Epithelial-mesenchymal Transition In Pancreatic Cancer Cells Research Articles

Curcumin inhibits the invasion and migration of pancreatic cancer cells by upregulating TFPI-2 to regulate ERK- and JNK-mediated epithelial-mesenchymal transition.

Pancreatic cancer (PC) is one of the most deadly human malignancies. Curcumin is a natural polyphenolic compound with wide-ranging pharmacological effects. Growing evidence suggests that curcumin has anticancer activity against PC, but the mechanism remains incompletely elucidated. This study aimed to investigate the effects and mechanisms of curcumin on the invasion and migration of PC cells. Effect of curcumin on tissue factor pathway inhibitor (TFPI)-2 mRNA expression in PC cells was initially identified using qRT-PCR. Cytotoxicity of curcumin was assessed with MTT assays and IC50 was calculated. Involvement of ERK and JNK pathways, as well as protein expression of TFPI-2 and epithelial-mesenchymal transition (EMT)-related markers, were detected using immunoblotting. Invasion and migration of PC cells were examined using Transwell assays. TFPI-2 expression was manipulated by transfection with siRNA and shRNA. Rescue assays were used to validate the effect of curcumin on cell invasion and migration via TFPI-2. Curcumin increased the expression of TFPI-2 mRNA and protein in PC cells and attenuated cell invasion and migration. Curcumin also inhibited ERK and JNK pathways and EMT in PC cells. Knockdown of TFPI-2 partially reversed the inhibition of ERK and JNK pathways and EMT by curcumin. Mechanistically, curcumin upregulated TFPI-2, thereby inhibiting the ERK and JNK pathways, leading to the inhibition of EMT in PC cells. Collectively, curcumin inhibits ERK- and JNK-mediated EMT through upregulating TFPI-2, which in turn suppresses the migration and invasion of PC cells. These findings provide new insights into the antitumor mechanism of curcumin.

Somatostatin Inhibited the EMT of Pancreatic Cancer Cells by Mediating the TGF-β/Smad Signaling Pathway.

Pancreatic cancer (PC), a commonly recognized malignancy, arises within the digestive tract. Somatostatin (SOM) is a regulatory peptide that acts on secretion in vivo. Several studies have shown that SOM has inhibitory effects on various cancers. This work aims to probe the inhibitory effect, and mechanism of SOM action, on the epithelial-mesenchymal transition (EMT) of PC cells. First, the effects of SOM and transforming growth factor-β (TGF-β) on the proliferation of PC cells was determined by Cell Counting Kit-8 (CCK-8) assay. Next, we assessed the impact of SOM and TGF-β on the metastasis and apoptosis of PC cells using transwell assays and flow cytometry. Finally, we evaluated the effects of SOM and TGF-β on the expression of EMT-related proteins, apoptosis-related proteins, and proteins related to the TGF-β/Smad signaling pathway in PC cells using western blot analysis. SOM suppressed the growth and metastasis of PC cells, and facilitated their apoptosis (p < 0.05). Moreover, SOM reversed pro-apoptotic effects of TGF-β (p < 0.05). Specifically, SOM increased the expression of Cysteine-aspartic acid protease 3 (Caspase-3) and Bcl-2-associated X protein (Bax) proteins while reducing the expression of B-cell lymphoma 2 (Bcl-2) protein (p < 0.05). SOM also reversed the TGF-β-induced EMT process. The TGF-β1, Smad2, and Smad3 proteins in PC cells treated with SOM were significantly down-regulated (p < 0.05). SOM suppressed the EMT progression in PC cells through its regulation of the TGF-β/Smad signaling pathway.

