Expression Of EMT-related Markers Research Articles

MCM6 promotes metastasis of hepatocellular carcinoma via MEK/ERK pathway and serves as a novel serum biomarker for early recurrence

BackgroundThe high incidence of recurrence and metastasis of hepatocellular carcinoma (HCC) necessitate the discovery of new predictive biomarkers of invasion and prognosis. Minichromosome maintenance complex component 6 (MCM6), which has been reported to up-regulate in multiple malignancies, was considered to be a novel diagnoses biomarker in HCC. However, its functional contributions and prognostic value remain unclear.MethodsThe expression of MCM6 was analyzed in 70 HCC tissues and 5 HCC cell lines by immunohistochemistry and real-time RT-PCR. The roles of MCM6 in HCC cell proliferation, migration and invasion were explored by CCK8, Wound healing and Transwell assays, respectively. Western blotting and Immunofluorescence staining were conducted to detect the protein expressions of ERK signaling pathway and EMT-related markers. To verify the above findings in vivo, we established subcutaneous xenograft tumor and orthotopic xenograft tumor models in nude mice. Finally, Enzyme-linked immunosorbent assay was used to evaluate the serum MCM6 level.ResultsMCM6 was significantly up-regulated in HCC tissues. Increased MCM6 expression was associated with aggressive clinicopathological features and worse prognosis in HCC patients. These results were consistent with our analyses of The Cancer Genome Atlas database (TCGA). Furthermore, knockdown of MCM6 significantly decreased proliferative and migratory/invasive capability of HCC cells in vitro, as well as decreased tumor volume, weight and the number of pulmonary metastases in vivo. Mechanistic analyses indicated that MCM6 promoted EMT and activated MEK/ERK signaling. More importantly, serum MCM6 levels in HCC patients were significantly higher than those in cirrhosis and healthy controls (P < 0.0001), and allowed distinguishing early recurrence with high accuracy (AUC = 0.773).ConclusionsOur findings indicate that MCM6 predicts poor prognosis and promotes metastasis in HCC. Postoperative serum MCM6 level could be valuable to detect preclinical early recurrence, indicative of a need for more careful surveillance and aggressive therapeutic intervention.

Microfluidic co-culture of pancreatic tumor spheroids with stellate cells as a novel 3D model for investigation of\xa0stroma-mediated cell motility and drug resistance

BackgroundPancreatic stellate cells (PSCs), a major component of the tumor microenvironment in pancreatic cancer, play roles in cancer progression as well as drug resistance. Culturing various cells in microfluidic (microchannel) devices has proven to be a useful in studying cellular interactions and drug sensitivity. Here we present a microchannel plate-based co-culture model that integrates tumor spheroids with PSCs in a three-dimensional (3D) collagen matrix to mimic the tumor microenvironment in vivo by recapitulating epithelial-mesenchymal transition and chemoresistance.MethodsA 7-channel microchannel plate was prepared using poly-dimethylsiloxane (PDMS) via soft lithography. PANC-1, a human pancreatic cancer cell line, and PSCs, each within a designated channel of the microchannel plate, were cultured embedded in type I collagen. Expression of EMT-related markers and factors was analyzed using immunofluorescent staining or Proteome analysis. Changes in viability following exposure to gemcitabine and paclitaxel were measured using Live/Dead assay.ResultsPANC-1 cells formed 3D tumor spheroids within 5 days and the number of spheroids increased when co-cultured with PSCs. Culture conditions were optimized for PANC-1 cells and PSCs, and their appropriate interaction was confirmed by reciprocal activation shown as increased cell motility. PSCs under co-culture showed an increased expression of α-SMA. Expression of EMT-related markers, such as vimentin and TGF-β, was higher in co-cultured PANC-1 spheroids compared to that in mono-cultured spheroids; as was the expression of many other EMT-related factors including TIMP1 and IL-8. Following gemcitabine exposure, no significant changes in survival were observed. When paclitaxel was combined with gemcitabine, a growth inhibitory advantage was prominent in tumor spheroids, which was accompanied by significant cytotoxicity in PSCs.ConclusionsWe demonstrated that cancer cells grown as tumor spheroids in a 3D collagen matrix and PSCs co-cultured in sub-millimeter proximity participate in mutual interactions that induce EMT and drug resistance in a microchannel plate. Microfluidic co-culture of pancreatic tumor spheroids with PSCs may serve as a useful model for studying EMT and drug resistance in a clinically relevant manner.

