Expression Of EMT-related Markers Research Articles

LncRNA FBXL19-AS1 promotes proliferation and metastasis via regulating epithelial-mesenchymal transition in non-small cell lung cancer.

To detect the relative expression of long non-coding ribonucleic acid (lncRNA) F-box and leucine-rich repeat protein 19-antisense RNA 1 (FBXL19-AS1) in tissues and cells of non-small-cell lung cancer (NSCLC), and investigate the mechanism of lncRNA FBXL19-AS1 in promoting NSCLC cell proliferation and metastasis by regulating epithelial-mesenchymal transition (EMT) via in vitro experiments. The relative expression of lncRNA FBXL19-AS1 in NSCLC tissues and cells was detected via quantitative Real Time-Polymerase Chain Reaction (qRT-PCR). The colony formation assay was performed to study the impact of interference with lncRNA FBXL19-AS1 expression on NSCLC cell proliferation. The flow cytometry was applied to determine the influence of si-FBXL19-AS1 on the cycle distribution of NSCLC cells. After the interference with lncRNA FBXL19-AS1 expression, the transwell assay was utilized to measure the changes in the migratory and invasive abilities of NSCLC cells, while the expression changes in EMT-related molecular markers was detected via Western blotting. The results of qRT-PCR showed that the expression of lncRNA FBXL19-AS1 in NSCLC tissues and cells was up-regulated. According to the results of the colony formation assay, the proliferative capacity of NSCLC cells was decreased after the interference with lncRNA FBXL19-AS1 expression. In flow cytometry, it was indicated that the cell cycle was arrested at the G0/G1 phase in the experimental group compared with that in the control group. The transwell assay results showed that the migratory and invasive abilities of NSCLC cells were weakened after the interference with lncRNA FBXL19-AS1 expression. The results of the Western blotting assay revealed that the expressions of EMT-related molecular markers (E-cadherin, N-cadherin, etc.) were changed. The expression of lncRNA FBXL19-AS1 in NSCLC tissues and cells is up-regulated, and the highly expressed lncRNA FBXL19-AS1 can promote NSCLC cell proliferation and metastasis by regulating the EMT.

Nicotine promotes cervical metastasis of oral cancer by regulating peroxiredoxin 1 and epithelial-mesenchymal transition in mice.

BackgroundTobacco is a major risk factor for oral squamous cell carcinoma (OSCC). However, the role of nicotine in OSCC is not completely understood.Materials and methodsTo analyze the mechanisms of nicotine-induced cervical metastasis, we investigated whether nicotine induced invasion, migration, and epithelial–mesenchymal transition (EMT) via regulating peroxiredoxin 1 (Prx1) in CAL 27 cells. In addition, we established a mouse model to confirm the roles of nicotine in regulating Ets1/Prx1/EMT signaling in OSCC metastasis.ResultsWe showed that nicotine induced CAL 27 cell invasion, migration, EMT, and Prx1 and Ets1 expression. Prx1 knockdown inhibited cell invasion, migration, and EMT. Ets1 silencing downregulated Prx1 expression and EMT. Prx1 and Ets1 were shown to interact in CAL 27 cells treated with nicotine, and nicotine could significantly upregulate the binding of the transcription factor Ets1 to the Prx1 gene promoter region. Additionally, an in vivo study showed that nicotine induced tumor metastasis and EMT. Prx1 knockdown inhibited cervical metastasis rates and EMT progression. No significant differences in metastasis rates and EMT-related marker expression levels were observed between vehicle- and nicotine-treated mice.ConclusionThe results indicate that nicotine promotes cervical lymph node metastasis through regulating Ets1/Prx1/EMT signaling during OSCC pathogenesis; consequently, Prx1 may represent a potential target for the prevention and treatment of OSCC.