The Impact of Epithelial–Mesenchymal Transition and Metformin on Pancreatic Cancer Chemoresistance: A Pathway towards Individualized Therapy

Globally, pancreatic ductal adenocarcinoma remains among the most aggressive forms of neoplastic diseases, having a dismal prognostic outcome. Recent findings elucidated that epithelial–mesenchymal transition (EMT) can play an important role in pancreatic tumorigenic processes, as it contributes to the manifestation of malignant proliferative masses, which impede adequate drug delivery. An organized literature search with PubMed, Scopus, Microsoft Academic and the Cochrane library was performed for articles published in English from 2011 to 2021 to review and summarize the latest updates and knowledge on the current understanding of EMT and its implications for tumorigenesis and chemoresistance. Furthermore, in the present paper, we investigate the recent findings on metformin as a possible neoadjuvant chemotherapy agent, which affects EMT progression and potentially provides superior oncological outcomes for PDAC patients. Our main conclusions indicate that selectively suppressing EMT in pancreatic cancer cells has a promising therapeutic utility by selectively targeting the chemotherapy-resistant sub-population of cancer stem cells, inhibiting tumor growth via EMT pathways and thereby improving remission in PDAC patients. Moreover, given that TGF-β1-driven EMT generates the migration of tumor-initiating cells by directly linking the acquisition of abnormal cellular motility with the maintenance of tumor initiating potency, the chemoprevention of TGF-β1-induced EMT may have promising clinical applications in the therapeutic management of PDAC outcomes.

MicroRNA-1252-5p, regulated by Myb, inhibits invasion and epithelial-mesenchymal transition of pancreatic cancer cells by targeting NEDD9.

MicroRNAs (miRNAs) are known to be involved in the development and progression of pancreatic cancer (PAC). The expression levels and roles of miR-1252-5p in PAC remain unclear. Quantitative real-time PCR and in situ hybridization were used to detect miR-1252-5p expression in PAC cells and human tissues. We studied the gain and loss of function of miR-1252-5p in the PAC cell lines in vitro and in vivo. The direct targets of miR-1252-5p were analyzed using public databases and a dual-luciferase reporter assay. Expression levels of miR-1252-5p are downregulated in PAC cell lines and tissue samples, and its expression is negatively associated with adverse clinical features and poor prognosis. In vitro and in vivo experiments show that miR-1252-5p overexpression inhibits the proliferation, migration, invasion, and epithelial-mesenchymal transition of PAC cells, and miR-1252-5p knockdown enhances these biological behaviors. MiR-1252-5p negatively regulates neural precursor cell expressed, developmentally downregulated 9 (NEDD9) by directly binding its 3'- untranslated region. Further mechanism research revealed that the SRC/STAT3 pathway is involved in miR-1252-5p/NEDD9 mediation of PAC's biological behaviors. We also verified that Myb inhibited miR-1252-5p by directly binding at its promoter. MiR-1252-5p may act as a tumor-suppressing miRNA in PAC and may be a potential therapeutic target for PAC patients.

MiR-338-5p Inhibits EGF-Induced EMT in Pancreatic Cancer Cells by Targeting EGFR/ERK Signaling.

The epidermal growth factor (EGF) pathway plays critical roles during cancer cell epithelial-mesenchymal transition (EMT) process and metastasis. Epidermal growth factor receptor (EGFR), as one of the important receptors of EGF, undergoes autophosphorylation with the stimulation of EGF and activates MAPK/ERK, PI3K/Akt/mTOR, and other pathways. Here, we identified EGFR was a target of miR-338-5p. Upon EGF treatment, overexpression of miR-338-5p not only downregulated EGFR expression and inhibited MAPK/ERK signaling, but also inhibited EMT and metastasis process of pancreatic cancer (PC) cells. In the clinical pathological analysis, miR-338-5p was significantly down-regulated in 44 pairs PC tissues and its expression was negatively associated with lymph node metastasis and AJCC stage. Furthermore, Overexpression of EGFR partially reversed the protective effect of miR-338-5p overexpression on EGF-mediated migration and invasion in PC cells. Taken together, miR-338-5p controls EGF-mediated EMT and metastasis in PC cells by targeting EGFR/ERK pathways. Here, we hope to provide new insights into the molecular mechanisms of pancreatic cancer, and may help facilitating development of EGFR-based therapies for human cancer.

Monitoring Epithelial-Mesenchymal Transition of Pancreatic Cancer Cells via Investigation of Mitochondrial Dysfunction.

