Hallmark Of Epithelial-mesenchymal Transition Research Articles

Dissecting the effects of androgen deprivation therapy on cadherin switching in advanced prostate cancer: A molecular perspective.

Prostate cancer is one of the most often diagnosed malignancies in males and its prevalence is rising in both developed and developing countries. Androgen deprivation therapy has been used as a standard treatment approach for advanced prostate cancer for more than 80 years. The primary aim of androgen deprivation therapy is to decrease circulatory androgen and block androgen signaling. Although a partly remediation is accomplished at the beginning of treatment, some cell populations become refractory to androgen deprivation therapy and continue to metastasize. Recent evidences suggest that androgen deprivation therapy may cause cadherin switching, from E-cadherin to N-cadherin, which is the hallmark of epithelial-mesenchymal transition. Diverse direct and indirect mechanisms are involved in this switching and consequently, the cadherin pool changes from E-cadherin to N-cadherin in the epithelial cells. Since E-cadherin represses invasive and migrative behaviors of the tumor cells, the loss of E-cadherin disrupts epithelial tissue structure leading to the release of tumor cells into surrounding tissues and circulation. In this study, we review the androgen deprivation therapy-dependent cadherin switching in advanced prostate cancer with emphasis on its molecular basis especially the transcriptional factors regulated through TFG-β pathway.

AEBP1 Is One of the Epithelial-Mesenchymal Transition Regulatory Genes in Colon Adenocarcinoma.

Epithelial-mesenchymal transition (EMT) is involved in various tumor processes, including tumorigenesis, tumor cell migration and metastasis, tumor stemness, and therapeutic resistance. Therefore, it is important to identify the genes most associated with EMT and develop them as therapeutic targets. In this work, we first analyzed EMT hallmark gene expression profiles among 10,535 pan-cancer samples from The Cancer Genome Atlas (TCGA) and divided them into EMT high and EMT low groups according to the metagene scores. Then, we identified 12 genes that were most associated with high EMT metagene score (R > 0.9) in 329 colon adenocarcinoma (COAD) patients. Among them, only 4 genes (AEBP1, KCNE4, GFPT2, and FAM26E) had statistically significant differences in prognosis (P < 0.05). Next, we selected AEBP1 as a candidate and showed that AEBP1 mRNA levels and EMT biomarkers strongly coexpressed in 329 COAD samples. In addition, AEBP1 was highly expressed and associated with poor clinical outcomes and prognosis in COAD patients. Finally, to explore whether AEBP1-mediated EMT was related to the tumor microenvironment (TME), we examined AEBP1 expression levels at the single-cell levels. Our results showed that AEBP1 levels were extremely high in tumor-associated fibroblasts, which may induce EMT. AEBP1 expression was also positively correlated with the expression of fibroblast biomarkers and also with EMT metascores, suggesting that AEBP1-mediated EMT may be associated with the stimulation of fibroblast activation. Therefore, AEBP1 may be a promising target for EMT inhibition, which reduces cancer metastasis and drug resistance in COAD patients.

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.

MCPIP1 inhibits Wnt/\u03b2-catenin signaling pathway activity and modulates epithelial-mesenchymal transition during clear cell renal cell carcinoma progression by targeting miRNAs

Epithelial-mesenchymal transition (EMT) refers to the acquisition of mesenchymal properties in cells participating in tumor progression. One hallmark of EMT is the increased level of active β-catenin, which can trigger the transcription of Wnt-specific genes responsible for the control of cell fate. We investigated how Monocyte Chemotactic Protein-1-Induced Protein-1 (MCPIP1), a negative regulator of inflammatory processes, affects EMT in a clear cell renal cell carcinoma (ccRCC) cell line, patient tumor tissues and a xenotransplant model. We showed that MCPIP1 degrades miRNAs via its RNase activity and thus protects the mRNA transcripts of negative regulators of the Wnt/β-catenin pathway from degradation, which in turn prevents EMT. Mechanistically, the loss of MCPIP1 RNase activity led to the upregulation of miRNA-519a-3p, miRNA-519b-3p, and miRNA-520c-3p, which inhibited the expression of Wnt pathway inhibitors (SFRP4, KREMEN1, CXXC4, CSNK1A1 and ZNFR3). Thus, the level of active nuclear β-catenin was increased, leading to increased levels of EMT inducers (SNAI1, SNAI2, ZEB1 and TWIST) and, consequently, decreased expression of E-cadherin, increased expression of mesenchymal markers, and acquisition of the mesenchymal phenotype. This study revealed that MCPIP1 may act as a tumor suppressor that prevents EMT by stabilizing Wnt inhibitors and decreasing the levels of active β-catenin and EMT inducers.

