Downregulation Of E-cadherin Expression Research Articles

AFF4 Predicts the Prognosis of Colorectal Cancer Patients and Suppresses Colorectal Cancer Metastasis via Promoting CDH1 Expression.

IntroductionAF4/FMR2 family member 4 (AFF4) is a core component of super elongation complex (SEC) and regulates the transcription elongation of many genes. AFF4 depletion or amplification is associated with multiple cancers, but its role in colorectal cancer (CRC) has not been investigated so far.MethodsqRT-PCR and Western blot analyzed AFF4 expression in the paired clinical CRC tissues. The patients’ overall survival curve was determined using the Kaplan-Meier plotter. In vitro experiments, such as cell proliferation, migration, and invasion, were used to preliminarily ascertain the role of AFF4 in CRC. A CRC cell liver metastasis animal model was well established. Livers were harvested and examined histologically by a series of indicators, such as tumor nodules, liver weight, ALT/AST activity, and tumor cell identification by hematoxylin-eosin (HE) staining.ResultsWe firstly examined the expression of AFF4 in colorectal cancer and normal tissues by collecting paired CRC tissues and adjacent normal tissues, revealing that AFF4 was significantly downregulated in CRC patients and lower expression of AFF4 was correlated with poor prognosis. Next, we observed that presence or absence of AFF4 in CRC cells had no effect on cancer cell proliferation, while AFF4 depletion significantly promoted the migration or invasion of CRC cells in vitro. Furthermore, we confirmed that AFF4 deficiency enhanced the metastatic capacity of CRC cells in vivo. Mechanistically, we found that AFF4 upregulated the transcription of CDH1 gene, which encodes E-cadherin and suppresses the epithelial-mesenchymal transition (EMT). Knockdown of AFF4 interfered with CDH1 transcription, resulting in downregulation of E-cadherin expression and the progression of CRC. Moreover, restored CDH1 expression could rescue the phenotype of CRC cells without AFF4.ConclusionsCollectively, our data demonstrated that AFF4 served as a significant novel regulator of CRC via CDH1 transcriptional regulation and a potential effective therapy target for patients with CRC.

Oxysterol-Binding Protein 2 Promotes Pancreatic Ductal Adenocarcinoma Progression Through Epithelial-Mesenchymal Transition.

Oxysterol-binding protein 2 (OSBP2) is crucial for promoting the growth and development of cancers; however, its effects on pancreatic ductal adenocarcinoma (PDAC) are still unclear. Here, we report that OSBP2 is an efficient tumor-associated protein to lead to extremely malignant characteristics in PDAC. We discovered that increased OSBP2 expression in primary tumors was associated with shorter survival in PDAC patients. Therefore, we used immunohistochemistry (IHC) to analyze the levels of OSBP2 expression in PDAC tissues and adjacent paracancerous tissues. We used wound healing and Transwell assays to evaluate the effects of OSBP2 on PDAC cell (ASPC-1 and BXPC-3) migration and invasion, respectively, and CCK-8 and Annexin V/PI double staining to evaluate the effects of OSBP2 on PDAC cell proliferation and apoptosis, respectively. Western blotting was used to analyze the effect of OSBP2 on the PDAC cell phenotype. We also explored the effect of OSBP2 on chemosensitivity to gemcitabine (GEM) and 5-fluorouracil (5-FU). We validated these findings in an in vivo mouse model. The data show that OSBP2 overexpression promoted PDAC cell migration, invasion, proliferation and chemotherapy resistance, and decreased apoptosis. OSBP2 overexpression downregulated E-cadherin expression and upregulated N-cadherin, vimentin, Snail, Slug, ZEB1, and β-catenin expression. Taken together, our findings indicated that OSBP2 was overexpressed in PDAC and that upregulation of OSBP2 may promote PDAC progression. Therefore, OSBP2 may have potential diagnostic and therapeutic value in PDAC.