In this protocol, we introduced a method of measuring mitochondrial dysfunction to confirm the epithelial–mesenchymal transition (EMT) in pancreatic cancer cells under a hypoxic environment. There are many expertized and complicated methods to verify EMT. However, our methods have indicated that EMT can be identified by examining changes in reactive oxygen species (ROS) generation and membrane potential in mitochondria. To demonstrate whether the changes in the indicators of mitochondrial dysfunction are correlative to EMT, cell morphology, and expression of E-cadherin and N-cadherin were additionally observed. The results verified that a decrease in membrane potential and an increase in ROS in mitochondria were associated with EMT of pancreatic cancer cells. This protocol would be useful as a basis for providing an additional indicator for changes in the tumor microenvironment of pancreatic cancer cells relating to EMT under a hypoxic environment.

MiR‑539 suppresses the proliferation, migration, invasion and epithelial mesenchymal transition of pancreatic cancer cells through targeting SP1.

MicroRNA (miR)-539 has inhibitory effects on certain types of cancer, but its role in pancreatic cancer (PCa) remains unclear. The present study investigated the effects of miR-539 on PCa, and aimed to determine possible therapeutic targets for the treatment of PCa. The expression of miR-539 in PCa tissues, paired normal adjacent tissues and PCa cell lines (CAPAN-2, BxPC3, CFPAC1, SW1990 and PANC1), and human non-cancerous pancreatic cells (hTRET-HPNE) was determined and compared. The effects of upregulation and downregulation of miR-539 on proliferation, apoptosis, cell cycle, invasion, migration and epithelial-mesenchymal transition (EMT) of PCa cells were investigated. Additionally, the target gene of miR-539 was predicted and its effects on PCa cells were further investigated. The results revealed low expression of miR-539 in PCa tissues and cell lines. Additionally, increasing miR-539 expression inhibited the proliferation, migration, invasion and EMT of PCa cells and induced apoptosis by blocking G1 phase of the cell cycle, while reducing miR-539 expression had the opposite results. Furthermore, specificity protein 1 (SP1) was found to be the target gene of miR-539. SP1 promoted the proliferation, migration, invasion and EMT transformation of PCa cells, but these effects were reversed by high expression of miR-539. Additionally, miR-539 suppressed the proliferation, metastasis, invasion and EMT transformation of PCa cells through targeting SP1. Therefore, miR-539 overexpression may contribute toward development of novel therapeutic strategies for PCa in the future.

Phillygenin, a MELK Inhibitor, Inhibits Cell Survival and Epithelial-Mesenchymal Transition in Pancreatic Cancer Cells.

IntroductionPancreatic cancer (PC) is one of the leading causes of cancer, with the lowest 5-year survival rate of all cancer types. Given the fast metastasis of PC and its resistance to surgery, radiotherapy, chemotherapy, and combinations thereof, it is imperative to develop more effective anti-PC drugs. Phillygenin (PHI) has been reported to exert anti-cancer, anti-bacterial, and anti‐inflammatory properties. However, the mechanism of PHI in the development of PC is still unclear.MethodsThe cytotoxicity of PHI in pancreatic cancer cells was evaluated by MTT assay, and clonogenic assay was used to test the anti-proliferation of PHI. The pro-apoptotic effect of PHI was detected by flow cytometry analysis. The changes of epithelial–mesenchymal transition (EMT) in pancreatic cancer cells treated with PHI were determined by Western blot. Transwell assay was used to test the migration and invasion of PC cells after treatment with PHI. Molecular docking was used to predict the potential binding site of candidate target with PHI.ResultsPHI could inhibit the proliferation, migration, and EMT of PC cells (PANC-1 and SW1990) and induce its apoptosis. Analysis of the Cancer Genome Atlas database indicated that elevated MELK levels correlated with poor overall survival (OS) and disease-free survival (DFS) of PC patients. In addition, molecular modeling showed that PHI may potentially target the catalytic domain of maternal embryonic leucine zipper kinase (MELK). Overexpression of MELK muted the anti-PC effects of PHI.ConclusionPHI holds promise as a potent candidate drug for the treatment of PC via targeted MELK.