28P Validation of cytokeratin (CK18) protein expression in epithelial cell lines and in circulating tumor cells (CTCs)

CTCs predicts an unfavourable prognosis and outcomes in cancers. Lowering of cytokeratin 18 expression is a hallmark of epithelial mesenchymal transition (EMT). Homogeneity and validation of CK18 expression in cancer cell lines and CTCs originating from distinct solid tumors is indeterminate and may contribute to non-specific counts. We hypothesize that the expression of CK18 in varied cell lines may differ quantitatively, and additionally may exhibit similar trends in CTCs enumerated from different tumor types.

Macrophage-Related SPP1 as a Potential Biomarker for Early Lymph Node Metastasis in Lung Adenocarcinoma.

Lymph node metastasis is a major factor that affects prognosis in patients with lung adenocarcinoma (LUAD). In some cases, lymph node metastasis has already occurred when the primary tumors are still small (i.e., early T stages), however, relevant studies on early lymph node metastasis are limited, and effective biomarkers remain lacking. This study aimed to explore new molecular biomarker for early lymph node metastasis in LUAD using transcriptome sequencing and experimental validation. Here, we performed transcriptome sequencing on tissues from 16 matched patients with Stage-T1 LUAD (eight cases of lymph node metastasis and eight cases of non-metastasis), and verified the transcriptome profiles in TCGA, GSE68465, and GSE43580 cohorts. With the bioinformatics analysis, we identified a higher abundance of M0 macrophages in the metastatic group using the CIBERSORT algorithm and immunohistochemistry (IHC) analysis and the enrichment of the epithelial–mesenchymal transition (EMT) pathway was identified in patients with higher M0 infiltration levels. Subsequently, the EMT hallmark gene SPP1, encoding secreted phosphoprotein 1 (SPP1), was identified to be significantly correlated with macrophage infiltration and M2 polarization, and was determined to be a key risk indicator for early lymph node metastasis. Notably, SPP1 in the blood, as detected by enzyme-linked immunosorbent assay (ELISA) showed a superior predictive capability for early lymph node metastasis [area under the curve (AUC) = 0.74]. Furthermore, a long non-coding RNA (lncRNA, AC037441), negatively correlated with SPP1 and macrophage infiltration, had also been identified and validated to be involved in the regulation of early lymph node metastasis. In conclusion, we revealed the potential role of macrophages in lymph node metastasis and identified the macrophage-related gene SPP1 as a potential biomarker for early lymph node metastasis in LUAD.

Role of NELF-B in supporting epithelial-mesenchymal transition and cell proliferation during hepatocellular carcinoma progression.

Negative elongation factor-B (NELF-B), also known as cofactor of BRCA1 (COBRA1), is one of the four subunits of the NELF complex. It interacts with BRCA1, in addition to other transcription complexes in various tissues. The NELF complex represses the transcription of several genes by stalling RNA polymerase II during the early phase of transcription elongation. The role of NELF-B in liver cancer and hepatocellular carcinoma (HCC), the most prevalent type of liver cancer, remains to be elucidated. It has been previously demonstrated that silencing of NELF-B inhibits the proliferation and migration of HepG2 cells. The present study aimed to investigate the consequences of ectopic expression and silencing of NELF-B in liver cancer HepG2 and SNU449 cell lines. Functional assays were performed to examine the effects on gene and protein expression, viability, migration and invasion of cells. Overexpression of NELF-B did not alter the proliferation and migration of HepG2 cells, or the expression of tested genes, indicating that overexpression alone may not be sufficient for altering these features in HepG2 cells. By contrast, knockdown of NELF-B in SNU449 cells resulted in decreased cell proliferation, together with induction of apoptosis and decreased expression levels of Ki-67 and survivin, which are markers of proliferation and inhibition of apoptosis, respectively. Additionally, silencing of NELF-B resulted in a significant decrease in the hallmarks of epithelial-mesenchymal transition (EMT), including cell migration and invasion, and decreased the expression levels of EMT markers, such as N-cadherin, vimentin and β-catenin. Decreased expression levels of forkhead box F2 transcription factor and increased mRNA levels of trefoil factor 1, a putative tumor suppressor, were also detected following the silencing of NELF-B. The current results demonstrated that NELF-B enhanced the manifestation of most hallmarks of cancer, including cell proliferation, migration, invasion and inhibition of apoptosis, indicating its critical role in the progression of HCC.