EHMT2 is a metastasis regulator in breast cancer

Various modes of epigenetic regulation of breast cancer proliferation and metastasis have been investigated, but epigenetic mechanisms involved in breast cancer metastasis remain elusive. Thus, in this study, EHMT2 (a histone methyltransferase) was determined to be significantly overexpressed in breast cancer tissues and in Oncomine data. In addition, knockdown of EHMT2 reduced cell migration/invasion and regulated the expression of EMT-related markers (E-cadherin, Claudin 1, and Vimentin). Furthermore, treatment with BIX-01294, a specific inhibitor of EHMT2, affected migration/invasion in MDA-MB-231 cells. Therefore, our findings demonstrate functions of EHMT2 in breast cancer metastasis and suggest that targeting EHMT2 may be an effective therapeutic strategy for preventing breast cancer metastasis.

MiR-200c/141 Regulates Breast Cancer Stem Cell Heterogeneity via Targeting HIPK1/β-Catenin Axis.

Increasing evidence demonstrates the existence of two inter-convertible states of breast cancer stem cells (BCSCs) with distinct behaviors in proliferation and mobility, and the BCSC heterogeneity is accurately regulated by sophisticated mechanisms including microRNAs. The microRNA-200 family including miR-200c/141 cluster was reported to affect cancer cell invasion and metastasis by regulating epithelial to mesenchymal transition (EMT). However, the effect of miR-200 family on BCSC heterogeneity is uncertain. Thus, we investigated whether the miR-200c/141 cluster had different effects on breast tumor growth and metastasis by switching the two states of BCSC.Methods: The spontaneous mammary tumor mouse model with miR-200c/141 conditional knockout was utilized for analyzing the role of miR-200c/141 cluster in vivo. The effect of miR-200c/141 cluster on BCSCs was performed by CD24/CD29 staining and ALDEFLUOR assay. miR-200c/141 target expression and EMT-related marker expression were verified in tumor sections, primary cells and breast cancer cell lines by qRT-PCR or western blotting. Statistical analysis was determined using two-way ANOVA and Student's t-test. All values were presented as the mean ± s.e.m.Results: The deletion of miR-200c/141 cluster regulated BCSC heterogeneity and promoted the EMT-like BCSC generation, which resulted in increased tumor metastasis and inhibited tumor growth by directly upregulating the target gene homeodomain-interacting protein kinase 1 (HIPK1) and sequential β-catenin activation.Conclusions: Our results indicated that miR-200c/141 played biphasic roles in breast tumor progression via affecting the BCSC heterogeneity, suggesting targeting BCSC heterogeneity to simultaneously restrict breast cancer initiation and metastasis could be a promising therapeutic strategy for breast cancer.

Transcription factor E2F1 promotes EMT by regulating ZEB2 in small cell lung cancer

BackgroundEpithelial-mesenchymal transition (EMT) is an early event in tumour invasion and metastasis, and widespread and distant metastasis at early stages is the typical biological behaviour in small cell lung cancer (SCLC). Our previous reports showed that high expression of the transcription factor E2F1 was involved in the invasion and metastasis of SCLC, but the role of E2F1 in the process of EMT in SCLC is unknown.MethodsImmunohistochemistry was performed to evaluate the expressions of EMT related markers. Immunofluorescence was used to detect the expressions of cytoskeletal proteins and EMT related markers when E2F1 was silenced in SCLC cell lines. Adenovirus containing shRNA against E2F1 was used to knock down the E2F1 expression, and the dual luciferase reporter system was employed to clarify the regulatory relationship between E2F1 and ZEB2.ResultsIn this study, we observed the remodelling of cytoskeletal proteins when E2F1 was silenced in SCLC cell lines, indicating that E2F1 was involved in the EMT in SCLC. Depletion of E2F1 promoted the expression of epithelial markers (CDH1 and CTNNB1) and inhibited the expression of mesenchymal markers (VIM and CDH2) in SCLC cell lines, verifying that E2F1 promotes EMT occurrence. Next, the mechanism by which E2F1 promoted EMT was explored. Among the CDH1 related inhibitory transcriptional regulators ZEB1, ZEB2, SNAI1 and SNAI2, the expression of ZEB2 was the highest in SCLC tissue samples and was highly consistent with E2F1 expression. ChIP-seq data and dual luciferase reporter system analysis confirmed that E2F1 could regulate ZEB2 gene expression.ConclusionOur data supports that E2F1 promotes EMT by regulating ZEB2 gene expression in SCLC.

MgCl2 and ZnCl2 promote human umbilical vein endothelial cell migration and invasion and stimulate epithelial-mesenchymal transition via the Wnt/β-catenin pathway.