Downregulation of TRIM33 Promotes Survival and Epithelial–Mesenchymal Transition in Gastric Cancer

Among all malignancies worldwide, gastric cancer is the fifth most common cancer with the third highest mortality rate. One of the main reasons for the low survival rate is the recurrence and metastasis that occurs in many patients after surgery. Numerous studies have shown that abnormal TRIM33 expression is associated with the progression of malignant tumors. TRIM33 can function either as a tumor suppressor or tumor promoter in different cancers. Our data showed that TRIM33 was highly expressed in stomach cancer, and in human gastric cancer tissues, low expression of TRIM33 was associated with poor prognosis in patients with gastric cancer. To clarify the function of TRIM33 in survival and epithelial–mesenchymal transition in gastric cancer cells, we investigated the effect of TRIM33 knockdown in several gastric cancer cell lines. Downregulation of TRIM33 in BGC-823 and SGC-7901 cells enhanced the proliferation, colony formation, and migratory ability of these gastric cancer cells. It also promoted epithelial–mesenchymal transition; transfection of cells with siRNA targeting TRIM33 led to the upregulation of vimentin and N-Cadherin expression, and downregulation of E-Cadherin expression. Meanwhile, the transforming growth factor beta pathway was activated: levels of transforming growth factor beta were elevated and the expressions of p-Smad2, Smad2, Smad3, and Smad4 were activated. To confirm the role of TRIM33 in vivo, a xenograft model was established in nude mice. Immunohistochemical analysis identified that the protein levels of TRIM33, p-Smad2, Smad2, Smad3, Smad4, vimentin, and N-Cadherin were increased, and E-Cadherin levels were decreased, in xenograft tumors from the si-TRIM33 group. Taken together, these results suggest that TRIM33 may be a potential marker for the diagnosis and prognosis of gastric cancer. Furthermore, it may also serve as a novel target for gastric cancer treatment.

Targeted intervention of eIF4A1 inhibits EMT and metastasis of pancreatic cancer cells via c-MYC/miR-9 signaling

BackgroundOwing to the lack of effective treatment options, early metastasis remains the major cause of pancreatic ductal adenocarcinoma (PDAC) recurrence and mortality. However, the molecular mechanism of early metastasis is largely unknown. We characterized the function of eukaryotic translation initiation factors (eIFs) in epithelial-mesenchymal-transition (EMT) and metastasis in pancreatic cancer cells to investigate whether eIFs and downstream c-MYC affect EMT and metastasis by joint interference.MethodsWe used The Cancer Genome Atlas (TCGA) and Genome Tissue Expression (GTEx) databases to analyze eIF4A1 expression in PDAC tissues and further validated the findings with a microarray containing 53 PDAC samples. Expression regulation and pharmacological inhibition of eIF4A1 and c-MYC were performed to determine their role in migration, invasion, and metastasis in pancreatic cancer cells in vitro and in vivo.ResultsElevated eIF4A1 expression was positively correlated with lymph node infiltration, tumor size, and indicated a poor prognosis. eIF4A1 decreased E-cadherin expression through the c-MYC/miR-9 axis. Loss of eIF4A1 and c-MYC decreased the EMT and metastasis capabilities of pancreatic cancer cells, whereas upregulation of eIF4A1 attenuated the inhibition of EMT and metastasis induced by c-MYC downregulation. Treatment with the eIF4A1 inhibitor rocaglamide (RocA) or the c-MYC inhibitor Mycro3 either alone or in combination significantly decreased the expression level of EMT markers in pancreatic cancer cells in vitro. However, the efficiency and safety of RocA alone were not inferior to those of the combination treatment in vivo.ConclusionOverexpression of eIF4A1 downregulated E-cadherin expression through the c-MYC/miR-9 axis, which promoted EMT and metastasis of pancreatic cancer cells. Despite the potential feedback loop between eIF4A1 and c-MYC, RocA monotherapy is a promising treatment inhibiting eIF4A1-induced PDAC metastasis.

The Herbal Formula JI017 Induces ER Stress via Nox4 in Breast Cancer Cells.

Chemotherapy is a powerful anti-tumor therapeutic strategy; however, resistance to treatment remains a serious concern. To overcome chemoresistance, combination therapy with anticancer drugs is a potential strategy. We developed a novel herbal extract, JI017, with lower toxicity and lesser side effects. JI017 induced programmed cell death and excessive unfolded protein response through the release of intracellular reactive oxygen species (ROS) and calcium in breast cancer cells. JI017 treatment increased the expression of endoplasmic reticulum (ER) stress markers, including p-PERK, p-eIF2α, ATF4, and CHOP, via the activation of both exosomal GRP78 and cell lysate GRP78. The ROS inhibitors diphenyleneiodonium and N-acetyl cysteine suppressed apoptosis and excessive ER stress by inhibiting Nox4 in JI017-treated breast cancer cells. Furthermore, in paclitaxel-resistant breast cancer cell lines, MCF-7R and MDA-MB-231R, a combination of JI017 and paclitaxel overcame paclitaxel resistance by blocking epithelial-mesenchymal transition (EMT) processes, such as the downregulation of E-cadherin expression and the upregulation of HIF-1α, vimentin, Snail, and Slug expression. These findings suggested that JI017 exerts a powerful anti-cancer effect in breast cancer and a combination therapy of JI017 and paclitaxel may be a potential cancer therapy for paclitaxel resistant breast cancer.