EMT-Induced Gemcitabine Resistance in Pancreatic Cancer Involves the Functional Loss of Equilibrative Nucleoside Transporter 1.

Epithelial-mesenchymal transition (EMT) in cancer cells drives cancer chemoresistance, yet the molecular events of EMT that underpin the acquisition of chemoresistance are poorly understood. Here, we demonstrate a loss of gemcitabine chemosensitivity facilitated by human equilibrative nucleoside transporter 1 (ENT1) during EMT in pancreatic cancer and identify that cadherin switching from the epithelial (E) to neuronal (N) type, a hallmark of EMT, contributes to this loss. Our findings demonstrate that N-cadherin decreases ENT1 expression, membrane localization, and gemcitabine transport, while E-cadherin augments each of these. Besides E- and N-cadherin, another epithelial cell adhesion molecule, EpCAM, played a more prominent role in determining ENT1 membrane localization. Forced expression of EpCAM opposed cadherin switching with restored ENT1 expression, membrane localization, and gemcitabine transport in EMT-committed pancreatic cancer cells. In gemcitabine-treated mice, EpCAM-positive tumors had high ENT1 expression and reduced metastasis, whereas tumors with N-cadherin expression resisted gemcitabine treatment and formed extensive secondary metastatic nodules. Tissue microarray profiling and multiplexed IHC analysis of pancreatic cancer patient-derived primary tumors revealed EpCAM and ENT1 cell surface coexpression is favored, and ENT1 plasma membrane expression positively predicted median overall survival times in patients treated with adjuvant gemcitabine. Together, our findings identify ENT1 as an inadvertent target of EMT signaling mediated by cadherin switching and provide a mechanism by which mesenchymal pancreatic cancer cells evade gemcitabine therapy during EMT.

RNF20 Is Critical for Snail-Mediated E-Cadherin Repression in Human Breast Cancer.

BackgroundE-cadherin, a hallmark of epithelial-mesenchymal transition (EMT), is often repressed due to Snail-mediated epigenetic modification; however, the exact mechanism remains unclear. There is an urgent need to understand the determinants of tumor aggressiveness and identify potential therapeutic targets in breast cancer.Experimental designWe studied the association of RNF20 with Snail and G9a by co-immunoprecipitation. We employed quantitative real-time PCR, ChIP, transwell assay, colony formation assay, and mammosphere assay to dissect the molecular events associated with the repression of E-cadherin in human breast cancer. We used a proteogenomic dataset that contains 105 breast tumor samples to determine the clinical relevance of RNF20 by Kaplan-Meier analyses.ResultsIn this study, we identified that Snail interacted with RNF20, an E3 ubiquitin-protein ligase responsible for monoubiquitination of H2BK120, and G9a, a methyltransferase for H3K9me2. RNF20 expression led to the inhibition of E-cadherin expression in the human breast cancer cells. Mechanically, we showed that RNF20 and H3K9m2 were enriched on the promoter of E-cadherin and knockdown of Snail reduced the enrichment of RNF20, showing a Snail-dependent manner. RNF20 expression enhanced breast cancer cell migration, invasion, tumorsphere and colony formation. Clinically, patients with high RNF20 expression had shorter overall survival.ConclusionRNF20 expression contributes to EMT induction and breast cancer progression through Snail-mediated epigenetic suppression of E-cadherin expression, suggesting the importance of RNF20 in breast cancer.

Cyclin F Downregulation Affects Epithelial-Mesenchymal Transition Increasing Proliferation and Migration of the A-375 Melanoma Cell Line.

