Subsequent Epithelial-mesenchymal Transition Research Articles

PD-1 Carried on Small Extracellular Vesicles Leads to OSCC Metastasis

Immune checkpoint molecule PD-1, expressed on the cell surface, impairs antigen-driven activation of T cells and thus plays a critical role in tumorigenesis, progression, and the poor prognosis of oral squamous cell carcinoma (OSCC). In addition, increasing evidence indicates that PD-1 carried on small extracellular vesicles (sEVs) also mediates tumor immunity, although their contributions to OSCC are yet unclear. Here, we investigated the biological functions of sEV PD-1 in patients with OSCC. The cell cycle, proliferation, apoptosis, migration, and invasion of CAL27 cell lines treated with or without sEV PD-1 were examined in vitro. We performed mass spectrometry to investigate the underlying biological process, combined with an immunohistochemical study of SCC7-bearing mice models and OSCC patient samples. In vitro data demonstrated that sEV PD-1 induced senescence and subsequent epithelial-mesenchymal transition (EMT) in CAL27 cells by ligating with tumor cell surface PD-L1 and activating the p38 mitogen-activated protein kinase (MAPK) pathway. Comprehensive immunohistochemical analysis of the xenograft mice models and OSCC patient samples revealed a very close correlation between the level of circulating sEV PD-1 and lymph node metastasis. These results demonstrate that circulating sEV PD-1 triggers senescence-initiated EMT in a PD-L1-p38 MAPK-dependent manner, contributing to tumor metastasis. It also suggests that the inhibition of sEV PD-1 may be a promising therapeutic target for the treatment of OSCC.

Phosphorylation of USP29 by CDK1 Governs TWIST1 Stability and Oncogenic Functions.

Triple-negative breast cancer (TNBC) is a highly lethal malignancy with limited therapy options. TWIST1, a key transcriptional factor of epithelial-mesenchymal transition (EMT), contributes to self-renewal of cancer stem-like cells (CSCs), chemo-resistance, metastasis, and TNBC-related death. However, the mechanism by which TWIST1 is deregulated in TNBC remains elusive. Here, USP29 is identified as a bona fide deubiquitinase of TWIST1. The deubiquitination of TWIST1 catalyzed by USP29 is required for its stabilization and subsequent EMT and CSC functions in TNBC, thereby conferring chemotherapeutic resistance and metastasis. Furthermore, the results unexpectedly reveal that CDK1 functions as the direct USP29 activator. Mechanistically, CDK1-mediated phosphorylation of USP29 is essential for its deubiquitinase activity toward TWIST1 and TWIST1 driven-malignant phenotypes in TNBC, which could be markedly mitigated by the genetic ablation or pharmacological inhibition of CDK1. Moreover, the histological analyses show that CDK1 and USP29 are highly upregulated in TNBC samples, which positively correlate with the expression of TWIST1. Taken together, the findings reveal a previously unrecognized tumor-promoting function and clinical significance of the CDK1-USP29 axis through stabilizing TWIST1 and provide the preclinical evidence that targeting this axis is an appealing therapeutic strategy to conquer chemo-resistance and metastasis in TNBC.

Afatinib Reverses EMT via Inhibiting CD44-Stat3 Axis to Promote Radiosensitivity in Nasopharyngeal Carcinoma