Previous studies have demonstrated that magnesium and zinc ions promote the migration and epithelial-mesenchymal transition (EMT) of cancer/endothelial cells. However, the impact of MgCl2 and ZnCl2 on the migration, invasion and EMT of human umbilical vein endothelial cells (HUVECs) and the involved mechanisms remain unclear. In the present study, HUVECs were incubated with various doses of MgCl2 and ZnCl2. The optimum concentrations of MgCl2 and ZnCl2 were selected by MTT assay. The migration and invasion capabilities of HUVECs were analyzed by Transwell assays. Subsequently, the expression of matrix metalloproteinase (MMP)-2 and MMP-9 mRNA and protein were determined by reverse transcription-quantitative polymerase chain reaction, western blotting and ELISA. MMP-2 and MMP-9 activities were measured by gelatin zymography. Immunofluorescence staining was performed to investigate cytoskeletal dynamics using Acti-stain™ 488 Fluorescent Phalloidin. Subsequently, the expression of EMT-related markers at the mRNA and protein levels and the activation of Wnt/β-catenin signaling were analyzed. The results identified increases in MMP-2 and MMP-9 expression and activity, indicating that MgCl2 and ZnCl2 promoted HUVEC migration and invasion. In addition, MgCl2 and ZnCl2 treatment induced cytoskeleton remodeling and stimulated EMT via activation of the Wnt/β-catenin signaling pathway, characterized by a decrease in E-cadherin and increases in N-cadherin, vimentin and Snail. These results suggest that MgCl2 and ZnCl2 may enhance the migration and invasion capabilities of HUVECs and promote EMT through the Wnt/β-catenin pathway.

Long Non-Coding RNA (LncRNA) HOXA11-AS Promotes Breast Cancer Invasion and Metastasis by Regulating Epithelial-Mesenchymal Transition.

BackgroundTo detect the expression of lncRNA HOXA11-AS and its biological effect in breast cancer.Material/MethodsIn this study, fluorescent quantitative real-time PCR (qRT-PCR), MTT assay and clone formation assay, flow cytometry, Transwell assay and wound healing assay, immunofluorescence, and Western blot analysis were conducted to detect the expression of lncRNA HOXA11-AS, cell proliferation activity, cell apoptosis rate and cell cycle distribution, the changes of cell invasion and metastasis capacity, and the expressions of molecular markers of epithelial-mesenchymal transition (EMT), respectively. Additionally, a nude mouse metastatic tumor model was established to study the influence of lncRNA HOXA11-AS on invasion and metastasis capacity of breast cancer cells.ResultsThe qRT-PCR experiment results showed that HOXA11-AS expression in breast cancer tissue of 50 patients was relatively higher than that in tissue adjacent to cancer. MTT assay suggested that tumor cell proliferation capacity was suppressed followed by the knockdown of lncRNA HOXA11-AS expression in MDA-MB-231 and MCF-7 cells; flow cytometry results demonstrated that interfering in lncRNA HOXA11-AS could induce tumor cell apoptosis and promote cell cycle progression to be arrested in G1/G0 stage; experiments in vivo/vitro manifested that interfering in lncRNA HOXA11-AS could inhibit tumor cell invasion and migration capacity by affecting the expressions of EMT-related molecular markers (E-cadherin, N-cadherin, Vimentin).ConclusionsHigh expression of lncRNA HOXA11-AS promotes breast cancer invasion and metastasis by affecting EMT, and interfering in lncRAN HOXA11-AS expression provides a theoretical basis and important molecular target for inhibiting the distant metastasis of breast cancer in clinical practice.

Abstract 5809: Fluorescence-guided spatiotemporal dynamics of epithelial-mesenchymal transition under inflammatory microenvironment during colorectal cancer progression