Fibrillin-1, a novel TGF-beta-induced factor, is preferentially expressed in metaplastic carcinoma with spindle sarcomatous metaplasia

TGF-β induces epithelial-mesenchymal transition (EMT), which is involved in tumour progression. This study aims to identify and characterise novel factors potentially related to TGF-β-mediated tumour aggression in breast cancer. We treated the human mammary epithelial cell line MCF10A with TGF-β and observed TGF-β-dependent upregulation of FBN1, involving demethylation of CpG sites, in MCF10A cells undergoing EMT. The biological importance of fibrillin-1, encoded by FBN1, was evaluated through immunohistochemistry on 225 breast cancer specimens of various subtypes. Fibrillin-1 expression was observed only in metaplastic carcinoma of the breast (MCB) (51.7%), and the expression was observed in spindle sarcomatous metaplasia (SSM), but not in other metaplasia, including matrix-producing, pleomorphic, and squamous metaplasia, and carcinomatous components of both MCB and non-MCB. Fibrillin-1 expression was also restricted to the SSM of non-mammary carcinosarcomas of various organs. Overall, fibrillin-1 expression was enriched in MCB and non-mammary carcinosarcoma with SSM (93.7% and 93.3%, respectively), but not in MCBs and non-mammary carcinosarcoma without SSM. FBN1 knockdown in MDA-MB-231 cells with high FBN1 expression did not compromise migration, invasion, and tumourigenesis, and did not alter the expression of other EMT-related markers. In conclusion, fibrillin-1 is a novel TGF-β-induced marker. Fibrillin-1 expression in SSM, but not in other metaplasia and carcinomatous components, in both MCBs and non-mammary carcinosarcomas, together with the inability of FBN1-knockdown to compromise migration and invasion, indicates that fibrillin-1 is a marker induced solely in spindle metaplasia during EMT and does not induce EMT nor lead to tumour aggressiveness.

Resveratrol Suppresses Epithelial-Mesenchymal Transition in GBM by Regulating Smad-Dependent Signaling.

Glioblastoma (GBM) is the most common and malignant intracranial tumor in adults. Despite continuous improvements in diagnosis and therapeutic method, the prognosis is still far away from expectations. The invasive phenotype of GBM is the main reason for the poor prognosis. Epithelial-mesenchymal transition (EMT) is recognized as a participator in this invasive phenotype. Resveratrol, a natural plant-derived compound, is reported to be able to regulate EMT. In the present study, we used TGF-β1 to induce EMT and aimed to evaluate the effect of resveratrol on EMT and to explore the underline mechanism in GBM. Western blotting was used to detect the expression of EMT-related markers, stemness markers, and Smad-dependent signaling. Wound healing assay and transwell invasion assay were performed to evaluate the migratory and invasive ability of GBM cells. Gliosphere formation assay was used to investigate the effect of resveratrol on the ability of self-renewal. Xenograft experiment was conducted to examine the effect of resveratrol on EMT and Smad-dependent signaling in vivo. Our data validated that resveratrol suppressed EMT and EMT-associated migratory and invasive ability via Smad-dependent signaling in GBM cells. We also confirmed that resveratrol obviously inhibited EMT-induced self-renewal ability of glioma stem cells (GSCs) and inhibited EMT-induced cancer stem cell markers Bmi1 and Sox2, suggesting that resveratrol is able to suppress EMT-generated stem cell-like properties in GBM cells. Furthermore, we also showed the inhibitory effect of resveratrol on EMT in xenograft experiments in vivo. Overall, our study reveals that resveratrol suppresses EMT and EMT-generated stem cell-like properties in GBM by regulating Smad-dependent signaling and provides experimental evidence of resveratrol for GBM treatment.

KAI1 reverses the epithelial-mesenchymal transition in human pancreatic cancer cells

BackgroundEpithelial-mesenchymal transition (EMT) plays an important role in pancreatic cancer (PC). In the present study, we investigated the effects of KAI1 gene overexpression on the EMT of human PC cell lines, MIA PaCa-2 and PACN-1. MethodsPlasmids overexpressing KAI1 and pCMV were transfected into MIA PaCa-2 and PACN-1 cells, respectively. After selection of differently transfected cells by G418, KAI1 protein levels were examined by Western blotting, and transfected cells were renamed as MIA PaCa-2-K, MIA PaCa-2-p, PACN-1-K and PACN-1-p. Wound healing and Transwell migration assays were then performed comparing the two groups of cells. EMT-related markers were analyzed by Western blotting. ResultsThe percentage of wound closure significantly decreased in MIA PaCa-2-K cells compared with MIA PaCa-2-p and MIA PaCa-2 cells after 24, 48 and 72 h (P < 0.05). In PACN-1-K cells, the percentage of wound closure significantly decreased as well (P < 0.05). Numbers of invading MIA PaCa-2, MIA PaCa-2-p and MIA PaCa-2-K cells were determined as 48.0 ± 15.4, 50.0 ± 12.4, and 12.0 ± 3.8, respectively. The corresponding numbers of invading PACN-1, PACN-1-p and PACN-1-K cells were 29.0 ± 10.6, 31.0 ± 11.4, and 8.0 ± 4.2, respectively. KAI1 overexpression induced a significant upregulation of E-cadherin and also significant downregulation of Snail, vimentin, matrix metalloproteinase 2 (MMP2) and MMP9 (all P < 0.05) in PC cells. ConclusionsKAI1 reversed EMT-related marker expression and inhibited migration and invasion of PC cells. Thus, KAI1 might represent a novel potential therapeutic target for PC.