Afatinib, a second-generation tyrosine kinase inhibitor (TKI), exerts its radiosensitive effects in nasopharyngeal carcinoma (NPC). However, the detailed mechanism of afatinib-mediated sensitivity to radiation is still obscure in NPC. Quantitative phosphorylated proteomics and bioinformatics analysis were performed to illustrate the global phosphoprotein changes. The activity of the CD44-Stat3 axis and Epithelial-Mesenchymal Transition (EMT)-linked markers were evaluated by Western blotting. Wound healing and transwell assays were used to determine the levels of cell migration upon afatinib combined IR treatment. Cell proliferation was tested by CCK-8 assay. A pharmacological agonist by IL-6 was applied to activate Stat3. The xenograft mouse model was treated with afatinib, radiation or a combination of afatinib and radiation to detect the radiosensitivity of afatinib in vivo. In the present study, we discovered that afatinib triggered global protein phosphorylation alterations in NPC cells. Further, bioinformatics analysis indicated that afatinib inhibited the CD44-Stat3 signaling and subsequent EMT process. Moreover, functional assays demonstrated that afatinib combined radiation treatment remarkably impeded cell viability, migration, EMT process and CD44-Stat3 activity in vitro and in vivo. In addition, pharmacological stimulation of Stat3 rescued radiosensitivity and biological functions induced by afatinib in NPC cells. This suggested that afatinib reversed the EMT process by blocking the activity of the CD44-Stat3 axis. Collectively, this work identifies the molecular mechanism of afatinib as a radiation sensitizer, thus providing a potentially useful combination treatment and drug target for NPC radiosensitization. Our findings describe a new function of afatinib in radiosensitivity and cancer treatment.

HIF-1α induced NID1 expression promotes pulmonary metastases via the PI3K-AKT pathway in salivary gland adenoid cystic carcinoma

BackgroundThis study aimed to investigate the potential role of nidogen 1 (NID1), a basement membrane component, in the growth and metastasis of salivary gland adenoid cystic carcinoma (SACC) and the underlying molecular mechanism. MethodsHigh-throughput next-generation sequencing was used to compare the gene expression profiles of SACC with and without lung metastasis. Luciferase gene reporter assays were used to measure the NID1 promoter activity. BALB/c nude mice were used to establish a lung metastasis model of SACC to evaluate the prometastatic activity of NID1. ChIP and dual-luciferase reporter assays were performed to confirm the HIF-1α-binding site in the NID1 promoter. ResultsNID1 expression in SACC was significantly increased and associated with lung metastasis (P = 0.011). The elevated NID1 expression was a predictor of poor outcomes in patients with SACC (P < 0.05). Overexpression of NID1 promoted cancer cell migration and invasion through PI3K/AKT pathway activation and subsequent epithelial–mesenchymal transition (EMT), as indicated by the upregulation of N-cadherin and vimentin. Furthermore, in vivo live monitoring of a mouse model of lung cancer demonstrated the pro-metastatic role of NID1 in SACC cell lung metastasis. Hypoxia-inducible factor 1α (HIF‐1α) upregulation via transfection of an HIF-1α-overexpressing plasmid enhanced HIF-1α binding to the NID1 promoter and the subsequent transcriptional activity and expression of NID1. ConclusionHIF‐1α-activated NID1 overexpression promotes SACC cell metastasis via PI3K/AKT pathway activation and EMT. Thus, NID1 could be a novel biomarker and therapeutic target for preventing metastasis and treating patients with SACC in future.

Perioperative Suppression of Schwann Cell Dedifferentiation Reduces the Risk of Adenomyosis Resulting from Endometrial-Myometrial Interface Disruption in Mice.

We have recently demonstrated that endometrial–myometrial interface (EMI) disruption (EMID) can cause adenomyosis in mice, providing experimental evidence for the well-documented epidemiological finding that iatrogenic uterine procedures increase the risk of adenomyosis. To further elucidate its underlying mechanisms, we designed this study to test the hypothesis that Schwann cells (SCs) dedifferentiating after EMID facilitate the genesis of adenomyosis, but the suppression of SC dedifferentiation perioperatively reduces the risk. We treated mice perioperatively with either mitogen-activated protein kinase kinase (MEK)/extracellular-signal regulated protein kinase (ERK) or c-Jun N-terminal kinase (JNK) inhibitors or a vehicle 4 h before and 24 h, 48 h and 72 h after the EMID procedure. We found that EMID resulted in progressive SCs dedifferentiation, concomitant with an increased abundance of epithelial cells in the myometrium and a subsequent epithelial–mesenchymal transition (EMT). This EMID-induced change was abrogated significantly with perioperative administration of JNK or MEK/ERK inhibitors. Consistently, perioperative administration of a JNK or a MEK/ERK inhibitor reduced the incidence by nearly 33.5% and 14.3%, respectively, in conjunction with reduced myometrial infiltration of adenomyosis and alleviation of adenomyosis-associated hyperalgesia. Both treatments significantly decelerated the establishment of adenomyosis and progression of EMT, fibroblast-to-myofibroblast trans-differentiation and fibrogenesis in adenomyotic lesions. Thus, we provide the first piece of evidence strongly implicating the involvement of SCs in the pathogenesis of adenomyosis induced by EMID.