Lost miR-141 and upregulated TM4SF1 expressions associate with poor prognosis of pancreatic cancer: regulation of EMT and angiogenesis by miR-141 and TM4SF1 via AKT

ABSTRACT Background Transmembrane-4-L-six-family-1 (TM4SF1) functions to regulate cell growth and mobility and TM4SF1 expression was upregulated in pancreatic cancer. This study further investigated the role of TM4SF1 in regulating pancreatic cancer epithelial-mesenchymal transition (EMT) and angiogenesis and the underlying molecular events. Methods Tissue specimens were collected from 90 pancreatic cancer patients for immunohistochemical and qRT-PCR analysis of miR-141 and TM4SF1 levels, respectively. Pancreatic cancer cell lines were used for in vitro assays, while nude mice were used for the in vivo assay. Results TM4SF1 expression was upregulated, whereas miR-141 expression was lost in pancreatic cancer tissues, both of which was associated with advanced clinicopathological features and poor survival of pancreatic cancer patients. Furthermore, miR-141 was able to target and reduce TM4SF1 expression in pancreatic cancer cells and miR-141 expression inhibited pancreatic cancer cell EMT in vitro and Matrigel plug angiogenesis and lung metastasis in nude mice. At the gene level, miR-141 directly targeted and reduced TM4SF1 expression and in turn induced E-cadherin expression and reduced VEGF-A expression by suppressing activation of the AKT signaling pathway. Conclusions This study demonstrated that upregulated TM4SF1 and lost miR-141 expression were associated with advanced clinicopathological features and poor survival of pancreatic cancer patients. Lost miR-141 expression but induced TM4SF1 expression altered expression of VEGF-A and E-cadherin and promoted pancreatic cancer cell EMT and angiogenesis via the AKT signaling pathway, suggesting that targeting of miR-141 and TM4SF1 may be a potential therapeutic strategy to control pancreatic cancer.

Mechanism of miR-210 involved in epithelial–mesenchymal transition of pancreatic cancer cells under hypoxia

Purpose: To investigate the possible mechanism of miR-210 involved in epithelial–mesenchymal transition (EMT) of pancreatic cancer cells under hypoxia. Methods: In this study, we used the following approaches. Hypoxic microenvironment was stimulated in vitro, and the CCK-8 assay was used to analyze cell viability. The MiRNA expression level was measured by qRT-PCR. HOXA9, EMT-related proteins, and NF-κB activities were examined by immunoblotting assay. Dual luciferase reporter assay was used to assess whether HOXA9 was a target of miR-210.Results: Under hypoxia condition, miR-210, HIF-1α and NF-κB were increased, and the HOXA9 was reduced in PANC-1 cells. When miR-210 was overexpressed in normoxic PANC-1 cells, EMT epithelial markers of E-cadherin and β-catenin were down-regulated, and mesenchymal markers of vimentin and N-cadherin were up-regulated to promote cell migration/invasive ability, and the HOXA9 level was decreased. After HOXA9 level decreased, the sensitivity to chemotherapeutic drug of gemcitabine was reduced, NF-κB expression level and cell migration/invasive ability was enhanced. Whereas, miR-210 antagonist into hypoxic PANC-1 cells, which up-regulated E-cadherin, β-catenin level, and down-regulated vimentin and N-cadherin levels to decrease cell migration/invasive ability, and increase the HOXA9. Furthermore, increasing HOXA9 level decreased NF-κB expression level and cell migration/invasive ability, enhanced the sensitivity to gemcitabine. At last, miRDB and TargetScan predicted that HOXA9 was a target of miR-210, and dual luciferase reporter assay verified this hypothesis.Conclusion: MiR-210 inhibited the expression of HOXA9 to activate the NF-κB signaling pathway and mediated the occurrence of EMT of pancreatic cancer cells induced by HIF-1α under hypoxia.