Resveratrol Inhibits the Migration and Metastasis of MDA-MB-231 Human Breast Cancer by Reversing TGF-β1-Induced Epithelial-Mesenchymal Transition.

Metastasis is a major cause of death in patients with breast cancer. In the process of cancer development, epithelial-mesenchymal transition (EMT) is crucial to promoting the invasion and migration of tumor cells. In a previous study, the role of resveratrol in migration and metastasis was investigated in MDA-MB-231 (MDA231) human breast cancer cells and a xenograft-bearing mouse model. Additionally, the related mechanism was explored. In the present study, in vitro Transwell assays showed that resveratrol can inhibit the migration of transforming growth factor (TGF)-β1-induced MDA231 cells in a concentration-dependent manner. An enzyme-linked immunosorbent assay (ELISA) showed that resveratrol can reduce the secretion of matrix metalloproteinase (MMP)-2 and MMP-9. Immunofluorescence was performed to confirm the expression of EMT-related markers. Immunofluorescence assays confirmed that resveratrol changed the expression of the EMT-related markers E-cadherin and vimentin. Western blot analysis demonstrated that resveratrol decreased the expression levels of MMP-2, MMP-9, Fibronectin, α-SMA, P-PI3K, P-AKT, Smad2, Smad3, P-Smad2, P-Smad3, vimentin, Snail1, and Slug, as well as increased the expression levels of E-cadherin in MDA231 cells. In vivo, resveratrol inhibited lung metastasis in a mouse model bearing MDA231 human breast cancer xenografts without marked changes in body weight or liver and kidney function. These results indicate that resveratrol inhibits the migration of MDA231 cells by reversing TGF-β1-induced EMT and inhibits the lung metastasis of MDA231 human breast cancer in a xenograft-bearing mouse model.

IGFBP7 contributes to epithelial-mesenchymal transition of HPAEpiC cells in response to radiation.

Radiation-induced lung injury (RILI) frequently occurs in patients with thoracic malignancies. In response to radiation, alveolar epithelial cells (AEC) undergo epithelial-mesenchymal transition (EMT) and contribute to the pathogenesis of RILI. Insulin-like growth factor binding protein 7 (IGFBP7) is reported as a downstream mediator of transforming growth factor-β1 (TGF-β1) pathway, which plays a crucial role in radiation-induced EMT. In the present study, the levels of IGFBP7 and TGF-β1 were simultaneously increased in experimental RILI models and radiation-treated AEC (human pulmonary alveolar epithelial cells [HPAEpic]). The expression of IGFBP7 in radiation-treated HPAEpic cells was obviously inhibited by the specific inhibitor of TGF-β receptor antagonist SB431542 and TGF-β1 neutralizing antibody, and time-dependently enhanced by TGF-β1 treatment. Moreover, IGFBP7 knockdown significantly attenuated the effects of radiation on morphology change, cell migration, expression of EMT-related markers (E-cadherin, α-SMA, and Vimentin), and phosphorylation of extracellular-signal-regulated kinase (ERK). The effects of IGFBP7 overexpression on the expression of EMT-related markers were partially reversed by the ERK inhibitor PD98059. In conclusion, IGFBP7, was enhanced by TGF-β1, may be involved in radiation-induced EMT of AEC via the ERK signaling pathway, thus contributing to the pathogenesis of RILI.