Helicobacter pylori cytotoxin-associated gene A virulence and its association with the epithelial-mesenchymal transition in gastric cancer

Gastric cancer is currently one of the most prevalent malignancies worldwide with a high mortality rate. Helicobacter pylori (H. pylori) infection significantly contributes to the onset and progression of gastric cancer mainly due to the induction of chronic inflammatory responses. The pathogenicity of H. pylori is associated with a vast number of virulence factors among which cytotoxin-associated gene A (CagA) plays a crucial role. We conducted a literature review of PubMed, Web of Science, and Scopus on September 1st, 2021. There were no limits regarding the year and the language of publication. Articles included in this review concerned human and animal studies. The following search string was applied during the search: (gastric cancer) AND (epithelial-mesenchymal transition) AND (Helicobacter pylori) AND (cytotoxin-associated gene A). The final analysis included 135 articles independently reviewed by the authors. H. pylori CagA-positive strains seem to be more virulent compared to the CagA-negative strains. CagA pathogenicity includes the increased secretion of pro-inflammatory cytokines, induction of cancer stem cell-like properties, apoptosis prevention, or overactivation of particular oncogenic pathways. H. pylori might induce epithelial-mesenchymal transition (EMT) via numerous pathways, among which CagA-related pathogenicity is considered to be of high significance. Mechanisms associated with CagA action are involved in the maintenance of chronic H. pylori infection, subsequent EMT induction, and further onset and progression of gastric cancer. Because of a huge number of H. pylori strains with different virulence mechanisms, the clinical outcome of patients is also associated with the particular type of strain that infected a patient.

UBE2T promotes β-catenin nuclear translocation in hepatocellular carcinoma through MAPK/ERK-dependent activation.

Ubiquitin‐conjugating enzyme E2T (UBE2T) has been implicated in many types of cancer including hepatocellular carcinoma (HCC). Epithelial–mesenchymal transition (EMT) process plays a fundamental role during tumor metastasis and progression. However, the molecular mechanisms underlying EMT in HCC in accordance with UBE2T still remain unknown. In this study, we showed that UBE2T overexpression augmented the oncogenic properties and specifically EMT in HCC cell lines, while its silencing attenuated them. UBE2T affected the activation of EMT‐associated signaling pathways: MAPK/ERK, AKT/mTOR, and Wnt/β‐catenin. In addition, we revealed that the epithelial protein complex of E‐cadherin/β‐catenin, a vital regulator of signal transduction in tumor initiation and progression, was totally disrupted at the cell membrane. In particular, we observed that UBE2T overexpression led to E‐cadherin loss accompanied by a simultaneous elevation of both cytoplasmic and nuclear β‐catenin, while its silencing resulted in a strong E‐cadherin turnover at the cell membrane. Interestingly, chemical inhibition of the MAPK/ERK, AKT/mTOR, and Wnt/β‐catenin signaling pathways demonstrated that the nuclear translocation of β‐catenin and subsequent EMT was enhanced mainly by MAPK/ERK. Collectively, our findings demonstrate the UBE2T/MAPK‐ERK/β‐catenin axis as a critical regulator of cell state transition and EMT in HCC.

