Side population of pancreatic cancer cells predominates in TGF‐β‐mediated epithelial to mesenchymal transition and invasion

We report here side population (SP) cells, a cancer stem cell enriched fraction from pancreatic cancer cell line, have enormous superior potential of the epithelial to mesenchymal transition (EMT), invasion, and metastasis. In an isolated SP cell culture, the cells rapidly expressed and up‐regulated E‐cadherin, an epithelial phenotypic marker, and the cells formed tightly contacted cell cluster, which is a representative epithelial phenotypic appearance. When the SP cells were incubated in the presence of TGF‐β, SP cells changed their shape into mesenchymal‐like appearance including spindle shaped assembly. This alteration was associated with significant reduction of E‐cadherin expression level. TGF‐β induced EMT‐associated gene alteration such as reduction of E‐cadherin mRNA and induction of Snail mRNA and matrixmetalloproteinase (MMP)‐2 mRNA. Finally, SP cells exerted notable matrigel invasion activity in response to TGF‐β treatment, whereas MP cells did not respond to TGF‐β‐mediated invasion. In conclusion, these results suggest that SP cells from pancreatic cancer cell line possess superior potentials of phenotypic switch, i.e., EMT/MET, micro‐invasion, and in vivo metastasis, as compared to MP cells. Because micro‐invasion and metastasis are key mechanisms of cancer malignant potential, SP cells would be the attractive target for preventing cancer progression. © 2008 UICC     

Regulation of motility,invasion, and metastatic potential of squamous cell carcinoma by 1α,25‐dihydroxycholecalciferol

BACKGROUND:

The active metabolite of vitamin D 1α,25‐dihydroxycholecalciferol (1,25D₃) has exhibited broad‐spectrum antitumor activity in xenograft animal models. However, its activity against metastatic disease has not been extensively investigated.

METHODS:

Squamous cell carcinoma (SCC) or 1,25D₃‐resistant variant SCC‐DR cells were treated with 1,25D₃. Actin organization was examined by immunofluorescence assay. Cell migration was assessed by “wound” healing and chemotactic migration assays. Cell invasion was assessed by a Matrigel‐based invasion assay and in situ zymography. Matrix metalloproteinase 2 (MMP‐2) and MMP‐9 expression and secretion were examined by immunoblot analysis and an enzyme‐linked immunosorbent assay, respectively. E‐cadherin expression was assessed by flow cytometry, immunoblot analysis, and immunohistochemistry. Knockdown of E‐cadherin was achieved by small interfering RNA. An experimental metastasis mouse model was created by intravenous injection of tumor cells; and lung tumor development in the mice was assessed by magnetic resonance imaging, gross observation, and histology.

RESULTS:

SCC cellular morphology and actin organization were altered by 10 nM 1,25D₃. 1,25D₃ inhibited SCC cell motility and invasion, which were associated with reduced expression and secretion of MMP‐2 and MMP‐9, and 1,25D₃ promoted the expression of E‐cadherin. These findings were not observed in SCC‐DR cells. Knock down of E‐cadherin rescued 1,25D₃‐inhibited cell migration. Intravenous injection of SCC or SCC‐DR cells resulted in the establishment of extensive pulmonary lesions in saline‐treated C3H mice. Treatment with 1,25D₃ resulted in a marked reduction in the formation of lung tumor colonies in mice that were injected with SCC cells, but not in mice that were injected with SCC‐DR cells.

CONCLUSIONS:

1,25D₃ suppressed SCC cell motility, invasion, and metastasis, partially through the promotion of E‐cadherin‐mediated cell‐cell adhesion. Cancer 2013. © 2012 American Cancer Society.     