Methyl-CpG-Binding Protein 2 Drives Furin/TGF-β1/Smad Loop to Promote Epithelial-Mesenchymal Transition in Pancreatic Cancer Cells

Methyl-CpG binding protein 2 (MeCP2) has been characterized as an oncogene in several types of cancer. However, its precise role in pancreatic ductal adenocarcinoma (PDAC) remains unclear. Hence, this study was to evaluate the potential role of MeCP2 in pancreatic cancer progression. We found that MeCP2 was up-regulated in pancreatic cancer tissues, and enhanced the abilities of migration, invasion and proliferation in pancreatic cancer cells, and promoted tumorigenesis. Further evidence revealed that MeCP2 remarkably increased mesenchymal markers of Vimentin, N-cadherin, and Snail, and down-regulated epithelial marker expression of E-cadherin and ZO-1, indicating that MeCP2 promotes Epithelial-mesenchymal transition (EMT). Also, we found that MeCP2 improved the expression of Furin, and activated TGF-β1, and raised the levels of p-Smad2/3. Importantly, we demonstrated that MeCP2, as a coactivator, enhanced Samd3 binding to the promoter of furin to improve its transcription. Therefore, it is noted that MeCP2/Smads drives the expression of Furin to activate TGF-β1 and in turn phosphorates Smad2/3, which forms a positive feedback loop to promote EMT in pancreatic cancer cells. Funding Statement: This study was supported by grants from the Natural Science Foundation of China [81672402, 81472333, 81301693]; Natural Science Foundation of Jiangsu Provincal, China [BK20171305]; Research Programs of Jiangsu Provincial Commission of Health and Family Planning, China [H201434]; the Graduate Research and Innovation Projects of Jiangsu Province [5561280022]. Declaration of Interests: The authors declared that there were no conflicts of interest. Ethics Approval Statement: The protocol was approved by the Institutional Animal Care and Use Committee of Jiangsu University, Zhenjiang, China.

Upregulation of LASP2 inhibits pancreatic cancer cell migration and invasion through suppressing TGF-β-induced EMT.

LASP2 (LIM and SH3 protein 2), a member of the LIM-protein subfamily of the nebulin group, was first identified as a splice variant of the nebulin gene. In the past, investigators mainly focused on the impact of LASP2 on cardiac diseases because of its identification in the myocardium. Recently, several studies have reported that LASP2 is associated with the progression of various cancers. However, there have been no investigations on the expression and function of LASP2 in pancreatic cancer (PC). In this study, we performed the quantitative real-time polymerase chain reaction and Western blot analysis to detect the expression of LASP2 in PC tissues and cell lines. PC cells were transfected with LASP2 overexpression plasmid or the negative control in the presence or absence of tumor growth factor-β (TGF-β). The transwell assays were used to measure the effects of LASP2 on PC cell migration and invasion. The protein expression of epithelial-mesenchymal transition (EMT) markers was detected using Western blot assay. Our results demonstrated that LASP2 was downregulated in PC tissues and cell lines. In addition, upregulation of LASP2 inhibited the PC cell migration and invasion. We also found that LASP2 upregulation reversed TGF-β-induced EMT in PC cells. Taken together, we provided novel evidence supporting the tumor-suppressor role of LASP2 in PC and suggested it as a potential therapeutic target in PC treatment.

Role of miR-214-5p in the migration and invasion of pancreatic cancer cells.