Induction of lncRNA NORAD accounts for hypoxia-induced chemoresistance and vasculogenic mimicry in colorectal cancer by sponging the miR-495-3p/ hypoxia-inducible factor-1α (HIF-1α)

ABSTRACT Hypoxic microenvironment represents the hallmark of solid tumors including colorectal cancer (CRC) and facilitates angiogenesis and chemoresistance, leading to poor prognosis. lncRNA NORAD acts as an oncogenic gene to orchestrate cancer progression by regulating cell proliferation and migration. Notably, an emerging study corroborates the elevation of NORAD during hypoxic conditions in pancreatic cancer. Nevertheless, its biological role in hypoxia-evoked CRC remains unclear. Herein, enhanced expression of NORAD and hypoxia-inducible factor-1α (HIF-1α) was validated in CRC tissues. Furthermore, there was a positive association between NORAD and HIF-1α in CRC tissues. CRC cells exposed to hypoxia exhibited a stronger ability to form vasculogenic mimicry (VM) and resistance to 5-fluorouracil (5-FU), concomitant with higher expression of NORAD. NORAD knockdown restrained hypoxia-induced VM formation and VM marker VE-cadherin expression. Moreover, knockdown of NORAD counteracted CRC cell resistance to 5-FU by decreasing cell viability and increasing cell apoptosis. Additionally, NORAD loss reduced hypoxia-induced HIF-1α expression and subsequent epithelial-mesenchymal transition (EMT) by increasing E-cadherin and inhibiting N-cadherin expression. Intriguingly, HIF-1α overexpression reversed NORAD downregulation-mediated inhibition of VM formation and 5-FU resistance. There was a low expression of miR-495-3p in CRC tissues. Furthermore, NORAD could act as a competitive endogenous RNA of miR-495-3p to regulate HIF-1α. Importantly, inhibition of miR-495-3p muted the efficacy of NORAD loss in hypoxia-induced EMT, VM, and chemoresistance. Thus, the current data highlight that NORAD knockdown may antagonize hypoxia-triggered CRC malignancy by suppressing VM formation and chemoresistance by sponging miR-495-3p/HIF-1α to regulate EMT, supporting a promising therapeutic target for refractory hypoxia in CRC.

Yes-associated protein is essential for proliferative vitreoretinopathy development via the epithelial-mesenchymal transition in retinal pigment epithelial fibrosis.

This study was aim to investigate whether the progression of proliferative vitreoretinopathy (PVR) depended on the activation of Yes‐associated protein (YAP) and the subsequent epithelial‐mesenchymal transition (EMT) of retinal pigment epithelial (RPE) cell. The effect of YAP activation on retinal fibrosis in a PVR mouse model and in human ARPE‐19 cells in vitro was studied. After treated with transforming growth factor‐β2(TGF‐β2), the expressions of fibrogenic molecules, YAP activation and the TGF‐β2‐Smad signalling pathway in ARPE‐19 cells were detected by Western blot and immunocytochemical analyses. The effect of YAP on change in fibrosis and EMT was tested by knockdown experiment using verteporfin (YAP inhibitor). YAP was upregulated in the PVR mouse model and during TGF‐β2–induced RPE cell EMT. In an in vivo study, verteporfin attenuated PVR progression in a mouse model. Additionally, YAP knockdown retained phenotype of RPE cells and ameliorated TGF‐β2–induced migration, gel contraction and EMT in vitro. YAP knockdown inhibited the TGF‐β2–induced upregulation of connective tissue growth factor (CTGF), smooth muscle actin (SMA‐α) and fibronectin. YAP was essential for the TGF‐β2–induced nuclear translocation and phosphorylation of Smad2/3. Our work provides direct evidence that YAP is an essential regulator of EMT and profibrotic responses in PVR and indicates that YAP inhibition could be a potential target in PVR therapeutic intervention.

