Crosstalk between Beta-Catenin and Snail in the Induction of Epithelial to Mesenchymal Transition in Hepatocarcinoma: Role of the ERK1/2 Pathway

Epithelial to mesenchymal transition (EMT) is an integral process in the progression of many epithelial tumors. It involves a coordinated series of events, leading to the loss of epithelial features and the acquisition of a mesenchymal phenotype, resulting in invasion and metastasis. The EMT of hepatocellular carcinoma (HCC) cells is thought to be a key event in intrahepatic dissemination and distal metastasis. In this study, we used 12-O-tet-radecanoylphorbol-13-acetate (TPA) to dissect the signaling pathways involved in the EMT of HepG2 hepatocarcinoma cells. The spectacular change in phenotype induced by TPA, leading to a pronounced spindle-shaped fibroblast-like cell morphology, required ERK1/2 activation. This ERK1/2-dependent EMT process was characterized by a loss of E-cadherin function, modification of the cytoskeleton, the acquisition of mesenchymal markers and profound changes to extracellular matrix composition and mobility. Snail was essential for E-cadherin repression, but was not sufficient for full commitment of the TPA-triggered EMT. We found that TPA triggered the formation of a complex between Snail and β-catenin that activated the Wnt pathway. This study thus provides the first evidence for the existence of a complex network governed by the ERK1/2 signaling pathway, converging on the coregulation of Snail and the Wnt/β-catenin pathway and responsible for the onset and the progression of EMT in hepatocellular carcinoma cells.     

Cripto-1 as a Key Factor in Tumor Progression,Epithelial to Mesenchymal Transition and Cancer Stem Cells

Cripto-1 is an essential protein for human development that plays a key role in the early phase of gastrulation in the differentiation of an embryo as well as assists with wound healing processes. Importantly, Cripto-1 induces epithelial to mesenchymal transition to turn fixed epithelial cells into a more mobile mesenchymal phenotype through the downregulation of epithelial adhesion molecules such as E-cadherin, occludins, and claudins, and the upregulation of mesenchymal, mobile proteins, such as N-cadherin, Snail, and Slug. Consequently, Cripto-1’s role in inducing EMT to promote cell motility is beneficial in embryogenesis, but detrimental in the formation, progression and metastasis of malignant tumors. Indeed, Cripto-1 is found to be upregulated in most cancers, such as breast, lung, gastrointestinal, hepatic, renal, cervical, ovarian, prostate, and skin cancers. Through its role in EMT, Cripto-1 can remodel cancer cells to enable them to travel through the extracellular matrix as well as blood and lymphatic vessels to metastasize to different organs. Additionally, Cripto-1 promotes the survival of cancer stem cells, which can lead to relapse in cancer patients.     

The Epithelial–Mesenchymal Transition at the Crossroads between Metabolism and Tumor Progression

The transition between epithelial and mesenchymal phenotype is emerging as a key determinant of tumor cell invasion and metastasis. It is a plastic process in which epithelial cells first acquire the ability to invade the extracellular matrix and migrate into the bloodstream via transdifferentiation into mesenchymal cells, a phenomenon known as epithelial–mesenchymal transition (EMT), and then reacquire the epithelial phenotype, the reverse process called mesenchymal–epithelial transition (MET), to colonize a new organ. During all metastatic stages, metabolic changes, which give cancer cells the ability to adapt to increased energy demand and to withstand a hostile new environment, are also important determinants of successful cancer progression. In this review, we describe the complex interaction between EMT and metabolism during tumor progression. First, we outline the main connections between the two processes, with particular emphasis on the role of cancer stem cells and LncRNAs. Then, we focus on some specific cancers, such as breast, lung, and thyroid cancer.     