Effects of SDF-1/CXCR7 on the Migration, Invasion and Epithelial-Mesenchymal Transition of Gastric Cancer Cells.

We found that SDF-1/CXCR7 axis played an important role in the growth and proliferation of gastric cancer in the previous studies. The objectives of this study were to explore the effects of SDF-1/CXCR7 on the metastatic ability of gastric cancer cells and the possible mechanisms. CXCR7 expression in SGC-7901 gastric cancer cells was stably knocked down via lentiviral vectors. The cell migration and invasion abilities were detected by transwell migration and invasion assays. The expressions of matrix metalloproteinase 2 (MMP-2), MMP-9, vascular endothelial growth factor (VEGF), epithelial-mesenchymal transition (EMT) markers and Akt phosphorylation were detected with real-time PCR and/or western blot. We found that SDF-1 markedly enhanced the migration and invasion abilities of SGC-7901 gastric cancer cells; CXCR7 knockdown inhibited these effects. SDF-1/CXCR7 increased the expressions of MMP-2, MMP-9 and VEGF. SDF-1/CXCR7 also downregulated E-cadherin expression but upregulated N-cadherin, vimentin and Snail expressions, suggesting that SDF-1/CXCR7 could promote the development of EMT in gastric cancer cells. Furthermore, SDF-1/CXCR7 could promote Akt phosphorylation. Our results indicated that SDF-1/CXCR7 enhanced the migration, invasion and EMT of gastric cancer cells and thus CXCR7 supression may be a strategy for inhibiting gastric cancer metastasis.

SNX10-mediated LPS sensing causes intestinal barrier dysfunction via a caspase-5-dependent signaling cascade.

Altered intestinal microbial composition promotes intestinal barrier dysfunction and triggers the initiation and recurrence of inflammatory bowel disease (IBD). Current treatments for IBD are focused on control of inflammation rather than on maintaining intestinal epithelial barrier function. Here, we show that the internalization of Gram-negative bacterial outer membrane vesicles (OMVs) in human intestinal epithelial cells promotes recruitment of caspase-5 and PIKfyve to early endosomal membranes via sorting nexin 10 (SNX10), resulting in LPS release from OMVs into the cytosol. Caspase-5 activated by cytosolic LPS leads to Lyn phosphorylation, which in turn promotes nuclear translocalization of Snail/Slug, downregulation of E-cadherin expression, and intestinal barrier dysfunction. SNX10 deletion or treatment with DC-SX029, a novel SNX10 inhibitor, rescues OMV-induced intestinal barrier dysfunction and ameliorates colitis in mice by blocking cytosolic LPS release, caspase-5 activation, and downstream signaling. Our results show that targeting SNX10 may be a new therapeutic approach for restoring intestinal epithelial barrier function and promising strategy for IBD treatment.

Transcriptional repression of E-cadherin in nickel-exposed lung epithelial cells mediated by loss of Sp1 binding at the promoter.

E-cadherin plays a central role in the stability of epithelial tissues by facilitating cell-cell adhesion. Loss of E-cadherin expression is a hallmark of epithelial-mesenchymal transition (EMT), a major event in the pathogenesis of several lung diseases. Our earlier studies showed that nickel, a ubiquitous environmental toxicant, induced EMT by persistently downregulating E-cadherin expression in human lung epithelial cells and that the EMT remained irreversible postexposure. However, the molecular basis of persistent E-cadherin downregulation by nickel exposure is not understood. Here, our studies show that the binding of transcription factor Sp1 to the promoter of E-cadherin encoding gene, CDH1, is essential for its expression. Nickel exposure caused a loss of Sp1 binding at the CDH1 promoter, resulting in its downregulation and EMT induction. Loss of Sp1 binding at the CDH1 promoter was associated with an increase in the binding of ZEB1 adjacent to the Sp1 binding site. ZEB1, an EMT master regulator persistently upregulated by nickel exposure, is a negative regulator of CDH1. CRISPR-Cas9-mediated knockout of ZEB1 restored Sp1 binding at the CDH1 promoter. Furthermore, ZEB1 knockout rescued E-cadherin expression and re-established the epithelial phenotype. Since EMT is associated with a number of nickel-exposure-associated chronic inflammatory lung diseases including asthma, fibrosis and cancer and metastasis, our findings provide new insights into the mechanisms associated with nickel pathogenesis.