Tetratricopeptide repeat domain 36 deficiency mitigates renal tubular injury by inhibiting TGF-β1-induced epithelial–mesenchymal transition in a mouse model of chronic kidney disease

The damage of proximal tubular epithelial cells (PTECs) is considered a central event in the pathogenesis of chronic kidney disease (CKD) and deregulated repair processes of PTECs result in epithelial–mesenchymal transition (EMT), which in turn aggravates tubular injury and kidney fibrosis. In this study, we firstly revealed that the reduction of TTC36 is associated with unilateral ureteral obstruction (UUO)-induced CKD; besides, ablation of TTC36 attenuated tubular injury and subsequent EMT in UUO-treated mice kidneys. Consistently, TTC36 overexpression promoted EMT in TGF-β1-induced HK2 cells. Moreover, TTC36 elevated the protein expression of CEBPB, which was involved in the regulation of TGF-β/SMAD3 signaling, and augmented SMAD3 signaling and downstream genetic response were reduced by CEBPB silencing. Collectively, our results uncovered that TTC36 deficiency plays a protective role in tubular injury and renal fibrosis triggered by UUO; further, TTC36 overexpression exacerbated TGF-β/SMAD3 signaling via elevating the stability of SMAD3 and CEBPB, suggesting that TTC36 inhibition may be a potential strategy in the therapy of obstructive nephropathy.

S100A16 suppresses the proliferation, migration and invasion of colorectal cancer cells in part via the JNK/p38 MAPK pathway.

S100 calcium binding protein A16 (S100A16) is the most recent member of the S100 calcium-binding protein family. The function of S100A16 has been associated with various types of cancer; however, its role in colorectal cancer (CRC) remains unknown. Therefore, the aim of the present study was to investigate the role of S100A16 in CRC progression. The Oncomine dataset used in the current study revealed that the expression of S100A16 was decreased in CRC compared with normal colorectal tissues. Similar results were also determined via immunohistochemistry. In addition, a negative association was identified between S100A16 expression and the prognosis of patients with CRC. Further functional experiments revealed that S100A16 knockdown promoted the proliferation, migration and invasion of HCT116 and SW480 cells, and vice versa in Lovo cells. Epithelial-mesenchymal transition (EMT) was promoted and the JNK/p38 MAPK pathway was activated in HCT116 cells following S100A16 knockdown, as determined via western blotting. Furthermore, S100A16 silencing promoted the migration and invasion of cells. EMT was also reversed when cells were treated with the JNK inhibitor (SP600125) or the p38 inhibitor (SB203580). In summary, the results of the present study demonstrated that S100A16 suppressed the proliferation, migration and invasion of CRC cells partially via the JNK/p38 MAPK signalling pathway and subsequent EMT mediation.

Interferometric Measurement of TGF-β Induced Epithelial-Mesenchymal Transition of Tumor Cells

A three-dimensional profile reconstruction of live cells in dynamic cell cultures is a challenging problem due to the highly scattering nature of cell mediums. Furthermore, it is an interesting problem since these cultures present the optimal in vitro conditions that most closely resemble the cells’ natural conditions. In this paper, we report a holographic method used for imaging during the process of treatment of dynamic cell cultures with transforming growth factor beta (TGF-β) and the subsequent epithelial-mesenchymal transition (EMT). The imaging of dynamic cell cultures presents many challenges for holographic techniques due to the highly scattering and high speed nature of the environment. Here we report the algorithmic workflow we used for decreasing the imaging noise due to the presence of cell medium and achieving high speed reconstruction rates in real time. We also report the prominent morphological features we extracted from the obtained depth maps throughout the experiment. We conducted experiments on four different cell lines: ONCO-DG1, HCT-116, MDA-MB-231, and HUVEC. We observed the EMT process throughout a 48 h period after treatment with TGF-β with 6 h intervals for each sample. We show some examples of the reconstructed depth maps of tumor cells during the EMT phase. From these depth maps we extract some morphological parameters and report how they change after the EMT process is completed. The obtained results indicate that the proposed method presents certain advantages from an optical perspective particularly for applications where a dynamical medium is present. These advantages are lower signal-to-noise ratio (SNR) values and a simple setup compared to the setups used in similar studies. Future studies on this application could lead to the development of a model for the EMT process and its effects on cell to cell interactions.

MEK1 Inhibitor Combined with Irradiation Reduces Migration of Breast Cancer Cells Including miR-221 and ZEB1 EMT Marker Expression.