Abstract Background: Epithelial-mesenchymal transition (EMT) is a biological process, by which epithelial cancer cells acquire mesenchymal phenotype with malignant properties for invasion and metastasis, leading to poor prognosis. Inflammatory microenvironment has been shown to be responsible for the development and progression of colorectal cancer. However, the role of inflammatory microenvironment in the EMT-related tumor progression remains unclear. To explore the relationship between inflammatory microenvironment and EMT, a live imaging system for EMT is a promising strategy on the in vitro and in vivo experiments. In this study, we developed a fluorescence-guided live cell imaging system for the assessment of spatiotemporal dynamics of EMT, and investigated the potential of inflammatory microenvironment for the induction of EMT phenotype in human colorectal cancer. Methods: Two human colorectal cancer cell lines, HCT116 and RKO, were stably transfected with vimentin promoter-driven red fluorescence protein TurboFP635 expression vector. Both cell lines were treated with inflammatory cytokines, IL-1β (1 ng/ml) and TNF-α (20 ng/ml), or co-cultured with mouse macrophage cell line RAW264.7 in the presence of lipopolysaccharide (LPS) (200 ng/ml). The time-lapse live imaging was observed by confocal laser scanning microscope. Migration and invasion properties were examined by transwell chamber assays. The fluorescence intensity was measured by microplate reader and flow cytometric analysis. The expression of EMT-related markers was assessed by Western blot analysis and q-PCR. EMT-induced HCT116 and RKO cells were treated with anti-inflammatory agents, aspirin (1mM) and salicylic acid (1 mM), for the suppression of EMT. Results: Inflammatory cytokines (IL-1β and TNF-α) induced red fluorescence intensity and morphological change like mesenchymal phenotype in HCT116 and RKO cells. Removal of inflammatory cytokines attenuated red fluorescence intensity and morphological change in both cells. Inflammatory cytokines also induced the migration and invasion properties in association with EMT-related markers. Moreover, co-culture with LPS-stimulated inflammatory macrophages also induced red fluorescence intensity and morphological change as well as inflammatory cytokines. Anti-inflammatory agents significantly suppressed the fluorescence-related EMT phenotype under inflammatory microenvironment. Conclusions: These results suggest that inflammatory microenvironment has a great potential for the induction of EMT process during colorectal cancer progression. This unique fluorescence-guided EMT imaging system is useful method for the exploration of inflammation-mediated tumor progression. Citation Format: Takeshi Ieda, Hiroshi Tazawa, Satoru Kikuchi, Shinji Kuroda, Toshiaki Ohara, Kazuhiro Noma, Hiroyuki Kishimoto, Takeshi Nagasaka, Masahiko Nishizaki, Shunsuke Kagawa, Takeshi Imamura, Toshiyoshi Fujiwara. Fluorescence-guided spatiotemporal dynamics of epithelial-mesenchymal transition under inflammatory microenvironment during colorectal cancer progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5809. doi:10.1158/1538-7445.AM2017-5809

Long non-coding ribonucleic acid zinc finger antisense 1 promotes the progression of colonic cancer by modulating ZEB1 expression.

Long non-coding RNA zinc finger antisense 1 (ZFAS1) is frequently amplified in hepatocellular carcinoma and promotes metastasis by increasing zinc finger E-box binding homeobox 1 (ZEB1), which can potentiate the progression of epithelial-to-mesenchymal transition (EMT). However, the expression pattern and role of ZFAS1 in colonic cancer remains unknown. The present study aimed to investigate the role of ZFAS1 and its clinical significance in colonic cancer. Paired clinical colonic cancer tissue samples and clinicopathologic characteristics of 73 patients were analyzed. Quantitative real-time polymerase chain reaction analysis was used to evaluate expression levels of ZFAS1 in colonic cancer tissues, cell lines, and plasma. ZEB1 and EMT-related markers expression levels also were explored. Cell biology assays were used to explore the biologic consequences of ZFAS1 in regulating cell proliferation and invasion, as well as the roles in regulating EMT. Zinc finger antisense 1 was up-regulated in colonic cancer tissues compared with adjacent mucosa (P<0.01), and its expression level was significantly correlated with TNM stage, vascular invasion, and lymph node metastasis (P<0.05). ZFAS1 and ZEB1 were also increased in patients' plasma. Moreover, ZFAS1 promoted proliferation, invasion, and impeded apoptosis. Knockdown of ZFAS1 decreased expression of ZEB1 and increased the epithelial markers E-cadherin, ZO-1 while decreasing mesenchymal markers vimentin and N-cadherin. Long non-coding RNA ZFAS1 may function as an oncogene by modulating ZEB1 to induce EMT. Manipulation of ZFAS1 level may be a novel approach to suppress colonic cancer progression. In addition, ZFAS1 in plasma has the potential to be a diagnostic biomarker of colonic cancer.

Coexpression of growth differentiation factor 11 and reactive oxygen species in metastatic oral cancer and its role in inducing the epithelial to mesenchymal transition

The aim of this study was to investigate growth differentiation factor 11 (GDF11) and reactive oxygen species (ROS) expression in metastatic oral cancer and explored their roles in inducing epithelial to mesenchymal transition (EMT). The expression of GDF11, ROS, and EMT-related markers was evaluated in primary tumor tissues from patients with oral squamous cell carcinoma (OSCC). SCC-9cells, a human tongue carcinoma cell line, were treated with recombinant GDF11. Induction of EMT, expression of EMT-related markers, and the effect of ROS on EMT in SCC-9cells were analyzed. Overexpression of GDF11 and ROS was observed in patients with metastatic oral cancer. Downregulated expression of E-cadherin and upregulated expression of vimentin, δ-EF1, SIP-1, MMP-2, and MMP-9 were observed in patients with metastatic oral cancer, relative to the expression of these factors in patients with nonmetastatic oral cancer. With recombinant GDF11 treatment, obvious spindle-shaped cells appeared, and gene expressions of EMT-related markers were altered in SCC-9cells. Treatment with the powerful antioxidant N-acetylcysteine inhibited GDF11-induced EMT and cell migration. GDF11 induces EMT and cell migration with ROS involvement in SCC-9cells. Overexpression of GDF11 and ROS is associated with metastatic oral cancer. GDF11 and ROS may participate in metastasis of oral cancer through EMT.