BMAL1 Knockdown Leans Epithelial–Mesenchymal Balance toward Epithelial Properties and Decreases the Chemoresistance of Colon Carcinoma Cells

The circadian clock coordinates biological and physiological functions to day/night cycles. The perturbation of the circadian clock increases cancer risk and affects cancer progression. Here, we studied how BMAL1 knockdown (BMAL1-KD) by shRNA affects the epithelial–mesenchymal transition (EMT), a critical early event in the invasion and metastasis of colorectal carcinoma (CRC). In corresponding to a gene set enrichment analysis, which showed a significant enrichment of EMT and invasive signatures in BMAL1_high CRC patients as compared to BMAL1_low CRC patients, our results revealed that BMAL1 is implicated in keeping the epithelial–mesenchymal equilibrium of CRC cells and influences their capacity of adhesion, migration, invasion, and chemoresistance. Firstly, BMAL1-KD increased the expression of epithelial markers (E-cadherin, CK-20, and EpCAM) but decreased the expression of Twist and mesenchymal markers (N-cadherin and vimentin) in CRC cell lines. Finally, the molecular alterations after BMAL1-KD promoted mesenchymal-to-epithelial transition-like changes mostly appeared in two primary CRC cell lines (i.e., HCT116 and SW480) compared to the metastatic cell line SW620. As a consequence, migration/invasion and drug resistance capacities decreased in HCT116 and SW480 BMAL1-KD cells. Together, BMAL1-KD alerts the delicate equilibrium between epithelial and mesenchymal properties of CRC cell lines, which revealed the crucial role of BMAL1 in EMT-related CRC metastasis and chemoresistance.     

Involvement of the ERK/HIF-1α/EMT Pathway in XCL1-Induced Migration of MDA-MB-231 and SK-BR-3 Breast Cancer Cells

Chemokine–receptor interactions play multiple roles in cancer progression. It was reported that the overexpression of X-C motif chemokine receptor 1 (XCR1), a specific receptor for chemokine X-C motif chemokine ligand 1 (XCL1), stimulates the migration of MDA-MB-231 triple-negative breast cancer cells. However, the exact mechanisms of this process remain to be elucidated. Our study found that XCL1 treatment markedly enhanced MDA-MB-231 cell migration. Additionally, XCL1 treatment enhanced epithelial–mesenchymal transition (EMT) of MDA-MB-231 cells via E-cadherin downregulation and upregulation of N-cadherin and vimentin as well as increases in β-catenin nucleus translocation. Furthermore, XCL1 enhanced the expression of hypoxia-inducible factor-1α (HIF-1α) and phosphorylation of extracellular signal-regulated kinase (ERK) 1/2. Notably, the effects of XCL1 on cell migration and intracellular signaling were negated by knockdown of XCR1 using siRNA, confirming XCR1-mediated actions. Treating MDA-MB-231 cells with U0126, a specific mitogen-activated protein kinase kinase (MEK) 1/2 inhibitor, blocked XCL1-induced HIF-1α accumulation and cell migration. The effect of XCL1 on cell migration was also evaluated in ER^-/HER2⁺ SK-BR-3 cells. XCL1 also promoted cell migration, EMT induction, HIF-1α accumulation, and ERK phosphorylation in SK-BR-3 cells. While XCL1 did not exhibit any significant impact on the matrix metalloproteinase (MMP)-2 and -9 expressions in MDA-MB-231 cells, it increased the expression of these enzymes in SK-BR-3 cells. Collectively, our results demonstrate that activation of the ERK/HIF-1α/EMT pathway is involved in the XCL1-induced migration of both MDA-MB-231 and SK-BR-3 breast cancer cells. Based on our findings, the XCL1–XCR1 interaction and its associated signaling molecules may serve as specific targets for the prevention of breast cancer cell migration and metastasis.     