Simple SummaryCombined chemotherapy and radiotherapy are an effective treatment for invasive breast cancer. However, some studies suggest that such interventions may increase the risk of metastasis. Cell metastatic behavior is highly dependent on RAS-RAF-MEK pathway and its downstream target activation, including miR-221 overexpression and epithelial-to-mesenchymal transition (EMT). By using MEK1 inhibitor (TAK-733) in combination with radiation therapy for breast cancer cells, significant decrease in migration capacity, including reduction of miR-221 and EMT (ZEB1) marker expression was observed. miR-221 holds great potential as therapeutic biomarker and target for new drug developments, however more insight into efficiency of miR-221 inhibition needs to be followed in the future.The miR-221 expression is dependent on the oncogenic RAS-RAF-MEK pathway activation and influences epithelial-to-mesenchymal transition (EMT). The Cancer Genome Atlas (TCGA) database analysis showed high gene significance for ZEB1 with EMT module analysis and miR-221 overexpression within the triple-negative breast cancer (TNBC) and HER2+ subgroups when compared to luminal A/B subgroups. EMT marker expression analysis after MEK1 (TAK-733) inhibitor treatment and irradiation was combined with miR-221 and ZEB1 expression analysis. The interaction of miR-221 overexpression with irradiation and its influence on migration, proliferation, colony formation and subsequent EMT target activation were investigated. The results revealed that MEK1 inhibitor treatment combined with irradiation could decrease the migratory potential of breast cancer cells including reduction of miR-221 and corresponding downstream ZEB1 (EMT) marker expression. The clonogenic survival assays revealed that miR-221 overexpressing SKBR3 cells were more radioresistant when compared to the control. Remarkably, the effect of miR-221 overexpression on migration in highly proliferative and highly HER2-positive SKBR3 cells remained constant even upon 8 Gy irradiation. Further, in naturally miR-221-overexpressing MDA-MB-231 cells, the proliferation and migration significantly decrease after miR-221 knockdown. This leads to the assumption that radiation alone is not reducing migration capacity of miR-221-overexpressing cells and that additional factors play an important role in this context. The miR-221/ZEB1 activity is efficiently targeted upon MEK1 inhibitor (TAK-733) treatment and when combined with irradiation treatment, significant reduction in migration of breast cancer cells was shown.

RETRACTED: lncRNA lnc-TSI Inhibits Metastasis of Clear Cell Renal Cell Carcinoma by Suppressing TGF-β-Induced Epithelial-Mesenchymal Transition

The transforming growth factor-β (TGF-β)/Smads signal plays an important role in cancer metastasis by mediating the epithelial-mesenchymal transition (EMT) in cancer cells. lnc-TSI is a recently identified long noncoding RNA that negatively regulates the TGF-β/Smads signal. The present study was conducted to test the hypothesis that lnc-TSI inhibits metastasis in clear cell renal cell carcinoma (ccRCC) by regulating the TGF-β/Smad3 pathway. Herein, we show that lnc-TSI was upregulated in ccRCC cells and tissue and was associated with activation of the TGF-β/Smads signal. Depleting lnc-TSI enhanced tumor cell invasion and metastasis in vitro and ccRCC lung metastasis in vivo, whereas overexpressing lnc-TSI inhibited ccRCC cell invasion and tumor metastasis. Mechanistic studies indicated that lnc-TSI specifically inhibited the phosphorylation of Smad3 and subsequent EMT by binding with the MH2 domain of Smad3 to block the interaction between Smad3 and TGF-β receptor I in ccRCC cells. In a cohort of 150 patients with ccRCC, expression of lnc-TSI in tumors was negatively correlated with phosphorylated (p)Smad3 and activated EMT markers. Patients with expression of tumor lnc-TSI greater than or equal to the median at radical nephrectomy had a higher survival rate compared to those with lnc-TSI below the median during follow-up. These findings reveal a new regulatory mechanism of ccRCC metastasis and suggest a potential molecular target for the development of anti-cancer drugs.

Induction of Autophagy by Pterostilbene Contributes to the Prevention of Renal Fibrosis via Attenuating NLRP3 Inflammasome Activation and Epithelial-Mesenchymal Transition.