SphK1 modulates cell migration and EMT-related marker expression by regulating the expression of p-FAK in colorectal cancer cells.

Sphingosine kinase1(SphK1) plays an important role in colorectal carcinoma metastasis. However, whether SphK1 modulates epithelial-mesenchymal transition(EMT)-related marker expression and the underlying mechanisms remain unclear. In this study, in order to clarify this issue, we used various colorectal cancer(CRC) cell lines, Caco2, HT29, RKO and HCT116. Each of the cell lines was divided into 3groups as follows: the control group, SKI-Ⅱ(SphK1 inhibitor) group and PF-562271[focal adhesion kinase(FAK) inhibitor] group. The migratory ability of the cells was examined by Transwell chamber assay. The mRNA and protein expression levels of SphK1, FAK(p-FAK), Slug, vimentin, N-cadherin and E-cadherin were detected by PCR and western blot analysis, respectively. The results revealed that the suppression of SphK1 reduced the cell migratory ability, and decreased the expression of Slug, vimentin and N-cadherin; however, the expression of E-cadherin was increased. Moreover, the inhibition of SphK1 reduced the expression of p-FAK. The inhibition of FAK(p-FAK) also decreased the cell migratory ability, and decreased the expression of Slug, vimentin and N-cadherin, whereas the expression of E-cadherin was increased. Thus, our data suggest that SphK1 modulates the expression of EMT-related markers and cell migration by regulating the expression of p-FAK in CRC cells. Thus, SphK1 may play a functional role in mediating the EMT process in CRC.

USP22 drives colorectal cancer invasion and metastasis via epithelial-mesenchymal transition by activating AP4.

Ubiquitin specific peptidase 22 (USP22), a putative cancer stem cell marker, is overexpressed in liver metastases of colorectal cancer (CRC). However, the mechanism by which USP22 promotes CRC metastasis remains largely unknown. Here, we report that USP22 and AP4 are simultaneously overexpressed during TGF-β1-induced CRC cell epithelial-mesenchymal transition (EMT). USP22 up-regulation enhances CRC cell migration and invasion and EMT-related marker and AP4 expression, but these effects are partly blocked by AP4 knockdown. In addition, USP22 binds to the promoter region of AP4 to activate its transcription. In vivo, elevated USP22 expression promotes CRC cell metastasis to the lungs in nude mice, as evidenced by the fact that CRC metastatic nodules stain deeply positive for USP22 and AP4. In human CRC tissues, the genes encoding USP22 and AP4 are overexpressed in metastatic liver lesions compared with primary cancer tissues, and their overexpression is significantly associated with poor CRC patient survival. These findings indicate that USP22 and AP4 may serve as prognostic markers for predicting the risk of developing distant metastases in CRC.

Correlation between epithelial-mesenchymal transition related markers and poor clinical outcome of triple-negative breast cancer

Objective To investigate the expression of epithelial-mesenchymal transition (EMT) related molecular markers (vimentin, smooth muscle actin, osteonectin, N-cadherin, and E-cadherin loss) and EMT inducer zinc-finger E-box binding homeobox 1 (ZEB1) in triple-negative breast cancer (TNBC) samples and inquire the correlation between their expression and clinicopathological features of the tumors. Methods We analyzed immunohistochemical expression of EMT-related markers mentioned above in 173 TNBC samples and correlation between their expression with clinicopathological features of the tumors, including clinical outcome. Results The expression levels of vimentin were more frequent in basal-like TNBC (87/154, 56.5%) than in non-basal-like TNBC (3/17, 17.6%). The ZEB1 expression was correlated only with the expression of smooth muscle actin (P=0.002). The survival analyses revealed that vimentin (P=0.515), smooth muscle actin (P=0.739), osteonectin (P=0.094), E-cadherin (P=0.925) and N-cadherin (P=0.509) were not associated with patients’ clinical outcomes. However, the ZEB1 expression was found to be an independent prognostic factor for poor disease-free survival. Conclusion These findings indicate that the expression of EMT-related markers in TNBC can be a signature of a certain subgroup of TNBC. Key words: Triple-negative breast cancer; Epithelial-mesenchymal transition; Vimentin; Smooth muscle actin; Osteonectin; N-cadherin; E-cadherin