MiR-146a attenuates liver fibrosis by inhibiting transforming growth factor-β1 mediated epithelial-mesenchymal transition in hepatocytes

Epithelial-mesenchymal transition (EMT) has emerged as a vital process in embryogenesis, carcinogenesis, and tissue fibrosis. Transforming growth factor-beta 1 (TGF-β1)-mediated signaling pathways play important roles in the EMT process. MicroRNA-146a (miR-146a) has been suggested as a significant regulatory molecule in fibrogenesis. Therefore, the present study aimed to evaluate the effect of miR-146a on the EMT of hepatocytes and to investigate the role of overexpressing miR-146a on rat hepatic fibrosis. The results showed that the miR-146a level decreased during the EMT process of L02 hepatocytes induced by TGF-β1 in vitro. Moreover, miR-146a overexpression led to significant reduction of EMT-related markers expression in hepatocytes. Subsequent experiments revealed that miR-146a attenuated the EMT process in hepatocytes by targeting small mothers against decapentaplegic (SMAD) 4. Meanwhile, restoration of SMAD4 expression rescued the inhibitory effect of miRNA-146a on EMT. Further in vivo studies revealed that intravenous injection of miR-146a-expressing adenovirus (Ad-miR-146a) successfully restored the miR-146a levels and mitigated fibrogenesis in the livers of CCl4-treated rats. More importantly, after Ad-miR-146a treatment, inhibition of both EMT traits and SMAD4 expression was observed. The results of the present study showed that miR-146a/SMAD4 is a key signaling cascade that inhibits hepatocyte EMT, and the introduction of miR-146a might present a promising therapeutic option for liver fibrosis.

Ionizing Radiation Promotes Epithelial-to-Mesenchymal Transition in Lung Epithelial Cells by TGF-β-producing M2 Macrophages.

Ionizing radiation induces pulmonary fibrosis, which is a common dose-limiting complication in patients receiving radiotherapy. Fibrosis occurs through the accumulation of large amounts of ECM components, synthesized by myofibroblasts in damaged lung tissue. Epithelial cells serve as one of the cellular sources of myofibroblasts via the epithelial-to-mesenchymal transition (EMT) process. In this study, we investigated the role of TGF-β-secreting M2 macrophages in association with ionizing radiation-induced EMT. The lung epithelial cell line MLE12, was irradiated and the expression of EMT markers and chemokines was examined. Moreover, the mouse lung macrophage MH-S cell line was cultured with conditioned media from irradiated MLE12 cells, to examine the effects of the secreted factors on the migration ability of macrophages. For the murine pulmonary fibrosis model, mice were locally irradiated and the levels of M1 or M2 macrophage-related markers and cytokines were measured in bronchoalvelolar lavage (BAL) fluid and lung tissue. In MLE12 cells, irradiation directly induced expression of EMT-related markers and secretion of various chemokines, which lead to macrophage migration. Interestingly, the sub-population of macrophages recruited in the lung of mice after thoracic irradiation was M2 macrophages that expressed Arg-1 and CD206. M2 macrophages induced the MLE12 to undergo phenotypic conversion to form fibroblast-like cells, which leads to a down-regulation of epithelial markers and an up-regulation of new EMT-related markers. In thoracic irradiated mice, pro-inflammatory cytokines such as IL-1β, IL-4 and IL-10 were increased at 2 weeks, but returned to normal levels from 16 weeks or 24 weeks after irradiation. However, thoracic irradiation led to a rapid increase of TGF-β and IGF-1 levels, which lasted up to 24 weeks. It was confirmed that M2 macrophages secreted the high levels of TGF-β. Moreover, the elimination of TGF-β from M2 macrophages attenuated mesenchymal transition of MLE12. TGF-β-secreting M2 macrophages play an important regulatory role in mesenchymal transition of epithelial cells in the lung of irradiated mice, thus contributing to radiation-induced pulmonary fibrosis.

Hypoxia Stimulates the Epithelial–to–Mesenchymal Transition in Lung Cancer Cells Through Accumulation of Nuclear β-Catenin

Recent studies implied a significant role of hypoxia-inducible factor-1α (HIF1α) in cell transformation. This study aimed to assess the effects of HIF1α on the epithelial-to-mesenchymal transition (EMT) and tumorigenesis of lung adenocarcinoma cells. Invasion, migration and colony formation assays were used to evaluate cell transformation. Expression of EMT-related markers were analyzed by western blot, reverse-transcription polymerase chain reaction or zymography. A luciferase assay was carried out to access the transcriptional activity of β-catenin. Hypoxia enhanced migration, invasion and transformation of A549 lung adenocarcinoma cells. Hypoxic stimulation promoted the expression of EMT-related markers in lung cancer cells. The expression of HIF1α was found to be involved in hypoxia-mediated modulation of expression of snail family transcriptional repressors 1 (SNAI1) and 2 (SLUG). Hypoxia enhanced nuclear accumulation and transcriptional activity of β-catenin. β-Catenin promotes expression of EMT-related genes and eventually contributes to the metastatic process.