AKR1B10 promotes breast cancer cell proliferation and migration via the PI3K/AKT/NF-\u03baB signaling pathway

BackgroundAberrant expression of Aldo-Keto reductase family 1 member B10 (AKR1B10) was associated with tumor size and metastasis of breast cancer in our published preliminary studies. However, little is known about the detailed function and underlying molecular mechanism of AKR1B10 in the pathological process of breast cancer.MethodsThe relationship between elevated AKR1B10 expression and the overall survival and disease-free survival of breast cancer patients was analyzed by Kaplan–Meier Plotter database. Breast cancer cell lines overexpressing AKR1B10 (MCF-7/AKR1B10) and breast cancer cell lines with knockdown of AKR1B10 (BT-20/shAKR1B10) were constructed to analyze the impact of AKR1B10 expression on cell proliferation and migration of breast cancer. The expression levels of AKR1B10 were detected and compared in the breast cancer cell lines and tissues by RT-qPCR, western blot and immunohistochemistry. The proliferation of breast cancer cells was monitored by CCK8 cell proliferation assay, and the migration and invasion of breast cancer cells was observed by cell scratch test and transwell assay. The proliferation- and EMT-related proteins including cyclinD1, c-myc, Survivin, Twist, SNAI1, SLUG, ZEB1, E-cadherin, PI3K, p-PI3K, AKT, p-AKT, IKBα, p-IKBα, NF-κB p65, p-NF-κB p65 were detected by western blot in breast cancer cells. MCF-7/AKR1B10 cells were treated with LY294002, a PI3K inhibitor, to consider the impact of AKR1B10 overexpression on the PI3K/AKT/NF-κB signal cascade and the presence of NF-κB p65 in nuclear. In vivo tumor xenograft experiments were used to observe the role of AKR1B10 in breast cancer growth in mice.ResultsAKR1B10 expression was significantly greater in breast cancer tissue compared to paired non-cancerous tissue. The expression of AKR1B10 positively correlated with lymph node metastasis, tumor size, Ki67 expression, and p53 expression, but inversely correlated with overall and disease-free survival rates. Gene Ontology analysis showed that AKR1B10 activity contributes to cell proliferation. Overexpression of AKR1B10 facilitated the proliferation of MCF-7 cells, and induced the migration and invasion of MCF-7 cells in vitro in association with induction of epithelial-mesenchymal transition (EMT). Conversely, knockdown of AKR1B10 inhibited these effects in BT-20 cells. Mechanistically, AKR1B10 activated PI3K, AKT, and NF-κB p65, and induced nuclear translocation of NF-κB p65, and expression of proliferation-related proteins including c-myc, cyclinD1, Survivin, and EMT-related proteins including ZEB1, SLUG, Twist, but downregulated E-cadherin expression in MCF-7 cells. AKR1B10 silencing reduced the phosphorylation of PI3K, AKT, and NF-κB p65, the nuclear translocation of NF-κB p65, and the expression of proliferation- and migration-related proteins in BT-20 cells. LY294002, a PI3K inhibitor, attenuated the phosphorylation of PI3K, AKT, and NF-κB p65, and the nuclear translocation of NF-κB p65. In vivo tumor xenograft experiments confirmed that AKR1B10 promoted breast cancer growth in mice.ConclusionsAKR1B10 promotes the proliferation, migration and invasion of breast cancer cells via the PI3K/AKT/NF-κB signaling pathway and represents a novel prognostic indicator as well as a potential therapeutic target in breast cancer.

Pleckstrin-2 as a Prognostic Factor and Mediator of Gastric Cancer Progression.

Pleckstrin-2 (PLEK2) is a crucial mediator of cytoskeletal reorganization. However, the potential roles of PLEK2 in gastric cancer are still unknown. PLEK2 expression in gastric cancer was examined by western blotting and real-time PCR. Survival analysis was utilized to test the clinical impacts of the levels of PLEK2 in gastric cancer patients. In vitro and in vivo studies were used to estimate the potential roles played by PLEK2 in modulating gastric cancer proliferation, self-renewal, and tumourigenicity. Bioinformatics approaches were used to monitor the effect of PLEK2 on epithelial-mesenchymal transition (EMT) signalling pathways. PLEK2 expression was significantly upregulated in gastric cancer as compared with nontumour samples. Kaplan-Meier plotter analysis revealed that gastric cancer patients with higher PLEK2 levels had substantially poorer overall survival compared with gastric cancer patients with lower PLEK2 levels. The upregulation or downregulation of PLEK2 in gastric cancer cell lines effectively enhanced or inhibited cell proliferation and proinvasive behaviour, respectively. Additionally, we also found that PLEK2 enhanced EMT through downregulating E-cadherin expression and upregulating Vimentin expression. Our findings demonstrated that PLEK2 plays a potential role in gastric cancer and may be a novel therapeutic target for gastric cancer.