Conditioned Media of Adipose-Derived Stem Cells Suppresses Sidestream Cigarette Smoke Extract Induced Cell Death and Epithelial-Mesenchymal Transition in Lung Epithelial Cells

The role of the epithelial–mesenchymal transition (EMT) in lung epithelial cells is increasingly being recognized as a key stage in the development of COPD, fibrosis, and lung cancers, which are all highly associated with cigarette smoking and with exposure to second-hand smoke. Using the exposure of human lung cancer epithelial A549 cells and non-cancerous Beas-2B cells to sidestream cigarette smoke extract (CSE) as a model, we studied the protective effects of adipose-derived stem cell-conditioned medium (ADSC-CM) against CSE-induced cell death and EMT. CSE dose-dependently induced cell death, decreased epithelial markers, and increased the expression of mesenchymal markers. Upstream regulator analysis of differentially expressed genes after CSE exposure revealed similar pathways as those observed in typical EMT induced by TGF-β1. CSE-induced cell death was clearly attenuated by ADSC-CM but not by other control media, such as a pass-through fraction of ADSC-CM or A549-CM. ADSC-CM effectively inhibited CSE-induced EMT and was able to reverse the gradual loss of epithelial marker expression associated with TGF-β1 treatment. CSE or TGF-β1 enhanced the speed of A549 migration by 2- to 3-fold, and ADSC-CM was effective in blocking the cell migration induced by either agent. Future work will build on the results of this in vitro study by defining the molecular mechanisms through which ADSC-CM protects lung epithelial cells from EMT induced by toxicants in second-hand smoke.     

Resveratrol Sensitizes Tamoxifen in Antiestrogen-Resistant Breast Cancer Cells with Epithelial-Mesenchymal Transition Features

Tamoxifen resistance remains to be a huge obstacle in the treatment of hormone-dependent breast cancer, and this therefore highlights the dire need to explore the underlying mechanisms. The epithelial-mesenchymal transition (EMT) is a molecular process through which an epithelial cell transfers into a mesenchymal phenotype. Roles of EMT in embryo development, cancer invasion and metastasis have been extensively reported. Herein, we established tamoxifen-resistant MCF-7/TR breast cancer cells and showed that MCF-7/TR cells underwent EMT driven by enhanced endogenous TGF-β/Smad signaling. Ectopic supplement of TGF-β promoted in MCF-7 cells a mesenchymal and resistant phenotype. In parallel, we demonstrated that resveratrol was capable of synergizing with tamoxifen and triggering apoptosis in MCF-7/TR cells. Further Western blot analysis indicated that the chemosensitizing effects of resveratrol were conferred with its modulation on endogenous TGF-β production and Smad phosphorylation. In particular, 50 μM resveratrol had minor effects on MCF-7/TR cell proliferation, but could significantly attenuate endogenous TGF-β production and the Smad pathway, ultimately leading to reversion of EMT. Collectively, our study highlighted distinct roles of EMT in tamoxifen resistance and resveratrol as a potential agent to overcome acquired tamoxifen resistance. The molecular mechanism of resveratrol chemosensitizing effects is, at least in part, TGF-β/Smad-dependent.     

Concurrent Chemoradiotherapy-Driven Cell Plasticity by miR-200 Family Implicates the Therapeutic Response of Esophageal Squamous Cell Carcinoma

Esophageal squamous cell carcinoma (ESCC) is a common and fatal malignancy with an increasing incidence worldwide. Over the past decade, concurrent chemoradiotherapy (CCRT) with or without surgery is an emerging therapeutic approach for locally advanced ESCC. Unfortunately, many patients exhibit poor response or develop acquired resistance to CCRT. Once resistance occurs, the overall survival rate drops down rapidly and without proper further treatment options, poses a critical clinical challenge for ESCC therapy. Here, we utilized lab-created CCRT-resistant cells as a preclinical study model to investigate the association of chemoradioresistantresistance with miRNA-mediated cell plasticity alteration, and to determine whether reversing EMT status can re-sensitize refractory cancer cells to CCRT response. During the CCRT treatment course, refractory cancer cells adopted the conversion of epithelial to mesenchymal phenotype; additionally, miR-200 family members were found significantly down-regulated in CCRT resistance cells by miRNA microarray screening. Down-regulated miR-200 family in CCRT resistance cells suppressed E-cadherin expression through snail and slug, and accompany with an increase in N-cadherin. Rescuing expressions of miR-200 family members in CCRT resistance cells, particularly in miR-200b and miR-200c, could convert cells to epithelial phenotype by increasing E-cadherin expression and sensitize cells to CCRT treatment. Conversely, the suppression of miR-200b and miR-200c in ESCC cells attenuated E-cadherin, and that converted cells to mesenchymal type by elevating N-cadherin expression, and impaired cell sensitivity to CCRT treatment. Moreover, the results of ESCC specimens staining established the clinical relevance that higher N-cadherin expression levels associate with the poor CCRT response outcome in ESCC patients. Conclusively, miR-200b and miR-200c can modulate the conversion of epithelial–mesenchymal phenotype in ESCC, and thereby altering the response of cells to CCRT treatment. Targeting epithelial–mesenchymal conversion in acquired CCRT resistance may be a potential therapeutic option for ESCC patients.     