Chronic kidney disease (CKD) is recognized as a global public health problem. NLRP3 inflammasome activation has been characterized to mediate diverse aspect mechanisms of CKD through regulation of proinflammatory cytokines, tubulointerstitial injury, glomerular diseases, renal inflammation, and fibrosis pathways. Autophagy is a characterized negative regulation mechanism in the regulation of the NLRP3 inflammasome, which is now recognized as the key regulator in the pathogenesis of inflammation and fibrosis in CKD. Thus, autophagy is undoubtedly an attractive target for developing new renal protective treatments of kidney disease via its potential effects in regulation of inflammasome. However, there is no clinical useful agent targeting the autophagy pathway for patients with renal diseases. Pterostilbene (PT, trans-3,5-dimethoxy-4-hydroxystilbene) is a natural analog of resveratrol that has various health benefits including autophagy inducing effects. Accordingly, we aim to investigate underlying mechanisms of preventive and therapeutic effects of PT by reducing NLRP3 inflammasome activation and fibrosis through autophagy-inducing effects. The renal protective effects of PT were evaluated by potassium oxonate (PO)-induced hyperuricemia and high adenine diet-induced CKD models. The autophagy induction mechanisms and anti-fibrosis effects of PT by down-regulation of NLRP3 inflammasome are investigated by using immortalized rat kidney proximal tubular epithelial NRK-52E cells. To determine the role of autophagy induction in the alleviating of NLRP3 inflammasome activation and epithelial-mesenchymal transition (EMT), NRK-52E with Atg5 knockdown [NRK-Atg5-(2)] cells were applied in the study. The results indicated that PT significantly reduces serum uric acid levels, liver xanthine oxidase activity, collagen accumulation, macrophage recruitment, and renal fibrosis in CKD models. At the molecular levels, pretreatment with PT downregulating TGF-β-triggered NLRP3 inflammasome activation, and subsequent EMT in NRK-52E cells. After blockage of autophagy by treatment of Atg5 shRNA, PT loss of its ability to prevent NLRP3 inflammasome activation and EMT. Taken together, we suggested the renal protective effects of PT in urate nephropathy and proved that PT induces autophagy leading to restraining TGF-β-mediated NLRP3 inflammasome activation and EMT. This study is also the first one to provide a clinical potential application of PT for a better management of CKD through its autophagy inducing effects.

Mesenchymal-epithelial transition regulates initiation of pluripotency exit before gastrulation.

The pluripotent epiblast gives rise to all tissues and organs in the adult body. Its differentiation starts at gastrulation, when the epiblast generates mesoderm and endoderm germ layers through epithelial-mesenchymal transition (EMT). Although gastrulation EMT coincides with loss of epiblast pluripotency, pluripotent cells in development and in vitro can adopt either mesenchymal or epithelial morphology. The relationship between epiblast cellular morphology and its pluripotency is not well understood. Here, using chicken epiblast and mammalian pluripotency stem cell (PSC) models, we show that PSCs undergo a mesenchymal-epithelial transition (MET) prior to EMT-associated pluripotency loss. Epiblast MET and its subsequent EMT are two distinct processes. The former, a partial MET, is associated with reversible initiation of pluripotency exit, whereas the latter, a full EMT, is associated with complete and irreversible pluripotency loss. We provide evidence that integrin-mediated cell-matrix interaction is a key player in pluripotency exit regulation. We propose that epiblast partial MET is an evolutionarily conserved process among all amniotic vertebrates and that epiblast pluripotency is restricted to an intermediate cellular state residing between the fully mesenchymal and fully epithelial states.