Sema4C modulates the migration of primary tumor-associated lymphatic endothelial cells via an ERK-mediated pathway.

Although lymphatic endothelial cells (LECs) serve a positive role in tumor lymphatic metastasis, the regulation of LECs undergoing migration similar to that of tumor cells remains poorly understood. A previous study revealed that semaphorin 4C (Sema4C) could be a marker of LECs in cervical cancer. Thus, the present study aimed to understand the mechanism via which Sema4C could promote the development of tumor-associated characteristics in LECs in cervical cancer. Primary tumor-associated LECs (TLECs) were distinguished from cervical cancer by flow cytometry. The promigratory ability was assessed using the Transwell assay. Lentivirus infection was used to alter the expression of Sema4C in TLECs. Confocal laser scanning was used to determine the infection efficiency of lentivirus infection. Sema4C/ERK/E-cadherin pathway was measured by reverse transcription-quantitative PCR and western blotting. The co-localization of Sema4C and the lymphatic marker lymphatic vessel endothelial hyaluronan receptor 1 was verified. Primary tumor-associated LECs (TLECs) were isolated from a mouse xenograft cervical tumor model. It was revealed that overexpressing Sema4C stimulated the migratory ability of TLECs, downregulated E-cadherin expression and stimulated ERK phosphorylation, whereas knocking down Sema4C had the opposite effects. The treatment of PD98059 (ERK inhibitor) blocked the pro-migratory ability of TLECs, which indicated a dependence on the ERK signaling pathway. It was identified that the Sema4C/ERK/E-cadherin pathway may be critical for the migration of TLECs, which may promote lymph node metastasis. Therefore, Sema4C could be a promising target for the treatment of cervical cancer with lymphatic metastasis.

Pannexin 1 Mediates Gastric Cancer Cell Epithelial-Mesenchymal Transition via Aquaporin 5.

Pannexin 1 (PANX1) has been implicated in cancer emergence and progression. However, its roles in gastric cancer remain unclear. In the present study, the function and molecular mechanisms of PANX1 in gastric cancer were investigated in vitro. Two gastric cancer cell lines exhibiting low and high PANX1 expression (SNU-16 and HCG-27, respectively) were transfected using a PANX1-containing plasmid or PANX1 transcript-targeting short hairpin (sh)RNA. In addition, HCG-27 cells and PANX1-overexpressing SNU-16 cells were subjected to short interfering (si)RNA-mediated aquaporin 5 (AQP5) knockdown. In vitro cell migration (scratch) and transwell invasion assays were performed to evaluate the cell migratory and invasive abilities. Real-time fluorescence quantitative PCR was used to detect transcripts encoding epithelial-mesenchymal transition markers. Immunofluorescence and Western blotting were conducted to quantify corresponding proteins. In SNU-16 cells, PANX1 overexpression induced conversion from round (cobblestone-like) to elongated (spindle-like) morphologies and enhanced the cell migratory and invasive abilities. PANX1 knockdown had the opposite effect in HGC-27 cells. In PANX1-overexpressing SNU-16 cells, expression of SLUG, vimentin, and AQP5 was significantly upregulated, whereas expression of E-cadherin was downregulated. In HGC-27 cells, PANX1 knockdown showed the opposite effect. In both PANX1-overexpressing SNU-16 cells and untransfected HGC-27 cells, silencing of AQP5 expression significantly inhibited PANX1-induced upregulation of SLUG and vimentin expression, as well as downregulation of E-cadherin expression and enhanced migratory and invasive abilities. In summary, elevated PANX1 expression induces gastric cancer cell epithelial-mesenchymal transition and the associated promotion of migratory and invasive abilities by inducing expression of AQP5, which facilitates SLUG-mediated regulation of vimentin and E-cadherin expression.