BackgroundCyclins are well-known cell cycle regulators. The activation of cyclin-dependent kinases by cyclins allows orchestration of the complicated cell cycle machinery and drives the cell from the G1 phase to the end of the mitotic phase. In recent years, it has become evident that cyclins are involved in processes beyond the cell cycle. Cyclin F does not activate CDKs but forms part of the Skp1-Cul1-F-box (SCF) complex where it is responsible for protein target recognition and subsequent degradation in a proteasome-dependent manner.ResultsHere, we report that the downregulation of cyclin F in the A-375 melanoma cell line increases cell viability and colony formation in a cell cycle independent manner. Lower levels of cyclin F do not appear to affect the cell cycle, based on flow cytometry measuring BrdU incorporation and propidium iodide staining. By means of immunofluorescence staining and Western blot analysis, we observed changes in cell morphology-related markers which suggested ongoing epithelial-mesenchymal transition (EMT) in response to cyclin F downregulation. Increases in vimentin and N-cadherin protein levels, decreases in levels of epithelial markers such as ZO-1, along with changes in morphology to a spindle-like shape with the appearance of actin stress fibers, are all hallmarks of EMT. These changes are associated with increased invasive and migratory potential, based on 2D migration assays. Moreover, we observe an increase in RhoABC, talin and paxillin levels, the proteins involved in controlling cell signaling and motility. Lastly, upon knocking down cyclin F expression, we observed a decrease in thrombospondin-1 expression, suggesting a role of cyclin F in angiogenesis.ConclusionCyclin F depletion induces proliferation and EMT processes in the A-375 melanoma model.

PRP4 Kinase Domain Loss Nullifies Drug Resistance and Epithelial-Mesenchymal Transition in Human Colorectal Carcinoma Cells.

We have investigated the involvement of the pre-mRNA processing factor 4B (PRP4) kinase domain in mediating drug resistance. HCT116 cells were treated with curcumin, and apoptosis was assessed based on flow cytometry and the generation of reactive oxygen species (ROS). Cells were then transfected with PRP4 or pre-mRNA-processing-splicing factor 8 (PRP8), and drug resistance was analyzed both in vitro and in vivo. Furthermore, we deleted the kinase domain in PRP4 using GatewayTM technology. Curcumin induced cell death through the production of ROS and decreased the activation of survival signals, but PRP4 overexpression reversed the curcumin-induced oxidative stress and apoptosis. PRP8 failed to reverse the curcumin-induced apoptosis in the HCT116 colon cancer cell line. In xenograft mouse model experiments, curcumin effectively reduced tumour size whereas PRP4 conferred resistance to curcumin, which was evident from increasing tumour size, while PRP8 failed to regulate the curcumin action. PRP4 overexpression altered the morphology, rearranged the actin cytoskeleton, triggered epithelial-mesenchymal transition (EMT), and decreased the invasiveness of HCT116 cells. The loss of E-cadherin, a hallmark of EMT, was observed in HCT116 cells overexpressing PRP4. Moreover, we observed that the EMT-inducing potential of PRP4 was aborted after the deletion of its kinase domain. Collectively, our investigations suggest that the PRP4 kinase domain is responsible for promoting drug resistance to curcumin by inducing EMT. Further evaluation of PRP4-induced inhibition of cell death and PRP4 kinase domain interactions with various other proteins might lead to the development of novel approaches for overcoming drug resistance in patients with colon cancer.

Impact of intercellular connectivity on epithelial mesenchymal transition plasticity

Epithelial mesenchymal transition (EMT) in development, tissue repair and carcinogenesis involves cellular plasticity with varying degrees of epithelial and mesenchymal properties. Several recent studies have focused on EMT phenotypic dynamism; however, information on cellular interaction in the context of EMT is inadequate. In our previous study, we investigated EMT phenotypic plasticity and anticipated it as a population driven interactive process. Present study has characterized cellular connectivity as a representative of interactivity during EMT in epithelial normal and cancer cell. It has also explored dynamism of connectivity and phenotype employing Markov model. Further, plasticity was substantiated with cell surface microvilli and molecular marker. The study unveiled interplay between phenotype and connectivity too. Findings have revealed that intercellular connectivity fueled EMT plasticity and its dynamism was more prominent in cancer population. However, normal cells are more vibrant in transition and phenotypic plasticity. We have proposed connectivity plasticity as a hallmark of EMT and needs to be studied in depth. Present study also paves the way in translating in vitro EMT findings in histopathological practices.