To analyze the role of miR-214-5p in proliferation and metastasis of pancreatic cancer (PC) cells, as well as its underlying mechanism. 30 pairs of PC tissues and adjacent normal tissues were collected in our Department. The expression level of miR-214-5p was detected by quantitative Real-time-polymerase chain reaction (qRT-PCR). Biological information analysis and luciferase report gene assay were used to verify potential target genes of miR-214-5p. Cell counting kit-8 (CCK-8) and transwell methods were applied to observe the interference of miR-214-5p on invasion and migration of PC cells. Western blot (WB) assay was applied to determine the expression changes of Jagged 1 (JAG1) and epithelial-mesenchymal transition (EMT)-related genes in PC cells. QRT-PCR results showed that the expression level of miR-214-5p is significantly down-regulated in PC tissues and cells. Bioinformatics software and luciferase report gene assay identified that JAG1 is a target gene of miR-214-5p. The negative correlation between protein expressions of miR-214-5p and JAG1 was assessed by Western Blot assay. Furthermore, miR-214-5p could suppress cell proliferation, invasion and migration, and it also blocked the EMT in PC cells in vitro. Meanwhile, JAG1 overexpression reversed the inhibitory effects of miR-214-5p on proliferation, invasion and migration of PC cells. Overexpressing miR-214-5p could significantly inhibit malignant behavior of PC cells through targeted regulation of JAG1. Thus, miR-214-5p might be a potential therapeutic target for treatment of PC.

Betulinic acid inhibits stemness and EMT of pancreatic cancer cells via activation of AMPK signaling.

Cancer stem cells (CSCs), which are found in various types of human cancer, including pancreatic cancer, possess elevated metastatic potential, lead to tumor recurrence and cause chemoradiotherapy resistance. Alterations in cellular bioenergetics through the regulation of 5′ adenosine monophosphate-activated protein kinase (AMPK) signaling may be a prerequisite to stemness. Betulinic acid (BA) is a well-known bioactive compound with antiretroviral and anti-inflammatory potential, which has been reported to exert anticancer effects on various types of cancer, including pancreatic cancer. The present study aimed to investigate whether BA could inhibit pancreatic CSCs via regulation of AMPK signaling. The proliferation of pancreatic cancer cells was examined by MTT and colony formation assays. The migratory and invasive abilities of pancreatic cancer cells were assessed using wound-scratch and Transwell invasion assays. In addition, the expression levels of candidate genes were measured by reverse transcription-quantitative polymerase chain reaction and western blotting. The results revealed that BA inhibited the proliferation and tumorsphere formation of pancreatic cancer cells, suppressed epithelial-mesenchymal transition (EMT), migration and invasion, and reduced the expression of three pluripotency factors [SRY-box 2 (Sox2), octamer-binding protein 4 (Oct4) and Nanog]. Furthermore, immunohistochemical analysis confirmed that there was a significant inverse association between the expression levels of phosphorylated (P)-AMPK and Sox2 in pancreatic cancer, and it was revealed that BA may activate AMPK signaling. Notably, knockdown of AMPK reversed the suppressive effects of BA on EMT and stemness of pancreatic cancer cells. In addition, BA reversed the effects of gemcitabine on stemness and enhanced the sensitivity of pancreatic cancer cells to gemcitabine. Collectively, these results indicated that BA may effectively inhibit pluripotency factor expression (Sox2, Oct4 and Nanog), EMT and the stem-like phenotype of pancreatic cancer cells via activating AMPK signaling. Therefore, BA may be considered an attractive therapeutic candidate and an effective inhibitor of the stem-like phenotype in pancreatic cancer cells. Further investigation into the development of BA as an anticancer drug is warranted.

E‑cadherin is downregulated by microenvironmental changes in pancreatic cancer and induces EMT.

The aim of the present study was to research the effect of microenvironmental change on epithelial‑mesenchymal transition (EMT) in pancreatic cancer cells and to determine the correlation between E‑cadherin expression and the prognosis of pancreatic cancer patients. We established hypoxic, serum‑deficient and TGF‑β‑induced microenvironment models of pancreatic cancer cells and studied the changes in the mRNA and protein expression of EMT‑related molecules, E‑cadherin and vimentin, using western blot analysis and real‑time PCR. Furthermore, immunohistochemistry was used to investigate E‑cadherin expression in pancreatic cancer tissues, and survival analysis and COX regression analysis were conducted. In pancreatic cancer cells under hypoxic, serum‑starved and TGF‑β‑induced microenvironments, E‑cadherin protein and mRNA levels were significantly decreased (P<0.05), while vimentin protein and mRNA expression levels were significantly increased (P<0.05). The results of immunohistochemistry showed that the protein level of E‑cadherin in pancreatic cancer tissues was positively correlated with overall survival (P<0.01). The results of Cox regression analysis showed that E‑cadherin was an independent prognostic factor in pancreatic cancer. In conclusion, E‑cadherin expression was significantly decreased by microenvironment changes, and this decrease induced EMT in pancreatic cancer cells. E‑cadherin is an independent prognostic marker in pancreatic cancer patients.