The Significance of Epithelial-to-Mesenchymal Transition for Circulating Tumor Cells

Epithelial to mesenchymal transition (EMT) is a process involved in embryonic development, but it also plays a role in remote metastasis formation in tumor diseases. During this process cells lose their epithelial features and adopt characteristics of mesenchymal cells. Thereby single tumor cells, which dissolve from the primary tumor, are enabled to invade the blood vessels and travel throughout the body as so called “circulating tumor cells” (CTCs). After leaving the blood stream the reverse process of EMT, the mesenchymal to epithelial transition (MET) helps the cells to seed in different tissues, thereby generating the bud of metastasis formation. As metastasis is the main reason for tumor-associated death, CTCs and the EMT process are in the focus of research in recent years. This review summarizes what was already found out about the molecular mechanisms driving EMT, the consequences of EMT for tumor cell detection, and suitable markers for the detection of CTCs which underwent EMT. The research work done in this field could open new roads towards combating cancer.     

Long Non-Coding RNA MIR31HG Promotes the Transforming Growth Factor β-Induced Epithelial-Mesenchymal Transition in Pancreatic Ductal Adenocarcinoma Cells

The epithelial-to-mesenchymal transition (EMT) describes a biological process in which polarized epithelial cells are converted into highly motile mesenchymal cells. It promotes cancer cell dissemination, allowing them to form distal metastases, and also involves drug resistance in metastatic cancers. Transforming growth factor β (TGFβ) is a multifunctional cytokine that plays essential roles in development and carcinogenesis. It is a major inducer of the EMT. The MIR31 host gene (MIR31HG) is a newly identified long non-coding (lnc)RNA that exhibits ambiguous roles in cancer. In this study, a cancer genomics analysis predicted that MIR31HG overexpression was positively correlated with poorer disease-free survival of pancreatic ductal adenocarcinoma (PDAC) patients, which was associated with upregulation of genes related to TGFβ signaling and the EMT. In vitro evidence demonstrated that TGFβ induced MIR31HG expression in PDAC cells, and knockdown of MIR31HG expression reversed TGFβ-induced EMT phenotypes and cancer cell migration. Therefore, MIR31HG has an oncogenic role in PDAC by promoting the EMT.     

Possible Action of Olaparib for Preventing Invasion of Oral Squamous Cell Carcinoma In Vitro and In Vivo

Despite recent advances in treatment, the prognosis of oral cancer remains poor, and prevention of recurrence and metastasis is critical. Olaparib is a PARP1 inhibitor that blocks polyADP-ribosylation, which is involved in the epithelial–mesenchymal transition (EMT) characteristic of tumor recurrence. We explored the potential of olaparib in inhibiting cancer invasion in oral carcinoma using three oral cancer cell lines, HSC-2, Ca9-22, and SAS. Olaparib treatment markedly reduced their proliferation, migration, invasion, and adhesion. Furthermore, qRT-PCR revealed that olaparib inhibited the mRNA expression of markers associated with tumorigenesis and EMT, notably Ki67, Vimentin, β-catenin, MMP2, MMP9, p53, and integrin α2 and β1, while E-Cadherin was upregulated. In vivo analysis of tumor xenografts generated by injection of HSC-2 cells into the masseter muscles of mice demonstrated significant inhibition of tumorigenesis and bone invasion by olaparib compared with the control. This was associated with reduced expression of proteins involved in osteoclastogenesis, RANK and RANKL. Moreover, SNAIL and PARP1 were downregulated, while E-cadherin was increased, indicating the effect of olaparib on proteins associated with EMT in this model. Taken together, these findings confirm the effects of olaparib on EMT and bone invasion in oral carcinoma and suggest a new therapeutic strategy for this disease.     