Catechol inhibits epidermal growth factor-induced epithelial-to-mesenchymal transition and stem cell-like properties in hepatocellular carcinoma cells

Epithelial-mesenchymal transition (EMT) is a major cellular process in which epithelial cells lose cell polarity and cell-cell adhesion and become motility and invasiveness by transforming into mesenchymal cells. Catechol is one of the natural compounds present in fruits and vegetables and has various pharmacological and physiological activities including anti-carcinogenic effects. However, the effects of catechol on EMT has not been reported. Epidermal growth factor (EGF) is one of the growth factors and is known to play a role in inducing EMT. The present study showed that catechol suppressed not only the morphological changes to the mesenchymal phenotype of epithelial HCC cells, but also the reduction of E-cadherin and the increment of Vimentin, which are typical hallmark of EMT. In addition, catechol suppressed EMT-related steps such as migration, invasion, anoikis resistance acquisition, and stem cell-like characterization through the EGFR-AKT-ERK signaling pathway during liver cancer metastasis. Therefore, these results suggest that catechol may be able to regulate the early metastasis of liver cancer in vitro.

Epithelial-mesenchymal transition suppresses ERas to activate autophagy in retinal pigment epithelial cells in proliferative vitreoretinopathy.

Proliferative vitreoretinopathy (PVR) is a complex ocular disease that leads to detached retinas and irreversible vision loss. The epithelial-mesenchymal transition (EMT) of retinal pigment epithelial (RPE) cells plays a critical role in PVR occurrence. However, the core targets driven by the EMT process that lead to the pathogenesis of PVR remain unclear. In our study, the relationship between embryonic stem cell-expressed Ras (ERas) and EMT in RPE cells was investigated. The subretinal and epiretinal membrane specimens of human PVR were examined for ERas and hallmarks of autophagy and EMT using Western blotting and immunofluorescence. EMT was induced by transforming growth factor (TGF)-β1 or epidermal growth factor (EGF) in ARPE-19 cells. Autophagy was inhibited by U0126 or bafilomycin A1 in ARPE-19 cells. ERas was decreased and the classical autophagy biomarker microtubule associated protein 1 light chain 3 alpha (LC3) was upregulated in the subretinal and epiretinal membranes of PVR patients in vivo. Moreover, ERas was downregulated and autophagy was activated in RPE ARPE-19 cells in response to transforming growth factor (TGF)-β1 and epidermal growth factor (EGF) induction. Finally, overexpression of ERas in RPE cells inhibited autophagy via impaired formation of autophagosomes and lysosomes. Our study revealed the role of ERas in the pathogenesis of PVR through EMT and provided a novel therapeutic target for PVR prevention and treatment.

Monoubiquitination of p120-catenin is essential for TGFβ-induced epithelial-mesenchymal transition and tumor metastasis.

Disassembly of intercellular junctions is a hallmark of epithelial-mesenchymal transition (EMT). However, how the junctions disassemble remains largely unknown. Here, we report that E3 ubiquitin ligase Smurf1 targets p120-catenin, a core component of adherens junction (AJ) complex, for monoubiquitination during transforming growth factor β (TGFβ)-induced EMT, thereby leading to AJ dissociation. Upon TGFβ treatment, activated extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylates T900 of p120-catenin to promote its interaction with Smurf1 and subsequent monoubiquitination. Inhibition of T900 phosphorylation or ubiquitination of p120-catenin abrogates TGFβ-induced AJ dissociation and consequent tight junction (TJ) dissociation and cytoskeleton rearrangement, hence markedly blocking lung metastasis of murine breast cancer. Moreover, the T900 phosphorylation level of p120-catenin is positively correlated with malignancy of human breast cancer. Hence, our study reveals the underlying mechanism by which TGFβ induces dissociation of AJs during EMT and provides a potential strategy to block tumor metastasis.