The role and mechanism of capping actin protein of muscle Z-line alpha subunit 1 in invasion and metastasis of human pancreatic cancer

Objective To observe the expression of capping actin protein of muscle Z-line alpha subunit 1 (CAPZA1) in pancreatic cancer and its relationship with prognosis, and explore the role and mechanism of CAPZA1 in regulating the invasion and metastasis of pancreatic cancer cells. Methods Immunohistochemistry was used to detect the expression of CAPZA1 and epithelial-mesenchymal transition (EMT) markers in pancreatic cancer tissues. The statistical analysis was made on the prognosis of patients with pancreatic cancer. Western blotting and real-time reverse transcriptase-polymerase chain reaction (RT-qPCR) were used to detect the expression of CAPZA1 and EMT markers in pancreatic cancer cells. Transwell, and cell counting kit-8 (CCK-8) were used to detect migration, invasion and proliferation of pancreatic cancer cells. Results The expression of CAPZA1 was negatively correlated with the biological characteristics of pancreatic cancer cells and the prognosis of patients. The expression of CAPZA1 was negatively correlated with the invasion and metastasis potentials of pancreatic cancer cells. Inhibition of CAPZA1 expression could promote the invasion and metastasis of pancreatic cancer cells (192.0±13.8 vs. 153.0±17.1, P=0.004), while over-expression of CAPZA1 could inhibit the invasion and metastasis of pancreatic cancer cells (107.0±18.5 vs. 153.0±17.1, P=0.004). The expression of CAPZA1 was correlated with the expression of EMT markers E-cadherin (0.76±0.09 vs. 0.57±0.14, P=0.034), N-cadherin (0.53±0.12 vs. 0.74±0.10, P=0.017) and Vimentin (0.56±0.20 vs. 0.93±0.19, P=0.017). Conclusion CAPZA1 can inhibit the EMT of pancreatic cancer cells and reduce the invasion ability of them0 by regulating the assembly of cell skeleton. Key words: Pancreatic cancer; Capping actin protein of muscle Z-line alpha subunit 1; Cell skeleton; Invasion; Epithelial-mesenchymal transition

ASIC1 and ASIC3 contribute to acidity-induced EMT of pancreatic cancer through activating Ca2+/RhoA pathway

Extracellular acid can have important effects on cancer cells. Acid-sensing ion channels (ASICs), which emerged as key receptors for extracellular acidic pH, are differently expressed during various diseases and have been implicated in underlying pathogenesis. This study reports that ASIC1 and ASIC3 are mainly expressed on membrane of pancreatic cancer cells and upregulated in pancreatic cancer tissues. ASIC1 and ASIC3 are responsible for an acidity-induced inward current, which is required for elevation of intracellular Ca2+ concentration ([Ca2+]i). Inhibition of ASIC1 and ASIC3 with siRNA or pharmacological inhibitor significantly decreased [Ca2+]i and its downstream RhoA during acidity and, thus, suppressed acidity-induced epithelial–mesenchymal transition (EMT) of pancreatic cancer cells. Meanwhile, downregulating [Ca2+]i with calcium chelating agent BAPTA-AM or knockdown of RhoA with siRNA also significantly repressed acidity-induced EMT of pancreatic cancer cells. Significantly, although without obvious effect on proliferation, knockdown of ASIC1 and ASIC3 in pancreatic cancer cells significantly suppresses liver and lung metastasis in xenograft model. In addition, ASIC1 and ASIC3 are positively correlated with expression of mesenchymal marker vimentin, but inversely correlated with epithelial marker E-cadherin in pancreatic cancer cells. In conclusion, this study indicates that ASICs are master regulator of acidity-induced EMT. In addition, the data demonstrate a functional link between ASICs and [Ca2+]i/RhoA pathway, which contributes to the acidity-induced EMT.