Combination Effect of Cilengitide with Erlotinib on TGF-β1-Induced Epithelial-to-Mesenchymal Transition in Human Non-Small Cell Lung Cancer Cells

The epithelial-to-mesenchymal transition (EMT) is important for morphogenesis during development and is mainly induced by transforming growth factor (TGF)-β. In lung cancer, EMT is characterized by the transformation of cancer cells into a mobile, invasive form that can transit to other organs. Here, using a non–small cell lung cancer (NSCLC) cell line, we evaluated the EMT-related effects of the epidermal growth factor receptor inhibitor erlotinib alone and in combination with cilengitide, a cyclic RGD-based integrin antagonist. Erlotinib showed anti-proliferative and inhibitory effects against the TGF-β1–induced EMT phenotype in NSCLC cells. Compared with erlotinib alone, combination treatment with cilengitide led to an enhanced inhibitory effect on TGF-β1–induced expression of mesenchymal markers and invasion in non–small cell lung cancer A549 cells. These results suggest that cilengitide could improve anticancer drug efficacy and contribute to improved treatment strategies to inhibit and prevent EMT-based cancer progression.     

The Identification of Small Molecule Inhibitors That Reduce Invasion and Metastasis of Aggressive Cancers

Transformed epithelial cells can activate programs of epithelial plasticity and switch from a sessile, epithelial phenotype to a motile, mesenchymal phenotype. This process is linked to the acquisition of an invasive phenotype and the formation of distant metastases. The development of compounds that block the acquisition of an invasive phenotype or revert the invasive mesenchymal phenotype into a more differentiated epithelial phenotype represent a promising anticancer strategy. In a high-throughput assay based on E-cadherin (re)induction and the inhibition of tumor cell invasion, 44,475 low molecular weight (LMW) compounds were screened. The screening resulted in the identification of candidate compounds from the PROAM02 class. Selected LMW compounds activated E-cadherin promoter activity and inhibited cancer cell invasion in multiple metastatic human cancer cell lines. The intraperitoneal administration of selected LMW compounds reduced the tumor burden in human prostate and breast cancer in vivo mouse models. Moreover, selected LMW compounds decreased the intra-bone growth of xenografted human prostate cancer cells. This study describes the identification of the PROAM02 class of small molecules that can be exploited to reduce cancer cell invasion and metastases. Further clinical evaluation of selected candidate inhibitors is warranted to address their safety, bioavailability and antitumor efficacy in the management of patients with aggressive cancers.     

Particulate Matter (PM10) Promotes Cell Invasion through Epithelial–Mesenchymal Transition (EMT) by TGF-β Activation in A549 Lung Cells

Air pollution presents a major environmental problem, inducing harmful effects on human health. Particulate matter of 10 μm or less in diameter (PM₁₀) is considered an important risk factor in lung carcinogenesis. Epithelial–mesenchymal transition (EMT) is a regulatory program capable of inducing invasion and metastasis in cancer. In this study, we demonstrated that PM₁₀ treatment induced phosphorylation of SMAD2/3 and upregulation of SMAD4. We also reported that PM₁₀ increased the expression and protein levels of TGFB1 (TGF-β), as well as EMT markers SNAI1 (Snail), SNAI2 (Slug), ZEB1 (ZEB1), CDH2 (N-cadherin), ACTA2 (α-SMA), and VIM (vimentin) in the lung A549 cell line. Cell exposed to PM₁₀ also showed a decrease in the expression of CDH1 (E-cadherin). We also demonstrated that expression levels of these EMT markers were reduced when cells are transfected with small interfering RNAs (siRNAs) against TGFB1. Interestingly, phosphorylation of SMAD2/3 and upregulation of SMAD induced by PM₁₀ were not affected by transfection of TGFB1 siRNAs. Finally, cells treated with PM₁₀ exhibited an increase in the capacity of invasiveness because of EMT induction. Our results provide new evidence regarding the effect of PM₁₀ in EMT and the acquisition of an invasive phenotype, a hallmark necessary for lung cancer progression.     