SMAD7 and SERPINE1 as novel dynamic network biomarkers detect and regulate the tipping point of TGF-beta induced EMT

Epithelial–mesenchymal transition (EMT) is a complex nonlinear biological process that plays essential roles in fundamental biological processes such as embryogenesis, wounding healing, tissue regeneration, and cancer metastasis. A hallmark of EMT is the switch-like behavior during state transition, which is characteristic of phase transitions. Hence, detecting the tipping point just before mesenchymal state transition is critical for understanding molecular mechanism of EMT. Through dynamic network biomarkers (DNB) model, a DNB group with 37 genes was identified which can provide the early-warning signals of EMT. Particularly, we found that two DNB genes, i.e., SMAD7 and SERPINE1 promoted EMT by switching their regulatory network which was further validated by biological experiments. Survival analyses revealed that SMAD7 and SERPINE1 as DNB genes further acted as prognostic biomarkers for lung adenocarcinoma.

A Novel DNA Methylation-Based Signature Can Predict the Responses of MGMT Promoter Unmethylated Glioblastomas to Temozolomide.

Glioblastoma (GBM) is the most malignant glioma, with a median overall survival (OS) of 14–16 months. Temozolomide (TMZ) is the first-line chemotherapy drug for glioma, but whether TMZ should be withheld from patients with GBMs that lack O6-methylguanine-DNA methyltransferase (MGMT) promoter methylation is still under debate. DNA methylation profiling holds great promise for further stratifying the responses of MGMT promoter unmethylated GBMs to TMZ. In this study, we studied 147 TMZ-treated MGMT promoter unmethylated GBM, whose methylation information was obtained from the HumanMethylation27 (HM-27K) BeadChips (n = 107) and the HumanMethylation450 (HM-450K) BeadChips (n = 40) for training and validation, respectively. In the training set, we performed univariate Cox regression and identified that 3,565 CpGs were significantly associated with the OS of the TMZ-treated MGMT promoter unmethylated GBMs. Functional analysis indicated that the genes corresponding to these CpGs were enriched in the biological processes or pathways of mitochondrial translation, cell cycle, and DNA repair. Based on these CpGs, we developed a 31-CpGs methylation signature utilizing the least absolute shrinkage and selection operator (LASSO) Cox regression algorithm. In both training and validation datasets, the signature identified the TMZ-sensitive GBMs in the MGMT promoter unmethylated GBMs, and only the patients in the low-risk group appear to benefit from the TMZ treatment. Furthermore, these identified TMZ-sensitive MGMT promoter unmethylated GBMs have a similar OS when compared with the MGMT promoter methylated GBMs after TMZ treatment in both two datasets. Multivariate Cox regression demonstrated the independent prognostic value of the signature in TMZ-treated MGMT promoter unmethylated GBMs. Moreover, we also noticed that the hallmark of epithelial–mesenchymal transition, ECM related biological processes and pathways were highly enriched in the MGMT unmethylated GBMs with the high-risk score, indicating that enhanced ECM activities could be involved in the TMZ-resistance of GBM. In conclusion, our findings promote our understanding of the roles of DNA methylation in MGMT umethylated GBMs and offer a very promising TMZ-sensitivity predictive signature for these GBMs that could be tested prospectively.

G9a and histone deacetylases are crucial for Snail2-mediated E-cadherin repression and metastasis in hepatocellular carcinoma.

Functional E‐cadherin loss, a hallmark of epithelial‐mesenchymal transition (EMT), is important for metastasis. However, the mechanism of Snail2 in hepatocellular carcinoma (HCC) EMT and metastasis remains unclear. Here, we showed that Snail2 was upregulated in primary HCC, and significantly increased during transforming growth factor‐β‐induced liver cell EMT. Snail2‐overexpressing and knockdown cell lines have been established to determine its function in EMT in HCC. H3K9 methylation was upregulated and H3K4 and H3K56 acetylation were downregulated at the E‐cadherin promoter in Snail2‐overexpressing cancer cells. Furthermore, Snail2 interacted with G9a and histone deacetylases (HDACs) to form a complex to suppress E‐cadherin transcription. Snail2 overexpression enhanced migration and invasion in HCC cells, whereas G9a and HDAC inhibition significantly reversed this effect. Moreover, Snail2 overexpression in cancer cells increased tumor metastasis and shortened survival time in mice, whereas G9a and HDAC inhibitors extended survival. Our study not only reveals a critical mechanism underlying the epigenetic regulation of EMT but also suggests novel treatment strategies for HCC.