Neferine Attenuates Epithelial-Mesenchymal Transition of Alveolar Epithelial Cells via TGF-β Signaling Pathway

Background: Idiopathic pulmonary fibrosis (IPF) is an incurable, progressive, and fatal interstitial lung disease with the characteristics of lung tissue damage and an enhancement in extracellular matrix (ECM). Alveolar epithelial cells (AMs) are major target cells that can directly promote to occurring of pulmonary fibrosis by acquisition of a mesenchymal phenotype through epithelial- mesenchymal transition (EMT). Neferine, a component of Chinese herbs, has been thought to be involved in anti-fibrotic activity in experimental lung fibrosis. However, its mechanism is not clear. In this study, we explore the regulation of neferine in TGF-β-induced EMT in lung fibrosis model and illustrate its mechanism of action. Methods: The alveolar epithelial cell line A549 was stimulated with TGF-β1 with or without Neferine pretreatment in advance. Morphologic variations and expression of EMT-related markers, including E-cadherin, β-catenina-SMA and Vimentin were detected. Expressions of Smad2, p- Smad 2, Smad3 and p-Smad3 were measured. Results: TGF-β1–treated A549 cells were transformed into the mesenchymal morphology with less E-cadherin, β-catenin and more a-SMA, Vimentin expression. The addition of Neferine inhibited the TGF-β1–induced change of the mesenchymal phenotype. Furthermore, Neferine inhibited the TGF-β1–induced increase in the expression of p- Smad2 and p-Smad3. Conclusions: Our study illustrate that Neferine inhibits TGF-β1–induced EMT in lung fibrosis model via TGF-β signaling pathway.

Effect of PPM1H on malignant phenotype of human pancreatic cancer cells.

The objective of this study was to investigate the effect of silencing gene protein phosphatase 1H (PPM1H) on malignant phenotype of human pancreatic cancer cell line BxPC-3. In order to explore the function of PPM1H in pancreatic cancer cells, real-time PCR and western blotting were used to detect the expression of PPM1H in different pancreatic cancer cell lines. Human pancreatic cancer cell line BxPC-3 was treated with 10ng/ml TGF-β1 and 200ng/ml BMP2 for 72h, respectively, and the mRNA and protein expression levels of PPM1H and EMT-related markers (E-cadherin, vimentin) were detected by real-time PCR and western blotting, respectively. Using exogenous RNA interference technology to silence the PPM1H gene, the expression of PPM1H and EMT-related markers at mRNA and protein levels were detected by real-time PCR and western blotting. The cell migration and invasion were measured using Transwell assays. Finally, cell counting kit-8 (CCK-8) and flow cytometry were used to determine the effect of PPM1H on cell proliferation and apoptosis of BxPC-3 cells. The expression levels of PPM1H in all of the examined pancreatic cancer cell lines (BxPC-3, MIA-PACA2, PANC-1, SW1990, PANC-03.27) were lower than that of normal pancreatic ductal epithelial cells (HPDE6-C7) at both mRNA and protein levels. Both TGF-β1 and BMP2 treatment induced EMT and downregulation of PPM1H in BxPC-3 cells. By using RNA interference to transiently knock down PPM1H expression in BxPC-3 cells, we found that the expression of E-cadherin was downregulated while vimentin was up-regulated. The data suggested that silencing PPM1H gene can induce EMT in BxPC-3 cells. In addition, Transwell migration assays showed that silencing PPM1H gene can promote the invasion and metastasis of BxPC-3 cells. Cell proliferation and apotosis detection demonstrated that silencing PPM1H gene can promote the proliferation and inhibit apoptosis of BxPC-3 cells. In conclusion, PPM1H is aberrantly expressed in human pancreatic cancer cell lines and can be downregulated when EMT is induced by cytokine stimulation. Silencing PPM1H gene can induce EMT in BxPC-3 cells, and promote the invasion and metastasis of BxPC-3 cells. Moreover, silencing PPM1H gene can promote the proliferation and inhibit apoptosis of BxPC-3 cells. PPM1H may be a new tumor-suppressor factor for pancreatic cancer and provides new insight into molecular targets for gene therapy of pancreatic cancer.

Elk-3 Contributes to the Progression of Liver Fibrosis by Regulating the Epithelial-Mesenchymal Transition.