Abstract 2901: Pdcd4 inhibits slug translation to suppress E-cadherin expression and invasion in colon tumor cells

Abstract Programmed cell death 4 (Pdcd4) has been demonstrated to function as a tumor suppressor and protein translation inhibitor. We previously reported that knockdown of Pdcd4 down-regulated E-cadherin expression. E-cadherin is a key regulator for tumor cell invasion and metastasis, whose expression is controlled by transcription repressors Snail, Slug, ZEB1/2, and Twist. Both Slug and Snail have been shown to repress E-cadherin expression and contribute to tumor progression in various cancers including colon cancer. Here, we reported that knockdown of Pdcd4 increased Slug protein level but not SLUG mRNA level in colon tumor cells. Conversely, over-expression of Pdcd4 decreased Slug expression. Using sucrose gradient fractionation assays, Pdcd4 knockdown was found to increase the population of SLUG mRNA in the polysomal fractions. These results indicate that Pdcd4 translationally regulates Slug expression. To understand the mechanism by which Pdcd4 regulates Slug translation, the SLUG 5' untranslated region (5'UTR) was cloned and fused with luciferase reporter and named as 5'UTR-Slug-Luc. Knockdown of Pdcd4 significantly enhanced the translation of 5'UTR-Slug-Luc while ectopic expression of Pdcd4 repressed the translation of 5'UTR-Slug-Luc, suggesting that SLUG 5'UTR is an essential element for Pdcd4 to inhibit Slug translation. Moreover, cells treated with silvestrol, an inhibitor for translation initiation factor 4A (eIF4A), significantly suppressed Slug translation. In addition, ectopic expression of Pdcd4 inhibited Slug translation and thereby decreased E-cadherin expression. By contrast, Pdcd4(D418A), a mutant that does not bind with eIF4A, failed to suppress the expression of Slug and E-cadherin. These findings suggest that Pdcd4-inhibited Slug translation is through suppressing eIF4A. Furthermore, knockdown of Slug in Pdcd4 knockdown cells restored the E-cadherin expression and reversed the invasive capacity induced by Pdcd4 knockdown, indicating that up-regulation of Slug contributes to Pdcd4-knockdown enhanced invasion. Taken together, this study demonstrates that Pdcd4 inhibits translation of Slug to regulate E-cadherin expression and invasion in colon tumor cells. Citation Format: Qing Wang, Jiang Zhu, Hsin-Sheng Yang. Pdcd4 inhibits slug translation to suppress E-cadherin expression and invasion in colon tumor cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2901.

Silencing of E-cadherin expression leads to increased chemosensitivity to irinotecan and oxaliplatin in colorectal cancer cell lines.

Colorectal carcinoma (CRC) is a leading malignant disease in most developed countries. In advanced stages it presents with metastatic dissemination and significant chemoresistance. Despite intensive studies, no convincing evidence has been published concerning the association of cadherins and epithelial-mesenchymal transition (EMT) as a direct cause of acquired chemoresistance in CRC. The present study was designed to investigate the role of E-cadherin in EMT and its associated chemosensitivity/chemoresistance in four immortalized CRC cell lines representing various stages of CRC development (i.e. HT29 and Caco-2-early, SW480 and SW620 late). The expression of E-cadherin gene CDH1 was downregulated by the specific siRNA. Cell proliferation and chemosensitivity to irinotecan (IT) and oxaliplatin (OPT) were detected using WST-1 and x-CELLigence Real Time analysis. Expression of selected EMT markers were tested and compared using RT-PCR and western blot analysis in both variants (E-cadherin silenced and non-silenced) of each cell line. We have discovered that downregulation of E-cadherin expression has a diverse effect on both cell proliferation as well as the expression of EMT markers in individual tested CRC cell lines, with Caco-2 cells being the most responsive. On the other hand, reduced E-cadherin expression resulted in increased sensitivity of all cell lines to IT and mostly to OPT which might be related to changes in intracellular metabolism of these drugs. These results suggest dichotomy of E-cadherin involvement in the phenotypic EMT spectrum of CRC and warrants further mechanistic studies.

TAp63α Is Involved in Tobacco Smoke-Induced Lung Cancer EMT and the Anti-cancer Activity of Curcumin via miR-19 Transcriptional Suppression.