The Wnt11 Signaling Pathway in Potential Cellular EMT and Osteochondral Differentiation Progression in Nephrolithiasis Formation

The molecular events leading to nephrolithiasis are extremely complex. Previous studies demonstrated that calcium and transforming growth factor-β1 (TGF-β1) may participate in the pathogenesis of stone formation, but the explicit mechanism has not been defined. Using a self-created genetic hypercalciuric stone-forming (GHS) rat model, we observed that the increased level of serous/uric TGF-β1 and elevated intracellular calcium in primary renal tubular epithelial cells (PRECs) was associated with nephrolithiasis progression in vivo. In the setting of high calcium plus high TGF-β1 in vitro, PRECs showed great potential epithelial to mesenchymal transition (EMT) progression and osteochondral differentiation properties, representing the multifarious increased mesenchymal and osteochondral phenotypes (Zeb1, Snail1, Col2A1, OPN, Sox9, Runx2) and decreased epithelial phenotypes (E-cadherin, CK19) bythe detection of mRNAs and corresponding proteins. Moreover, TGF-β-dependent Wnt11 knockdown and L-type Ca²⁺ channel blocker could greatly reverse EMT progression and osteochondral differentiation in PRECs. TGF-β1 alone could effectively promote EMT, but it had no effect on osteochondral differentiation in NRK cells (Rat kidney epithelial cell line). Stimulation with Ca²⁺ alone did not accelerate differentiation of NRK. Co-incubation of extracellular Ca²⁺ and TGF-β1 synergistically promotes EMT and osteochondral differentiation in NRK control cells. Our data supplied a novel view that the pathogenesis of calcium stone development may be associated with synergic effects of TGF-β1 and Ca²⁺, which promote EMT and osteochondral differentiation via Wnt11 and the L-type calcium channel.     

Doxorubicin-Resistant TNBC Cells Exhibit Rapid Growth with Cancer Stem Cell-like Properties and EMT Phenotype,Which Can Be Transferred to Parental Cells through Autocrine Signaling

Emerging evidence suggests that breast cancer stem cells (BCSCs), and epithelial–mesenchymal transition (EMT) may be involved in resistance to doxorubicin. However, it is unlear whether the doxorubicin-induced EMT and expansion of BCSCs is related to cancer dormancy, or outgrowing cancer cells with maintaining resistance to doxorubicin, or whether the phenotypes can be transferred to other doxorubicin-sensitive cells. Here, we characterized the phenotype of doxorubicin-resistant TNBC cells while monitoring the EMT process and expansion of CSCs during the establishment of doxorubicin-resistant MDA-MB-231 human breast cancer cells (DRM cells). In addition, we assessed the potential signaling associated with the EMT process and expansion of CSCs in doxorubicin-resistance of DRM cells. DRM cells exhibited morphological changes from spindle-shaped MDA-MB-231 cells into round-shaped giant cells. They exhibited highly proliferative, EMT, adhesive, and invasive phenotypes. Molecularly, they showed up-regulation of Cyclin D1, mesenchymal markers (β-catenin, and N-cadherin), MMP-2, MMP-9, ICAM-1 and down-regulation of E-cadherin. As the molecular mechanisms responsible for the resistance to doxorubicin, up-regulation of EGFR and its downstream signaling, were suggested. AKT and ERK1/2 expression were also increased in DRM cells with the advancement of resistance to doxorubicin. Furthermore, doxorubicin resistance of DRM cells can be transferred by autocrine signaling. In conclusion, DRM cells harbored EMT features with CSC properties possessing increased proliferation, invasion, migration, and adhesion ability. The doxorubicin resistance, and doxorubicin-induced EMT and CSC properties of DRM cells, can be transferred to parental cells through autocrine signaling. Lastly, this feature of DRM cells might be associated with the up-regulation of EGFR.