Constitutively active MEK1 is sufficient to induce epithelial‐to‐mesenchymal transition in intestinal epithelial cells and to promote tumor invasion and metastasis

Constitutive activation of the MAP kinase kinase MEK1 induces oncogenic transformation in intestinal epithelial cells. Loss of cell–cell adhesion followed by the dissociation of epithelial structures is a prerequisite for increased cell motility and tumor invasion. This phenotypic switch is designated epithelial‐to‐mesenchymal transition (EMT). EMT also plays an important role in determining the dissemination of tumors. However, the role of MEK1 in intestinal EMT, tumor invasion and metastasis has not been elucidated. To determine the functions of activated MEK1 in intestinal tumorigenesis, we established intestinal epithelial cell lines that overexpress wild‐type MEK1 (wtMEK) or activated MEK1 (caMEK). Our results indicate that expression of caMEK is sufficient to induce EMT as confirmed with the induction of N‐cadherin, vimentin, Snail1 and Snail2, whereas a reduction in E‐cadherin, occludin, ZO‐1 and cortical F‐actin was noted. The Snail1 and Snail2 promoter analyses revealed that Egr‐1 and Fra‐1, an AP‐1 protein, are responsible for MEK1‐induced Snail1 and Snail2 expression, respectively. Cells expressing activated MEK1 clearly acquired an invasive capacity when compared to wtMEK‐expressing cells. Zymography studies confirmed elevated levels of MMP2 and MMP9 activities in media of caMEK‐expressing cells. Importantly, cells expressing activated MEK1 induced tumors with short latency in correlation with their ability to induce experimental metastasis in vivo and to express factors known to promote colorectal cancer cell metastasis. In conclusion, our results demonstrate, for the first time, that constitutive activation of MEK1 in intestinal epithelial cells is sufficient to induce an EMT associated with tumor invasion and metastasis. © 2009 UICC     

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.     

Mesenchymal stromal cells induce epithelial‐to‐mesenchymal transition in human colorectal cancer cells through the expression of surface‐bound TGF‐β

Mesenchymal stem/stromal cells (MSC) are multipotent precursors endowed with the ability to home to primary and metastatic tumor sites, where they can integrate into the tumor‐associated stroma. However, molecular mechanisms and outcome of their interaction with cancer cells have not been fully clarified. In this study, we investigated the effects mediated by bone marrow‐derived MSC on human colorectal cancer (CRC) cells in vitro and in vivo. We found that MSC triggered epithelial‐to‐mesenchymal transition (EMT) in tumor cells in vitro, as indicated by upregulation of EMT‐related genes, downregulation of E‐cadherin and acquisition of mesenchymal morphology. These effects required cell‐to‐cell contact and were mediated by surface‐bound TGF‐β newly expressed on MSC upon coculture with tumor cells. In vivo tumor masses formed by MSC‐conditioned CRC cells were larger and characterized by higher vessel density, decreased E‐cadherin expression and increased expression of mesenchymal markers. Furthermore, MSC‐conditioned tumor cells displayed increased invasiveness in vitro and enhanced capacity to invade peripheral tissues in vivo. Thus, by promoting EMT‐related phenomena, MSC appear to favor the acquisition of an aggressive phenotype by CRC cells.     

Tetraspanin CD63 acts as a pro‐metastatic factor via β‐catenin stabilization

The tetraspanin CD63 is implicated in pro‐metastatic signaling pathways but, so far, it is unclear, how CD63 levels affect the tumor cell phenotype. Here, we investigated the effect of CD63 modulation in different metastatic tumor cell lines. In vitro, knock down of CD63 induced a more epithelial‐like phenotype concomitant with increased E‐cadherin expression, downregulation of its repressors Slug and Zeb1, and decreased N‐cadherin. In addition, β‐catenin protein was markedly reduced, negatively affecting expression of the target genes MMP‐2 and PAI‐1. β‐catenin inhibitors mimicked the epithelial phenotype induced by CD63 knock down. Inhibition of β‐catenin upstream regulators PI3K/AKT or GSK3β could rescue the mesenchymal phenotype underlining the importance of the β‐catenin pathway in CD63‐regulated cell plasticity. CD63 knock down‐induced phenotypical changes correlated with a decrease of experimental metastasis whereas CD63 overexpression enhanced the tumor cell‐intrinsic metastatic potential. Taken together, our data show that CD63 is a crucial player in the regulation of the tumor cell‐intrinsic metastatic potential by affecting cell plasticity.     

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.     