Abstract 2475: Long noncoding RNA NNT-AS1 sponges miR-424 to promote the progression and metastasis of gastric cancer

Abstract Background: Long noncoding RNAs (lncRNAs) have been illustrated to function as important regulator in carcinogenesis and cancer progression. However, the roles of lncRNA NNT-AS1 in gastric cancer remain unclear. In present study, we investigate the expression level of NNT-AS1 in gastric cancer tissue and the role on gastric cancer cells tumorigenesis. Methods: NNT-AS1 expression level was measured using RT-PCR. The prognosis and survival rate of gastric cancer patients with high/low expression of NNT-AS1 was tracked and analyzed. Cellular experiments were performed to test the role of NNT-AS1 on gastric cancer phenotype, including proliferation analysis, transwell assay, EMT-related marker blots and xenograft mice assay. Bioinformatics online program and luciferase assay were performed to predict and verify the potential miRNAs targeting NNT-AS1. Results: NNT-AS1 expression level was significantly upregulated in 55 cases of gastric cancer tissue samples compared with adjacent normal tissue. Besides, the aberrantly enhanced NNT-AS1 expression predicted poor prognosis and lower survival rate. In vitro, NNT-AS1 knockdown suppressed the gastric cancer cells proliferation, invasion and EMT-related marker (N-cadherin and vimentin) expression. In vivo, lentivirus mediated NNT-AS1 silencing decreased the tumor growth. MiR-503 was verified to be the target miRNA of NNT-AS1 using bioinformatics online program and luciferase assay. Furthermore, miR-424 could reverse the role of NNT-AS1 on gastric cancer cells. Conclusion: In conclusion, our study indicates that NNT-AS1 sponges miR-424 to facilitate gastric cancer cells invasion and metastasis, revealing the oncogenic role of NNT-AS1 on gastric cancer cells. Citation Format: Beibei Chen, Qingfang Zhao, Lulu Guan, Huifang Lv, Liangyu Bie, Jinxi Huang, Jitian Li, Jianying Zhang, Xiaobing Chen. Long noncoding RNA NNT-AS1 sponges miR-424 to promote the progression and metastasis of gastric cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2475.

High expression of forkhead box protein C2 is associated with aggressive phenotypes and poor prognosis in clinical hepatocellular carcinoma

BackgroundHepatocellular carcinoma (HCC) is one of the major causes of tumor death; thus, the identification of markers related to its diagnosis and prognosis is critical. Previous studies have revealed that epithelial-to-mesenchymal transition (EMT) is involved in tumor invasion and metastasis, and the forkhead box protein C2 (FOXC2) has been shown to promote tumor cell proliferation, invasion, and EMT. In the present study, we examined the clinicopathological significance of FOXC2 and EMT-related markers in clinical HCC specimens and identified factors related to the diagnosis and prognosis of HCC.MethodsThe expression of FOXC2 and EMT-related markers was evaluated by immunohistochemistry in 84 cases of hepatocellular carcinoma.ResultsA high expression of FOXC2 was observed in 26 of 84 cases, and expression was significantly correlated with background liver cirrhosis, poor tumor differentiation, high serum AFP, and elevated cell proliferation markers. In addition, this high expression was related to the induction of the Cadherin switch and vimentin expression and was an independent predictor for poor prognosis.ConclusionThe high expression of FOXC2 in HCC is correlated with tumor malignancy and poor prognosis, suggesting that FOXC2 may be an important prognostic factor for HCC.

Abstract A51: Nicotine induces cervical lymph node metastasis of mouse tongue cancer by regulating Prx1 /EMT signaling