Methyl-CpG-binding domain 3 inhibits epithelial\u2013mesenchymal transition in pancreatic cancer cells via TGF-\u03b2/Smad signalling

Background:Methyl-CpG-binding domain 3 (MBD3) is an aberrant expression in human malignancies. However, the role of MBD3 in pancreatic cancer progression remains to be clarified. In this study, we investigated the effects of MBD3 on the epithelial–mesenchymal transition (EMT), and the underlying mechanism in pancreatic cancer cells.Methods:The abilities of migration and invasion were examined by transwell and BD Matrigel invasion assays. EMT and TGF-β/Smad signalling were evaluated.Results:First, we find that MBD3 expression is lower in pancreatic cancer tissues than that in non-tumour tissues, and patients with lower MBD3 levels survive significantly less than those with higher levels. Subsequently, we find that MBD3 knockdown promotes the abilities of migration and invasion, while MBD3 overexpression inhibits the above abilities. Also, MBD3 knockdown remarkably increases mesenchymal markers expression of Vimentin, α-SMA, Snail, N-cadherin, β-catenin, and downregulates epithelial markers expression of E-cadherin. On the contrary, MBD3 overexpression results in the opposite effects. Further evidence reveals that MBD3 knockdown up-regulates expression of TGF-β, and then activates p-Smad2 and p-Smad3, while MBD3 overexpression results in downregulation of TGF-β, p-Smad2, and p-Smad3.Conclusions:MBD3 inhibits EMT in pancreatic cancer cells probably via TGF-β/Smad signalling, and may be a new candidate target for diagnostics and prognosis of pancreatic cancer.

Impact by pancreatic stellate cells on epithelial-mesenchymal transition and pancreatic cancer cell invasion: Adding a third dimension in vitro

Pancreatic cancer is associated with a highly abundant stroma and low-grade inflammation. In the local tumour microenvironment, elevated glucose levels, the presence of tumour-associated stellate cells and macrophages are hypothesised to promote the tumour progression and invasion. The present study investigated the influence by the microenvironment on pancreatic cancer cell invasion in vitro. After co-culture with tumour-associated pancreatic stellate cells (TPSCs), pancreatic cancer cells displayed up to 8-fold reduction in levels of epithelial-mesenchymal transition (EMT) markers E-cadherin and ZO-1, while β-catenin and vimentin levels were increased. A 3D organotypic model showed that TPSCs stimulated pancreatic cancer cell invasion, both as single cell (PANC-1) and cohort (MIAPaCa-2) invasion. The combined presence of TPSCs and M2-like macrophages induced invasion of the non-invasive BxPC-3 cells. High glucose conditions further enhanced changes in EMT markers as well as the cancer cell invasion. In summary, co-culture with TPSCs induced molecular changes associated with EMT in pancreatic cancer cells, regardless of differentiation status, and the organotypic model demonstrated the influence of microenvironmental factors, such as glucose, stellate cells and macrophages, on pancreatic cancer cell invasion.

YAP overexpression promotes the epithelial-mesenchymal transition and chemoresistance in pancreatic cancer cells.

The expression of Yes-associated protein (YAP) has been reported to be dysregulated in pancreatic cancer. However, its contributions to tumor formation and progression remain to be elucidated. The present study demonstrated that YAP overexpression promoted the epithelial‑mesenchymal transition (EMT) in a manner associated with pancreatic cancer invasion in vitro. RNA interference‑mediated silencing of YAP attenuated cell invasion in vitro. Mechanistically, the present study demonstrated that YAP overexpression fosters pancreatic cancer progression by inducing the EMT in pancreatic cancer cells by activating the AKT cascade, which can counteract the effect of gemcitabine. These data suggested that the YAP acts synergistically to promote pancreatic cancer progression by hyperactivation of AKT signaling. The present study revealed YAP as a potential therapeutic target for pancreatic cancer and a biomarker for predicting gemcitabine treatment response.