As a key risk factor for lung cancer, tobacco smoke (TS) influences several cellular processes, including epithelial-mesenchymal transition (EMT). TAp63α is a crucial transcription factor involved in tumor progression. The present study was designed to investigate the potential role and underlying mechanisms of TAp63α in TS-induced lung cancer EMT. We found that compared to normal tissues, the tumor tissues collected from lung cancer patients showed a lower level of TAp63α expression, along with downregulated E-cadherin expression and upregulated Vimentin expression. Results of treatment with TAp63α and TAp63α siRNA as well as with tumor growth factor-β (TGF-β) showed that TAp63α acted as a tumor suppressor gene, and its upregulated expression suppressed lung cancer EMT. Significantly, TS exposure altered expression of EMT-related markers, enhanced cell migratory and invasive capacities, and decreased the TAp63α expression level in lung cancer cells. Overexpression of TAp63α significantly alleviated TS-stimulated lung cancer EMT. Mechanistically, TAp63α expression transcriptionally reduced the miR-19 level, which resulted in the suppression of lung cancer EMT. Additionally, as a natural compound possessing anti-cancer effects, curcumin inhibited TS-induced lung cancer EMT by increasing TAp63α expression and reducing miR-19 expression. Collectively, our results indicate that TAp63α inhibits TS-induced lung cancer EMT via transcriptionally suppressing miR-19 and the inhibitory effect of TAp63α on miR-19 mediates the anti-cancer action of curcumin. These findings provide new insights into novel targets for lung cancer prevention.

LncRNA-HCG18 regulates the viability, apoptosis, migration, invasion and epithelial-mesenchymal transition of papillary thyroid cancer cells via regulating the miR-106a-5p/PPP2R2A axis

The incidence of papillary thyroid cancer (PTC) has experienced a rapid increase in recent years. Long non-coding RNA-homo sapiens HLA complex group (HCG) 18 plays a regulatory role in cancers, but its role in PTC remained unknown. This study determined the expressions of HCG18, microRNA (miR)-106a-5p, and protein phosphatase 2 regulatory subunit B alpha (PPP2R2A) in PTC tissues and cells by qRT-PCR. ENCORI predicted the targeting relation between HCG18 and miR-106a-5p. TargetScan v7.2 predicted the targeting relation between miR-106a-5p and PPP2R2A. Dual-luciferase reporter assay was performed to validate the two targeting relations. The viability, migration, and invasion of PTC cells were detected by Cell Counting Kit-8, wound healing assay, and Transwell assay, respectively. The expressions of matrix metalloproteinase 2 (MMP-2), MMP-9, E-cadherin, N-cadherin, and Vimentin in TPC-1 and MDA-T68 cells were assessed by qRT-PCR and Western blot. It was found that HCG18 was down-regulated in PTC. Overexpressing HCG18 suppressed viability, migration, and invasion, promoted apoptosis, and inhibited miR-106a-5p expression in PTC cells. HCG18 interacted with miR-106a-5p, the expression of which was upregulated in PTC. Upregulating miR-106a-5p expression by lentivirus infection promoted viability, migration and invasion and inhibited apoptosis of PTC cells, reversed the effect of HCG18 on the biological behaviors of PTC cells, and promoted the expressions of MMP-2, MMP-9, E-cadherin, and Vimentin and downregulated E-cadherin expression in PTC cells. PPP2R2A, a direct target of miR-106a-5p, was downregulated in PTC, and HCG18 promoted PPP2R2A expression in PTC cells by sponging miR-106a-5p. Furthermore, PPP2R2A reversed the effects of miR-106a-5p on PTC cells. In conclusion, HCG18 suppressed viability, migration, invasion and epithelial–mesenchymal transition and promoted apoptosis of PTC cells via regulating the miR-106a-5p/PPP2R2A axis.

In Vitro Chemopreventive Potential of Phlorotannins-Rich Extract from Brown Algae by Inhibition of Benzo[a]pyrene-Induced P2X7 Activation and Toxic Effects.

Phlorotannins are polyphenols occurring exclusively in some species of brown algae, known for numerous biological activities, e.g., antioxidant, antiproliferative, antidiabetic, and antiallergic properties. Their effects on the response of human lung cells to benzo[a]pyrene (B[a]P) has not been characterized. Our objective was to in vitro evaluate the effects of a phlorotannin-rich extract obtained from the brown algae Ascophyllum nodosum and Fucus vesiculosus on B[a]P cytotoxic effects. The A549 cell line was incubated with B[a]P for 48 and 72 h in the presence or absence of the brown algae extract. Cytochrome P450 activity, activation of P2X7 receptor, F-actin disorganization, and loss of E-cadherin expression were assessed using microplate cytometry and fluorescence microscopy. Relative to control, incubation with the brown algae extract was associated with lower B[a]P-induced CYP1 activity, lower P2X7 receptor activation, and lower reactive oxygen species production. The brown algae extract inhibited the alterations of F-actin arrangement and the downregulation of E-cadherin expression. We identified a phlorotannins-rich extract that could be deeper investigated as a cancer chemopreventive agent to block B[a]P-mediated carcinogenesis.

