggest for the first time that FZD2 could promote clinically relevant EMT, CD44⁺ stem‐like properties, and the VM phenotype in HCC involving a potential Hippo signaling pathway‐dependent mechanism, and should be considered as a promising therapeutic target for the treatment of HCC.     

MAEL expression links epithelial‐mesenchymal transition and stem cell properties in colorectal cancer

MAEL plays a central role during spermatogenesis by repressing transposable elements and preventing their mobilisation, however, its role on cancers is unclear. In this study, MAEL expression was analysed in a tissue microarray containing 185 samples of primary colon cancer tumor samples and human colon cancer cell lines. The effect of MAEL on cell proliferation, tumorigenesis, metastasis and drug resistance was examined in vitro and in vivo. Immunoprecipitation assay, confocal immunofluorescent analysis and luciferase assay were used for mechanism study. As results, MAEL was significantly upregulated in colon cancer patient tissue samples, and elevated MAEL protein levels positively correlated with overall survival and disease free survival of colon cancer patients. Using in vitro and in vivo models, we demonstrated that MAEL expression was correlated with cell proliferation, invasion and drug resistance of colon cancer cells by inducing epithelial‐mesenchymal transition and stemness characteristics. Mechanistically, our study demonstrated that MAEL interacts with Snail and inhibit E‐cadherin promoter activity. Collectively, MAEL is an oncogene that plays an important role in the development and progression of colon cancer, which may be a novel potential therapeutic target for colon cancer.     

Cancer‐associated fibroblasts and epithelial‐mesenchymal transition in metastatic oral tongue squamous cell carcinoma

We examined cancer‐associated fibroblasts (CAFs) and a panel of immunohistochemical markers of epithelial‐mesenchymal transition (EMT) in 19 pair‐matched oral tongue squamous cell carcinoma (SCC) and metastatic tumors to regional lymph nodes (RLNs). α‐Smooth muscle actin (α‐SMA) was studied to identify CAFs. EMT was studied with syndecan‐1, Cadherin‐11, fibroblast‐specific protein (FSP)‐1, s ecreted p rotein a cidic and r ich in c ysteine (SPARC) and Twist. Triple immunostaining in RLNs was used to highlight the carcinoma cells (E‐cadherin and Ki‐67) and their relationship to the CAFs (α‐SMA). We found that metastatic RLNs hosted CAFs similarly as in pair‐matched primary tumors. Expression of EMT markers is common in both primary and metastatic tumors. We demonstrate that metastatic carcinoma cells (Ki‐67 positive) downregulate E‐cadherin expression at the periphery of cancer islands, where they are in direct contact with CAFs. The supporting connective tissue microenvironment also commonly expresses syndecan‐1, Cadherin‐11, FSP‐1, and SPARC. In conclusion, CAFs are common to both primary and metastatic SCC. We hypothesize that CAFs not only promote tumor invasion but also facilitate metastases, either by cometastasizing and/or being recruited to lymph nodes. Evidence of EMT is common within primary tumors and metastatic SCC and may be further modulated by CAFs.     

Expression of TNF‐α and CD44 is implicated in poor prognosis,cancer cell invasion,metastasis and resistance to the sunitinib treatment in clear cell renal cell carcinomas

Tumor necrosis factor‐α (TNF‐α) is involved in epithelial‐mesenchymal transition (EMT) and expression of CD44, a cancer stem cell marker, in several cancers. This study was performed to clarify the significance of TNF‐α and CD44 in clear cell renal cell carcinomas (ccRCCs). Expression of TNF‐α and CD44 was examined by immunohistochemistry in 120 ccRCCs. Involvement of TNF‐α in EMT and induction of CD44 was analyzed by monitoring expression of EMT‐related genes and CD44, and invasion in cultured ccRCC cell lines. TNF‐α and CD44 were immunolocalized mainly to carcinoma cells of high‐grade ccRCCs with positive correlations with primary tumor stage. A positive correlation was also obtained between TNF‐α and CD44 expression, and co‐upregulation of TNF‐α and CD44 was associated with primary tumor stage, distant metastasis, and poor prognosis. TNF‐α enhanced migration and invasion of ccRCC cells together with down‐regulation of E‐cadherin expression and up‐regulation of matrix metalloproteinase 9 and CD44 expression. TNF‐α also up‐regulated the expression of TNF‐α itself in ccRCC cells. Among the 25 ccRCC patients treated with sunitinib for metastatic disease, high CD44 expression was associated with poor treatment outcome. Importantly, residual carcinoma cells in the sunitinib‐treated metastatic ccRCCs were strongly positive for CD44, and the CD44 expression was significantly higher in the tumors from the sunitinib‐treated patients than in those from untreated ones. Our data show that TNF‐α plays an important role in progression of ccRCCs by inducing EMT and CD44 expression, and suggest that CD44 induced by TNF‐α may be involved in the resistance to the sunitinib treatment.     

microRNA‐219‐5p inhibits epithelial‐mesenchymal transition and metastasis of colorectal cancer by targeting lymphoid enhancer‐binding factor 1