Epithelial–mesenchymal transition in human gastric cancer cell lines induced by TNF‐α‐inducing protein of Helicobacter pylori

Helicobacter pylori strains produce tumor necrosis factor‐α (TNF‐α)‐inducing protein, Tipα as a carcinogenic factor in the gastric epithelium. Tipα acts as a homodimer with 38‐kDa protein, whereas del‐Tipα is an inactive monomer. H. pylori isolated from gastric cancer patients secreted large amounts of Tipα, which are incorporated into gastric cancer cells by directly binding to nucleolin on the cell surface, which is a receptor of Tipα. The binding complex induces expression of TNF‐α and chemokine genes, and activates NF‐κB (nuclear factor kappa‐light‐chain‐enhancer of activated B cells). To understand the mechanisms of Tipα in tumor progression, we looked at numerous effects of Tipα on human gastric cancer cell lines. Induction of cell migration and elongation was found to be mediated through the binding to surface nucleolin, which was inhibited by the nucleolin‐targeted siRNAs. Tipα induced formation of filopodia in MKN‐1 cells, suggesting invasive morphological changes. Tipα enhanced the phosphorylation of 11 cancer‐related proteins in serine, threonine and tyrosine, indicating activation of MEK‐ERK signal cascade. Although the downregulation of E‐cadherin was not shown in MKN‐1 cells, Tipα induced the expression of vimentin, a significant marker of the epithelial–mesenchymal transition (EMT). It is of great importance to note that Tipα reduced the Young's modulus of MKN‐1 cells determined by atomic force microscopy: This shows lower cell stiffness and increased cell motility. The morphological changes induced in human gastric cancer cells by Tipα are significant phenotypes of EMT. This is the first report that Tipα is a new inducer of EMT, probably associated with tumor progression in human gastric carcinogenesis.     

Novel regulatory program for norepinephrine‐induced epithelial–mesenchymal transition in gastric adenocarcinoma cell lines

Norepinephrine and epinephrine, catecholamine hormones that are major mediators for chronic stress‐induced cancers, are implicated in the progression of a number of cancer cells, including gastric adenocarcinoma. However, the underlying mechanisms of these hormones have not been well elucidated. Epithelial–mesenchymal transition (EMT) is a crucial event responsible for cancer cell invasion and metastasis. The hypothesis regarding whether the promotive effects of norepinephrine (NE) on cancer are in part due to its ability to induce an EMT program has not been proven. In this study, we show that NE does not only obviously induce EMT alterations in the morphological characteristics of gastric adenocarcinoma cells, but also increases the markers of EMT, including vimentin expression, and decreases E‐cadherin expression, further resulting in cell motility and invasiveness. We also reveal that these actions are mainly mediated through the activation of β₂‐AR–HIF‐1α–Snail signaling pathways. In summary, this study implies that NE induces EMT in gastric adenocarcinoma through the regulation of β₂‐AR–HIF‐1α–Snail activity. The data provide a new perspective on chronic stress in a negative social and psychological state, which may be a risk factor for cancer development and progression.     

Elevated expression of ΔNp63 in advanced esophageal squamous cell carcinoma

This study aims to explore the expression level of ΔNp63 in esophageal squamous cell carcinoma (ESCC). To investigate the association between ΔNp63 (p40) expression and ESCC biology, we compared the levels of ΔNp63 expression in normal and tumor tissues, with a specific focus on the diagnostic value of ΔNp63 in ESCC. We analyzed 160 consecutive patients with ESCC who underwent surgical resection without neoadjuvant chemotherapy at Gunma University Hospital (Maebashi, Japan) between September 2000 and January 2010. The clinicopathological characteristics and survival of patients were subclassified based on the expression of ΔNp63 as determined by immunohistochemistry, indicating that ΔNp63 was highly expressed in 75.6% (121/160) of ESCC patients. Clinicopathological analysis of ΔNp63 expression showed that ΔNp63‐positive tumors significantly correlated with two important clinical parameters: T factor (P = 0.0316) and venous invasion (P = 0.0195). The 5‐year overall survival rates of advanced ESCC patients with positive and negative expression of ΔNp63 were 35.6% and 71.7%, respectively. Multivariate analysis revealed that the expression of ΔNp63 was identified as an independent prognostic factor (P = 0.0049) in advanced ESCC. In line with this, ΔNp63α‐transduced ESCC cell lines increased tumor growth in a soft agar colony formation assay. We report here for the first time that ΔNp63 expression increases the oncogenic potential of ESCC and is an independent marker for predicting poor outcome in advanced ESCC. Our findings suggest that ΔNp63 could serve as a new diagnostic marker for ESCC and might be a relevant therapeutic target for the treatment of patients with this disease.     