Helicobacter pylori induces epithelial-mesenchymal transition in gastric carcinogenesis via the AKT/GSK3β signaling pathway.

Helicobacter pylori (H. pylori) is a main risk factor for gastric cancer (GC). Epithelial-mesenchymal transition (EMT) is involved in the development and progression of H. pylori-associated GC. However, the exact molecular mechanism of this process remains unclear. The AKT/GSK3β signaling pathway has been demonstrated to promote EMT in several types of cancer. The present study investigated whether H. pylori infection induced EMT, and promoted the development and metastasis of cancer in the normal gastric mucosa, and whether this process was dependent on AKT activation. The expression levels of the EMT-associated proteins, including E-cadherin and N-cadherin, were determined in 165 gastric mucosal samples of different disease stages by immunohistochemical analysis. The expression levels of E-cadherin, N-cadherin, AKT, phosphorylated (p-)AKT (Ser473), GSK3β and p-GSK3β (Ser9) were further determined in H. pylori-infected Mongolian gerbil gastric tissues and cells co-cultured with H. pylori by immunohistochemical analysis and western blotting. The results indicated that the expression levels of the epithelial marker E-cadherin were decreased, whereas the expression levels of the mesenchymal marker N-cadherin were increased during gastric carcinogenesis. Their expression levels were associated with H. pylori infection. Furthermore, H. pylori infection resulted in downregulation of E-cadherin expression and upregulation of N-cadherin expression in Mongolian gerbils and GES-1 cells. In addition, an investigation of the associated mechanism of action revealed that p-AKT (Ser473) and p-GSK3β (Ser9) were activated in GES-1 cells following co-culture with H. pylori. Furthermore, following pretreatment of the cells with the AKT inhibitor VIII, the expression levels of E-cadherin, N-cadherin, p-AKT and p-GSK3β did not show significant differences between GES-1 cells that were co-cultured with or without H. pylori. The levels of p-AKT and p-GSK3β were increased in H. pylori-infected Mongolian gerbils. In conclusion, the present study demonstrated that H. pylori infection activated AKT and resulted in the phosphorylation and inactivation of GSK3β, which in turn promoted early stage EMT. These effects were AKT-dependent. This mechanism may serve as a prerequisite for GC development.

ZEB1 and oncogenic Ras constitute a regulatory switch for stimulus-dependent E-cadherin downregulation.

E‐cadherin, an epithelial cell–specific cell adhesion molecule, has both promoting and suppressing effects on tumor invasion and metastasis. It is often downregulated during cancer progression through gene deletion/mutation, transcriptional repression, or epigenetic silencing. We describe a novel regulatory switch to induce stimulus‐dependent downregulation of mRNA encoding E‐cadherin (CDH1 mRNA) in KRAS‐mutated cancer cells. The regulatory switch consists of ZEB1 and oncogenic K‐Ras, does not target the promoter region of CDH1, and requires an external cue to temporally downregulate E‐cadherin expression. Its repressive effect is maintained as long as the external stimulus continues and is attenuated with cessation of the stimulus. Contextual external cues that turn this regulatory switch on include activation of protein kinase C or fibroblast growth factor signaling. The mode of action is distinct from that of EPCAM repression by ZEB1, which does not require an external cue. Thus, KRAS‐mutated cancer cells acquire a novel mode of regulating E‐cadherin expression depending on ZEB1, which could contribute to phenotypic plasticity of cancer cells during malignant progression.

Chronic stress promotes EMT-mediated metastasis through activation of STAT3 signaling pathway by miR-337-3p in breast cancer

Chronic stress could induce cancer metastasis by constant activation of the sympathetic nervous system, while cellular mechanism remains obscure. The aim of this research is to explore the metastasis associated negative effect of chronic stress. The analysis of transcriptome sequencing implied that activation of STAT3 signaling pathway by downregulated miR-337-3p might be a potential mechanism to induce epithelial to mesenchymal transition (EMT) of cancer cell and promote metastasis under chronic stress. We also verified this biological process in further experiments. Downregulation of miR-337-3p could downregulate E-cadherin expression and upregulate vimentin expression in vitro and in vivo. STAT3, related signal pathways of which are involved in metastasis regulation, was directly targeted by miR-337-3p. In conclusion, the above results denoted that activation of miR-337-3p/STAT3 axis might be a potential pathway for the increasing metastasis of breast cancer under chronic stress.