Aberrant expression of microRNAs (miRs) has been shown to play a critical role in the pathogenesis and progression of tumors. microRNA‐219‐5p (miR‐219‐5p) has been reported to be abnormally expressed in some types of human tumors. However, the mechanism between miR‐219‐5p and colorectal cancer (CRC) metastasis remains unclear. In the present study, miR‐219‐5p was found to be downregulated in CRC tissue compared with matched normal tissue. Through luciferase reporter assay, we demonstrated lymphoid enhancer‐binding factor 1 (LEF1) as a direct target of miR‐219‐5p. Overexpression of miR‐219‐5p could inhibit motility, migration and invasion of CRC cells, and inhibit epithelial‐mesenchymal transition (EMT). Furthermore, silencing LEF1 phenocopied this metastasis‐suppressive function. The recovery experiment showed that re‐expression of LEF1 rescued this suppressive effect on tumor metastasis and reversed the expression of EMT markers caused by miR‐219‐5p. Additionally, we demonstrated that miR‐219‐5p exerted this tumor‐suppressive function by blocking activation of the AKT and ERK pathways. Finally, a nude mice experiment showed that miR‐219‐5p reduced the lung metastasis ability of CRC cells. Taken together, our findings indicate that miR‐219‐5p inhibits metastasis and EMT of CRC by targeting LEF1 and suppressing the AKT and ERK pathways, which may provide a new antitumor strategy to delay CRC metastasis.     

miR‐25‐3p reverses epithelial‐mesenchymal transition via targeting Sema4C in cisplatin‐resistance cervical cancer cells

Acquisition of epithelial‐mesenchymal transition (EMT) has recently been proposed as an important contributor of drug resistance in cervical cancer cells. However, the underlying mechanisms are still unclear. MicroRNAs play a crucial role in regulating EMT. The aim of this study was to explore the potential role of miR‐25‐3p in regulating EMT in cisplatin‐resistant (CR) cervical cancer cells. To this end, we established stable CR cervical cancer cells, HeLa‐CR and CaSki‐CR, and investigated the function of miR‐25‐3p in regulating EMT. It is found that CR cervical cancer cells possessed more EMT characteristics and demonstrated higher migratory abilities and invasiveness. miR‐25‐3p downregulation was also seen in HeLa‐CR and CaSki‐CR cells. Of note, ectopic expression of miR‐25‐3p reversed the EMT phenotype and sensitized CR cells to cisplatin via targeting Sema4C. Furthermore, stable overexpression of miR‐25‐3p in HeLa‐CR cells suppressed tumor growth in mice, downregulated Sema4C and Snail, and upregulated E‐cadherin compared with the control group. These results suggest that miR‐25‐3p is an important regulator of cervical cancer EMT and chemoresistance. Thus, upregulation of miR‐25‐3p could be a novel approach to treat cervical cancers that are resistant to chemotherapy.     

TGFβ‐induced epithelial‐to‐mesenchymal transition in prostate cancer cells is mediated via TRPM7 expression

Growth factors, such as the transforming growth factor beta (TGFβ), play an important role in promoting metastasis of prostate cancer, thus understanding how TGFβ could induce prostate cancer cell migration may enable us to develop targeted strategies for treatment of advanced metastatic prostate cancer. To more clearly define the mechanism(s) involved in prostate cancer cell migration, we undertook a series of studies utilizing non‐malignant prostate epithelial cells RWPE1 and prostate cancer DU145 and PC3 cells. Our studies show that increased cell migration was observed in prostate cancer cells, which was mediated through epithelial‐to‐mesenchymal transition (EMT). Importantly, addition of Mg²⁺, but not Ca²⁺, increased cell migration. Furthermore, TRPM7 expression, which functions as an Mg²⁺ influx channel, was also increased in prostate cancer cells. Inhibition of TRPM7 currents by 2‐APB, significantly blocked cell migration in both DU145 and PC3 cells. Addition of growth factor TGFβ showed a further increase in cell migration, which was again blocked by the addition of 2‐APB. Importantly, TGFβ addition also significantly increased TRPM7 expression and function, and silencing of TRPM7 negated TGFβ‐induced cell migration along with a decrease in EMT markers showing loss of cell adhesion. Furthermore, resveratrol, which decreases prostate cancer cell migration, inhibited TRPM7 expression and function including TGFβ‐induced cell migration and activation of TRPM7 function. Together, these results suggest that Mg²⁺ influx via TRPM7 promotes cell migration by inducing EMT in prostate cancer cells and resveratrol negatively modulates TRPM7 function thereby inhibiting prostate cancer metastasis.     

Phloroglucinol suppresses metastatic ability of breast cancer cells by inhibition of epithelial‐mesenchymal cell transition

Metastasis is a challenging clinical problem and the primary cause of death in breast cancer patients. However, there is no therapeutic agent against metastasis of breast cancer cells. Here we report that phloroglucinol, a natural phlorotannin component of brown algae suppresses metastatic ability of breast cancer cells. Treatment with phloroglucinol effectively inhibited mesenchymal phenotypes of basal type breast cancer cells through downregulation of SLUG without causing a cytotoxic effect. Importantly, phloroglucinol decreased SLUG through inhibition of PI3K/AKT and RAS/RAF‐1/ERK signaling. In agreement with in vitro data, phloroglucinol was also effective against in vivo metastasis of breast cancer cells, drastically suppressing their metastatic ability to lungs, and extending the survival time of mice. Collectively, our findings demonstrate a novel anticancer activity of phloroglucinol against metastasis of breast cancer cells, implicating its clinical relevance.