Abstract Oral squamous cell carcinoma (OSCC) represents one of the most common malignant neoplasms worldwide, while tobacco is a major risk factor for OSCC. Nicotine has been reported to induce EMT and promote the progression in several malignant tumors. Our previous study showed that upregulation of Peroxiredoxin 1(PRX1) expression was observed and associated with nicotine-induced oxidative stress in OSCC. However, the effects of nicotine and the underlying molecular mechanisms on the development of OSCC metastases are not completely understood. Here, we established the cervical metastasis mouse models of tongue cancer by orthotopic transplantation of OSCC cell line siControl CAL 27 cells and siPRX1 CAL 27 cells labeled with GFP into the tongues of nude mice, in order to investigate the roles and mechanisms of nicotine in OSCC metastasis. Fluorescence stereomicroscopy was used to confirm the tumor formation and the development of cervical metastases, which the tumor cells showed distinct green fluorescence in mouse tongues and lymph nodes. The results showed that at day 13 after CAL 27 cell implantation, nicotine promoted OSCC growth in the tongues and metastatic rates of cervical lymph node in mice compared with control. Histologic examination and immunohistochemical staining with anti-keratin antibody further confirmed tumor formation and lymph node metastases. Nicotine treatment was shown to promote the downregulation of expression levels of E-cadherin, and upregulation of vimentin and Snail expression in tongues and lymph nodes, compared with those in the control mice. PRX1 knockdown led to the reduction in the metastasis rate, upregulation of E-cadherin expression, and downregulation of vimentin and Snail expression in tongues and lymph nodes. No significant differences were seen in the cervical lymph node metastatic rates and the expression of EMT-related markers between nicotine-treated and vehicle-treated mice after PRX1 knockdown. These results suggest that nicotine may induce cervical lymph node metastases through the regulation of Prx1/EMT signal pathway during OSCC pathogenesis. PRX1 plays a critical role in nicotine-induced metastasis of OSCC. Citation Format: Min Wang, Wenwen Niu, Moci Qi, Xiaofei Tang. Nicotine induces cervical lymph node metastasis of mouse tongue cancer by regulating Prx1 /EMT signaling [abstract]. In: Proceedings of the AACR Special Conference: Advances in Modeling Cancer in Mice: Technology, Biology, and Beyond; 2017 Sep 24-27; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(10 Suppl):Abstract nr A51.

Bone Marrow-Derived Mesenchymal Stem Cells Promoted Cutaneous Wound Healing by Regulating Keratinocyte Migration via β2-Adrenergic Receptor Signaling.

Mesenchymal stem cells (MSCs) play an important role in cutaneous wound healing; however, the functional mechanisms involved in the healing process are poorly understood. A series of studies indicate that keratinocytes that migrate into the wound bed rely on an epithelial-mesenchymal transition (EMT)-like process to initiate re-epithelialization. We therefore examined whether bone marrow-derived MSCs (BMSCs) could affect biological behavior and induce EMT-like characteristics in the human epidermal keratinocytes (HEKs) and in the immortalized human keratinocyte cell line HaCaT cells, and we investigated the signaling pathways of BMSC-mediated phenotypic changes. By assessing the expression of EMT-related markers including E-cadherin, α-SMA, and Snail family transcription factors by β2-adrenergic receptor (β2-AR) blockage using ICI-118,551, a β2-AR selective antagonist, or β2-AR small interfering RNA (siRNA), we showed an involvement of β2-AR signaling in the induction of EMT-like alterations in human keratinocytes in vitro. β2-AR signaling also affected collective and individual cell migration in human keratinocyte cell lines, which was attenuated by administration of ICI-118,551. Treating the cells with BMSC-conditioned media (BMSC-CM) not only recapitulated the effect of isoproterenol (ISO) on cell migration but also induced the expression of β2-AR and a panel of proteins associated with mesenchymal phenotype in HEKs and HaCaT cells. Similarly, a blockade of the β2-AR by either ICI-118,551 or β2-AR siRNAs reversed both responses of the epidermal keratinocyte cell lines relative to BMSC-CM exposure. These results were further verified in our vivo findings and indicated that the exogenous application of MSCs promoted cutaneous wound healing and endowed the keratinocytes surrounding the wound area with an increased migratory phenotype through activation of β2-AR signaling. Our findings suggest a biochemical mechanism underlying the function of MSCs in wound re-epithelization, which provides a reliable theoretical basis for the wide application of MSCs in the treatment of chronic wounds.

Small interfering RNA-mediated knockdown of fatty acid synthase attenuates the proliferation and metastasis of human gastric cancer cells via the mTOR/Gli1 signaling pathway.