Background/AimsThe role of Elk-3 in the epithelial-mesenchymal transition (EMT) during liver fibrogenesis remains unclear. Here, we determined the expression of Elk-3 in in vitro and in vivo models and in human liver fibrotic tissues. We also investigated the molecular relationships among Elk-3, early growth response-1 (Egr-1), and the mitogen activated protein kinases (MAPK) pathway during EMT in hepatocytes.MethodsWe established anin vitro EMT model in which normal mouse hepatocyte cell lines were treated with transforming growth factor (TGF)-β1 and a CCl4-induced liver fibrosis model. Characteristics of EMT were determined by evaluating the expression levels of related markers. The expression of Elk-3 and its target Egr-1 were analyzed using Western blotting. Gene silencing of Elk-3 was performed using an siRNA knockdown system.ResultsThe expression levels of mesenchymal markers were increased during TGF-β1-induced EMT of hepatocytes. The expression levels of Elk-3 and Egr-1 were significantly (p<0.05) increased during the EMT of hepatocytes, in CCl4-induced mouse liver fibrotic tissues, and in human liver cirrhotic tissues. Silencing of Elk-3 and inhibition of the Ras-Elk-3 pathway with an inhibitor suppressed the expression of EMT-related markers. Moreover, Elk-3 expression was regulated by p38 MAPK phosphorylation during EMT.ConclusionsElk-3 contributes to the progression of liver fibrosis by modulating the EMT via the regulation of Egr-1 under MAPK signaling.

Abstract 1613: A novel live imaging system for inflammation-induced epithelial-mesenchymal transition in colorectal cancers

Abstract Background: Epithelial-mesenchymal transition (EMT) is a biological process, by which epithelial cancer cells acquire the mesenchymal phenotype with malignant properties for invasion and metastasis, leading to poor prognosis. However, as EMT is a reversible process and depends on tumor microenvironment, the precise role of EMT in cancer progression remains unclear. Inflammatory microenvironment has been shown to be responsible for the development and progression of colorectal cancers. To evaluate the implication of EMT in the inflammation-mediated colorectal cancer progression, a live imaging system for EMT is needed on the in vitro and in vivo experiments. In this study, we generated the EMT-detectable colorectal cancer cells by introducing mesenchymal cell marker promoter-driven fluorescence protein expression vector, and investigated whether inflammation-induced EMT is detectable in vitro. Methods: To generate the EMT-detectable colorectal cancer cells HCT116-VIM635, human colorectal cancer cell line HCT116 was stably transfected with vimentin promoter-driven red fluorescence protein TurboFP635 expression vector. EMT was induced in HCT116-VIM635 cells by treatment with inflammatory cytokines, IL-1β (1 ng/ml) and TNF-α (20 ng/ml), or by co-culture with mouse macrophage cell line RAW264.7 in the presence of lipopolysaccharide (LPS) (200 ng/ml). The time-lapse live imaging was observed by confocal laser scanning microscope. Migration and invasion properties were examined by transwell chamber assays. The fluorescence intensity was measured by microplate reader and flow cytometric analysis. The expression of EMT-related markers was assessed by Western blot analysis. Results: Administration of IL-1β or TNF-α induced the TurboFP635 expression in consistent with morphological change like mesenchymal phenotype in HCT116-VIM635 cells. Removal of these inflammatory cytokines attenuated the TurboFP635 expression and morphological change in HCT116-VIM635 cells, suggesting the detection of reverse EMT process in these cells. Inflammatory cytokines also induced the migration and invasion properties and the expression of EMT-related markers in HCT116-VIM635 cells. Moreover, co-culture with RAW264.7 cells stimulated with LPS also induced the TurboFP635 expression and morphological change as well as inflammatory cytokines in HCT116-VIM635 cells. Conclusions: Our results suggest that this unique live imaging system for EMT has a great potential to detect reversible EMT process during inflammation-induced cancer progression. This system would be useful strategy to assess the role of EMT during inflammation-mediated cancer progression in vivo. Citation Format: Takeshi Ieda, Hiroshi Tazawa, Satoru Kikuchi, Shinji Kuroda, Toshiaki Ohara, Kazuhiro Noma, Hiroyuki Kishimoto, Takeshi Nagasaka, Masahiko Nishizaki, Shunsuke Kagawa, Toshiyoshi Fujiwara. A novel live imaging system for inflammation-induced epithelial-mesenchymal transition in colorectal cancers. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1613.

Aquaporin 1 Is an Independent Marker of Poor Prognosis in Lung Adenocarcinoma.