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.     

Repressive role of stabilized hypoxia inducible factor 1α expression on transforming growth factor β‐induced extracellular matrix production in lung cancer cells

Activation of transforming growth factor β (TGF‐β) combined with persistent hypoxia often affects the tumor microenvironment. Disruption of cadherin/catenin complexes induced by these stimulations yields aberrant extracellular matrix (ECM) production, characteristics of epithelial‐mesenchymal transition (EMT). Hypoxia‐inducible factors (HIF), the hallmark of the response to hypoxia, play differential roles during development of diseases. Recent studies show that localization of cadherin/catenin complexes at the cell membrane might be tightly regulated by protein phosphatase activity. We aimed to investigate the role of stabilized HIF‐1α expression by protein phosphatase activity on dissociation of the E‐cadherin/β‐catenin complex and aberrant ECM expression in lung cancer cells under stimulation by TGF‐β. By using lung cancer cells treated with HIF‐1α stabilizers or carrying doxycycline‐dependent HIF‐1α deletion or point mutants, we investigated the role of stabilized HIF‐1α expression on TGF‐β‐induced EMT in lung cancer cells. Furthermore, the underlying mechanisms were determined by inhibition of protein phosphatase activity. Persistent stimulation by TGF‐β and hypoxia induced EMT phenotypes in H358 cells in which stabilized HIF‐1α expression was inhibited. Stabilized HIF‐1α protein expression inhibited the TGF‐β‐stimulated appearance of EMT phenotypes across cell types and species, independent of de novo vascular endothelial growth factor A (VEGFA) expression. Inhibition of protein phosphatase 2A activity abrogated the HIF‐1α‐induced repression of the TGF‐β‐stimulated appearance of EMT phenotypes. This is the first study to show a direct role of stabilized HIF‐1α expression on inhibition of TGF‐β‐induced EMT phenotypes in lung cancer cells, in part, through protein phosphatase activity.     

Vasohibin‐2 is required for epithelial–mesenchymal transition of ovarian cancer cells by modulating transforming growth factor‐β signaling

Vasohibin‐2 (VASH2) is a homolog of VASH1, an endothelium‐derived angiogenesis inhibitor. Vasohibin‐2 is mainly expressed in cancer cells, and has been implicated in the progression of cancer by inducing angiogenesis and tumor growth. Although VASH2 has been recently reported to be involved in epithelial–mesenchymal transition (EMT), its precise roles are obscure. The aim of the present study was to clarify the role of VASH2 in the EMT of cancer cells in relation to transforming growth factor‐β (TGF‐β) signaling, which is a major stimulator of EMT. Decreased expression of VASH2 in ovarian cancer cells significantly repressed the expression of TGF‐β type I receptor, namely activin receptor‐like kinase 5. Transforming growth factor‐β1‐induced phosphorylation of Smad2 and Smad3 was markedly decreased in VASH2 knockdown cells while the expression of Smad2 and Smad3 was unchanged. Accordingly, the responses to TGF‐β1 shown by promoter assay and plasminogen activator inhibitor type 1 expression were significantly attenuated in VASH2 knockdown cells. Furthermore, knockdown of VASH2 in cancer cells abrogated the TGF‐β1‐induced reduced expression of epithelial markers including E‐cadherin, and the elevated expression of mesenchymal markers including fibronectin, ZEB2, and Snail2, suggesting that endogenous VASH2 is required for TGF‐β1‐induced EMT. In accordance with these results, the effects of TGF‐β1 on cell morphology, migration, invasion, and MMP2 expression were also abrogated when VASH2 was knocked down. These results indicate that VASH2 played a significant role in the EMT by modulating the TGF‐β signaling. We propose that VASH2 would be a novel molecular target for the prevention of EMT in cancers.