Fatty acid synthase (FASN), the main enzyme involved in de novo lipogenesis, is overexpressed in several types of tumor tissues. In addition, it is associated with tumor cell proliferation, metastasis, epithelial-mesenchymal transition (EMT) and a poor prognosis. However, the precise functions and internal mechanisms of FASN with regard to the proliferation, metastasis and EMT in gastric cancer (GC) cells remain elusive. The present study investigated FASN protein expression in 18 randomly selected pairs of GC tumors and matched normal tissues by western blot analysis. FASN-specific small interfering RNA (siRNA) was then transfected into SGC-7901 cells to examine the effect of FASN on proliferation and migration in vitro. Western blotting was used to detect the protein expression of FASN, EMT-related markers and key signaling molecules of the mechanistic target of rapamycin/zinc finger protein GLI1 (mTOR/Gli1) pathway. Reverse transcription-quantitative polymerase chain reaction was conducted to detect the mRNA expression of FASN and EMT-related markers. The FASN level was higher in the GC tissues compared with that in the surrounding normal tissues. Knockdown of FASN suppressed GC cell proliferation and metastasis in vitro. The silencing of FASN expression using siRNA reversed EMT at the protein and mRNA levels and decreased the expression of Gli1 via regulation of AMP-activated protein kinase/mTOR and protein kinase B/mTOR signaling in GC cells. Inhibition of FASN suppresses GC proliferation and metastasis through targeting of the mTOR/Gli1 signaling pathway, indicating that it may serve as a potential target for the treatment of GC.

YAP1 contributes to NSCLC invasion and migration by promoting Slug transcription via the transcription co-factor TEAD

Yes-associated protein 1 (YAP1) contributes to the development of multiple tumors, but the mechanism underlying YAP1 deregulation in non-small cell lung cancer (NSCLC) remains unclear. By performing immunohistochemistry (IHC) assays, we found that YAP1 was significantly upregulated in NSCLC compared with adjacent tissues; therefore, we sought to elucidate whether the upregulation of YAP1 contributes to NSCLC progression. MTT and transwell assays showed that YAP1 overexpression promoted proliferation, migration, and invasion in the NSCLC cell lines A549 and H460; YAP1 overexpression also promoted the significant differential expression of epithelial-mesenchymal transition (EMT)-related markers. Nevertheless, YAP1 knockdown alleviated TGF-β1-induced EMT and proliferation, migration, and invasion in NSCLC. Furthermore, western blotting showed that the co-transcription complex YAP1/TEAD was impaired by YAPS94A (a YAP1 mutant without the TEAD binding site), and verteporfin (a small molecular inhibitor of YAP1) inhibited A549 and H460 cell metastasis and EMT-related markers expression, indicating that TEAD mediated the NSCLC aggressiveness induced by YAP1. Moreover, sequence analysis and ChIP and luciferase assays confirmed that YAP1 transcriptionally activated Slug expression by binding to TEAD. Importantly, silencing YAP1 inhibited A549 cell tumorigenesis and EMT and downregulated Slug expression in vivo. Overall, our findings revealed that YAP1 is a driver of NSCLC metastasis because YAP1 promoted the EMT program by inducing Slug transcription.

Tumor-Associated Macrophages Derived TGF-β‒Induced Epithelial to Mesenchymal Transition in Colorectal Cancer Cells through Smad2,3-4/Snail Signaling Pathway

PurposeWe investigated the role of tumor-associated macrophages (TAMs) on the epithelial to mesenchymal transition (EMT) of colorectal cancer cells and determined the potential mechanism involved in the metastatic process.Materials and MethodsIn this study, flow cytometry was used to detect the expression of target proteins. We used transwell assay to evaluate the migration of cancer cells under specific conditions. Using real-time polymerase chain reaction, we examined the expressions of cytokines and EMT-related markers in mRNA level. Animal assay was performed for analysis in vivo and hematoxylin and eosin was used to visualize the effect of TAMs on tumor metastasis. We also used immunohistochemistry and Western blotting to detect the expression of target proteins.ResultsHere, we observed enrichment of TAMs in colorectal tumor tissues, resulting in high metastasis in clinical therapy. Moreover, those TAMs could facilitate the EMT progression of colorectal cancer cells, which is induced by the transforming growth factor-β (TGF-β) derived from TAMs, leading to the invasion and migration of cancer cells.ConclusionOur results demonstrated that TAMs contributed the EMT progression through a TGF-β/Smad2,3-4/Snail signaling pathway, and disrupting this pathway with TGF-β receptor inhibitor could suppress metastasis, readjusting our focus to the connection of TAMs and cancer metastasis.