Background:Aquaporin 1 (AQP1) overexpression has been shown to be associated with uncontrolled cell replication, invasion, migration, and tumor metastasis. We aimed to evaluate AQP1 expression in lung adenocarcinomas and to examine its association with clinicopathological features and prognostic significance. We also investigated the association between AQP1 overexpression and epithelial-mesenchymal transition (EMT) markers.Methods:We examined AQP1 expression in 505 cases of surgically resected lung adenocarcinomas acquired at the Seoul National University Bundang Hospital from 2003 to 2012. Expression of AQP1 and EMT-related markers, including Ecadherin and vimentin, were analyzed by immunohistochemistry and tissue microarray.Results:AQP1 overexpression was associated with several aggressive pathological parameters, including venous invasion, lymphatic invasion, and tumor recurrence. AQP1 overexpression tended to be associated with higher histological grade, advanced pathological stage, and anaplastic lymphoma kinase (ALK) translocation; however, these differences were not statistically significant. In addition, AQP1 overexpression positively correlated with loss of E-cadherin expression and acquired expression of vimentin. Lung adenocarcinoma patients with AQP1 overexpression showed shorter progression-free survival (PFS, 46.1 months vs. 56.2 months) compared to patients without AQP1 overexpression. Multivariate analysis confirmed that AQP1 overexpression was significantly associated with shorter PFS (hazard ratio, 1.429; 95% confidence interval, 1.033 to 1.977; p=.031).Conclusions:AQP1 overexpression was thereby concluded to be an independent factor of poor prognosis associated with shorter PFS in lung adenocarcinoma. These results suggested that AQP1 overexpression might be considered as a prognostic biomarker of lung adenocarcinoma.

High tumor-associated macrophages infiltration is associated with poor prognosis and may contribute to the phenomenon of epithelial-mesenchymal transition in gastric cancer.

BackgroundRecent studies show that epithelial–mesenchymal transition (EMT) and tumor-associated macrophages (TAMs) contribute to the progression and poor prognosis of carcinoma through multiple mechanisms. Both inflammation and changing of epithelium have a close relationship with tumorigenesis of gastric cancer. However, the relevance between EMT and TAMs is still unclear in gastric cancer and needs more scientific research. This study is designed to explore the relationship between EMT and TAMs in gastric cancer.Materials and methodsImmunohistochemistry was used to detect the expression of EMT-related proteins and TAM markers in cancer tissues and normal gastric tissues.ResultsHigh levels of EMT and TAMs infiltration are related to aggressive features and independent prognostic factors in gastric cancer, respectively. In addition, expression of the two indicators is associated with expression of transforming growth factor-β1 (TGF-β1). Infiltration of TAMs is also associated with EMT-related marker in gastric cancer.ConclusionOur results suggest that high levels of EMT and TAMs infiltration are related to aggressive features and independent prognostic factors in gastric cancer, respectively. A correlation was found between EMT- and TAM-related indicators, which may be associated with TGF-β signaling pathway. The level of TAMs infiltration plays an important role in gastric cancer, the markers of which can be used as prognostic indicators.

LncRNA-ATB mediated E-cadherin repression promotes the progression of colon cancer and predicts poor prognosis.

Long non-coding RNA-activated by TGF-β (lncRNA-ATB) promotes the invasion-metastasis cascade in hepatocellular carcinoma via downregulating E-cadherin (E-cad) and inducing epithelial-to- mesenchymal transition (EMT) and is clinically significant in human colon cancer. However, its molecular mechanisms in colon cancer progression remain unclear. This study aimed to elucidate the role of lncRNA-ATB and its clinical value in colon cancer. Expression levels of lncRNA-ATB in colon cancer tissues and colon cancer cell lines were evaluated using quantitative real-time polymerase chain reaction. The clinicopathological significance and prognostic value of lncRNA-ATB were investigated, and roles of lncRNA-ATB in regulating E-cad and other EMT-related markers expression and colon cancer progression were evaluated in vitro. Expression levels of lncRNA-ATB and E-cad in human plasma were evaluated. Long non-coding RNA-activated by TGF-β was upregulated in colon cancer tissues compared with adjacent mucosa (P < 0.001). LncRNA-ATB levels were also higher in metastatic cancer tissues (P < 0.001). Among the three highly invasive colon cancer cell lines, lncRNA-ATB levels were relatively higher with concurrent low levels of E-cad compared with levels in the three low-invasive cell lines. LncRNA-ATB expression correlated with pN stage (P < 0.01) and American Joint Committee on Cancer stage (P < 0.01). Striking differences were observed in overall survival and disease-free survival in cases with both high lncRNA-ATB expression and low E-cad expression. Reduction of lncRNA-ATB increased expression of epithelial markers E-cad, ZO-1, and decreased expression of mesenchymal markers ZEB1 and N-cadherin (N-cad), and significantly influenced colon cancer cell progression. Plasma lncRNA-ATB was upregulated in colon cancer patients one month after surgery (P < 0.05). Long non-coding RNA-activated by TGF-β may act on colon tumorigenesis by suppressing E-cad expression and promoting EMT process, and lncRNA-ATB inhibition may provide a promising therapeutic option for suppressing colon cancer progression.