APLNR is involved in ATRA-induced growth inhibition of nasopharyngeal carcinoma and may suppress EMT through PI3K-Akt-mTOR signaling.

The apelin receptor (APLNR) is a GPCR involved in many pathophysiological processes; however, the correlation between APLNR expression and nasopharyngeal carcinoma (NPC) has not been reported. In this study, we used cDNA microarray data to determine APLNR expression levels in NPC tissues. We found that APLNR expression was reduced in NPC tissues compared with noncancerous nasopharyngeal epithelial tissues. Subsequently, a large-scale sample of 1015 tissues was used to validate this discovery and explore the relationship between APLNR expression and prognosis of NPC. Expression levels of APLNR in NPC tissues were indeed down-regulated. Furthermore, positive expression of APLNR in NPC predicted a better prognosis (disease-free survival: P = 0.001; overall survival: P < 0.001). Moreover, ingenuity pathway analysis revealed that an indirect interaction existed between APLNR and retinoic acid (RA) in the cancer regulatory network. Consistently, after treatment with all-trans-RA (ATRA), we found that APLNR was significantly up-regulated in NPC cell lines (5-8F and HNE1), and proliferation of NPC cells was inhibited. Cell cycle arrest occurred in the G0/G1 phase. In contrast, knockdown of APLNR diminished ATRA-induced growth inhibition of NPC cells. In addition, we surprisingly found that APLNR also played an important role in migration and invasion of NPC. Wound-healing and Transwell assays revealed that APLNR overexpression led to reduced migratory and invasive properties in 2 NPC cell lines. Western blot results revealed that hallmarks of epithelial-mesenchymal transition (EMT) were altered as well, suggesting that APLNR was capable of inhibiting EMT in NPC cells. Our study further demonstrated that low expression of APLNR promoted EMT in NPC cells by activating the PI3K-protein kinase B-mammalian target of rapamycin signaling pathway. Taken together, our data suggest that APLNR could potentially predict prognosis for patients with NPC and inhibit proliferation, migration, invasion, and EMT in nasopharyngeal cancer cells.-Liu, Y., Liu, Q., Chen, S., Liu, Y., Huang, Y., Chen, P., Li, X., Gao, G., Xu, K., Fan, S., Zeng, Z., Xiong, W., Tan, M., Li, G., Zhang, W. APLNR is involved in ATRA-induced growth inhibition of nasopharyngeal carcinoma and may suppress EMT through PI3K-Akt-mTOR signaling.

Epithelial-mesenchymal transition markers screened in a cell-based model and validated in lung adenocarcinoma

BackgroundRe-capture of the differences between tumor and normal tissues observed at the patient level in cell cultures and animal models is critical for applications of these cancer-related differences. The epithelial-mesenchymal transition (EMT) process is essential for tumor migratory and invasive capabilities. Although plenty of EMT markers are revealed, molecular features during the early stages of EMT are poorly understood.MethodsA cell-based model to induce lung cell (A549) EMT using conditioned medium of in vitro cancer activated fibroblast (WI38) was established. High-throughput sequencing methods, including RNA-seq and miRNA-seq, and advanced bioinformatics methods were used to explore the transcriptome profile transitions accompanying the progression of EMT. We validated our findings with experimental techniques including transwell and immunofluorescence assay, as well as the TCGA data.ResultsWe have constructed an in vitro cell model to mimic the EMT in patients. We discovered that several new transcription factors were among the early genes (3 h) to respond to cancer micro-environmental cues which could play critical roles in triggering further EMT signals. The early EMT markers also included genes encoding membrane transporters and blood coagulation function. Three of the nine-examined early EMT hallmark genes, GALNT6, SPARC and HES7, were up-regulated specifically in the early stages of lung adenocarcinoma (LUAD) and confirmed by TCGA patient transcriptome data. In addition, we showed that miR-3613, a regulator of EGFR pathway genes, was constantly repressed during EMT progress and indicative of an epithelial miRNA marker.ConclusionsThe CAF-stimulated EMT cell model may recapture some of the molecular changes during EMT progression in clinical patients. The identified early EMT hallmark genes GALNT6, SPARC and HES7and miR-3613 provide new markers and therapeutic targets for LUAD for the further clinical diagnosis and drug screening.