Matrix stiffness and tumor-associated macrophages modulate epithelial to mesenchymal transition of human adenocarcinoma cells

The tumor microenvironment (TME) is gaining increasing attention in oncology, as it is recognized to be functionally important during tumor development and progression. Tumors are heterogeneous tissues that, in addition to tumor cells, contain tumor-associated cell types such as immune cells, fibroblasts, and endothelial cells. These other cells, together with the specific extracellular matrix (ECM), create a permissive environment for tumor growth. While the influence of tumor-infiltrating cells and mechanical properties of the ECM in tumor invasion and progression have been studied separately, their interaction within the complex TME and the epithelial -to-mesenchymal transition (EMT) is still unclear. In this work, we develop a 3D co-culture model of lung adenocarcinoma cells and macrophages in an interpenetrating network hydrogel, to investigate the influence of the macrophage phenotype and ECM stiffness in the induction of EMT. Rising ECM stiffness increases both tumor cell proliferation and invasiveness. The presence of tumor-associated macrophages and the ECM stiffness jointly contribute to an invasive phenotype, and modulate the expression of key EMT-related markers. Overall, these findings support the utility of in vitro 3D cancer models that allow one to study interactions among key components of the TME.

Nemopilema nomurai jellyfish venom exerts an anti-metastatic effect by inhibiting Smad- and NF-\u03baB-mediated epithelial\u2013mesenchymal transition in HepG2 cells

Epithelial–mesenchymal transition (EMT) is a key initial step in metastasis for malignant cancer cells to obtain invasive and motile properties. Inhibiting EMT has become a new strategy for cancer therapy. In our previous in vivo study, Nemopilema nomurai jellyfish venom (NnV) -treated HepG2 xenograft mice group showed that E-cadherin expression was strongly detected compared with non-treated groups. Therefore, this study aimed to determine whether NnV could inhibit the invasive and migratory abilities of HepG2 human hepatocellular carcinoma cells and to examine its effect on EMT. Our results revealed that transforming growth factor (TGF)-β1 induced cell morphological changes and downregulated E-cadherin and β-catenin expression, but upregulated N-cadherin and vimentin expression through the Smad and NF-κB pathways in HepG2 cells. Treatment of TGF-β1-stimulated HepG2 cells with NnV reversed the EMT-related marker expression, thereby inhibiting cell migration and invasion. NnV also significantly suppressed the activation of p-Smad3, Smad4, and p-NF-κB in a dose-dependent manner. These data indicated that NnV can significantly suppress cell migration and invasion by inhibiting EMT in HepG2 cells, and therefore might be a promising target for hepatocellular carcinoma therapeutics.

14, 15-EET induces breast cancer cell EMT and cisplatin resistance by up-regulating integrin \u03b1v\u03b23 and activating FAK/PI3K/AKT signaling

Background14,15-epoxyeicosatrienoic acid (14,15-EET) is an important lipid signaling molecule involved in the regulation of tumor metastasis, however, the role and molecular mechanisms of 14,15-EET activity in breast cancer cell epithelial-mesenchymal transition (EMT) and drug resistance remain enigmatic.MethodsThe 14, 15-EET level in serum and in tumor or non-cancerous tissue from breast cancer patients was measured by ELISA. qRT-PCR and western blot analyses were used to examine expression of integrin αvβ3. The role of 14, 15-EET in breast cancer cell adhesion, invasion was explored by adhesion and Transwell assays. The role of 14, 15-EET in breast cancer cell cisplatin resistance in vitro was determined by MTT assay. Western blot was conducted to detect the protein expressions of EMT-related markers and FAK/PI3K/AKT signaling. Xenograft models in nude mice were established to explore the roles of 14, 15-EET in breast cancer cells EMT and cisplatin resistance in vivo.ResultsIn the present study, we show that serum level of 14, 15-EET increases in breast cancer patients and 14, 15-EET level of tumor tissue is higher than that of non-cancerous tissue. Moreover, 14, 15-EET increases integrin αvβ3 expression, leading to FAK activation. 14, 15-EET induces breast cancer cell EMT via integrin αvβ3 and FAK/PI3K/AKT cascade activation in vitro. Furthermore, we find that 14, 15-EET induces breast cancer cells EMT and cisplatin resistance in vivo, αvβ3 integrin and the resulting FAK/PI3K/AKT signaling pathway are responsible for 14, 15-EET induced-breast cancer cells cisplatin resistance.ConclusionsOur findings suggest that inhibition of 14, 15-EET or inactivation of integrin αvβ3/FAK/PI3K/AKT pathway could serve as a novel approach to reverse EMT and cisplatin resistance in breast cancer cells.