Vitamin D deficiency exacerbates bleomycin-induced pulmonary fibrosis partially through aggravating TGF-\u03b2/Smad2/3-mediated epithelial-mesenchymal transition

BackgroundOur earlier report indicated that active vitamin D3 inhibited epithelial-mesenchymal transition (EMT) in bleomycin (BLM)-induced pulmonary fibrosis. The objective of this study was to further investigate whether vitamin D deficiency exacerbates BLM-induced pulmonary fibrosis.MethodsThis study consists of two independent experiments. Experiment 1, male mice were fed with vitamin D deficient (VDD) fodder. Experiment 2, Cyp27b1+/+, Cyp27b1+/− and Cyp27b1−/− mice were fed with standard diet. For pulmonary fibrosis, mice were intratracheally instilled with a single dose of BLM (1.5 mg/kg). Serum 25(OH) D level was measured. Pulmonary collagen deposition was assessed by Sirius red staining. EMT was measured and transforming growth factor-beta (TGF-β)/Smad3 signaling was evaluated in the lungs of BLM-treated mice.ResultsThe relative weight of lungs was elevated in BLM-treated mice. Col1α1 and Col1α2, two collagen protein genes, were upregulated, and collagen deposition, as determined by Sirius red staining, was observed in the lungs of BLM-treated mice. E-cadherin, an epithelial marker, was downregulated. By contrast, vimentin and α-SMA, two EMT markers, were upregulated in the lungs of BLM-treated mice. Pulmonary TGF-β/Smad3 signaling was activated in BLM-induced lung fibrosis. Further analysis showed that feeding VDD diet, leading to vitamin D deficiency, aggravated elevation of BLM-induced relative lung weight. Moreover, feeding VDD diet aggravated BLM-induced TGF-β/Smad3 activation and subsequent EMT in the lungs. In addition, feeding VDD diet exacerbated BLM-induced pulmonary fibrosis. Additional experiment showed that Cyp27b1 gene knockout, leading to active vitamin D3 deficiency, exacerbated BLM-induced pulmonary fibrosis. Moreover, Cyp27b1 gene knockout aggravated pulmonary TGF-β/Smad2/3 activation and subsequent EMT in BLM-induced lung fibrosis.ConclusionVitamin D deficiency exacerbates BLM-induced pulmonary fibrosis partially through aggravating TGF-β/Smad2/3-mediated EMT in the lungs.

MicroRNA-204-3p modulates epithelial-mesenchymal transition by targeting paired box gene 2 in human melanoma A-375 cells.

BackgroundPaired box 2 gene promoted the progression of subsequent epithelial-mesenchymal transition (EMT) in malignant melanoma, which is the most dangerous type of skin cancer. We aimed to investigate the functional role of miR-204-3p in metastatic melanoma.MethodsTo study the effects of overexpressed miR-204-3p on the melanoma cells, miR-204-3p mimic vector were transfected into A-375 cells for detecting the cell viability using CCK-8 kit. Wound healing and Transwell assays were performed to assess the migration and invasion capacities of melanoma cells. TargetScan and miRTarBase were used to predict the potential target of miR-204-3p, which was subsequently verified by dual-luciferase assay. The expressions of several EMT-associated genes were determined by quantitative real-time polymerase chain (qRT-PCR) and Western blot.ResultsThe expression of miR-204-3p in several melanoma cell lines was lower than that in HaCaT cells. After transfecting with the miR-204-3p mimic vector, the cell viability of A-375 cells decreased over time and significantly reduced at 48 h (P<0.01). Overexpression of miR-204-3p noticeably suppressed the migration and invasion abilities of A-375 cells (P<0.01). PAX2 and guanine nucleotide binding protein (G protein) q polypeptide (GNAQ) were the potential targets of miR-204-3p, which could effectively silence PAX2 rather than GNAQ. MiR-204-3p overexpression could upregulate the epithelial cadherin (E-cadherin) and downregulate the vimentin, matrix metalloproteinase 2 (MMP2) and MMP-9 at transcriptional and posttranscriptional levels.ConclusionsOur results revealed that miR-204-3p had inhibitory effects on the migratory and invasive capacities and EMT progression in malignant melanoma through targeting PAX2.

MicroRNA-212-3p inhibits the Proliferation and Invasion of Human Hepatocellular Carcinoma Cells by Suppressing CTGF expression

MicroRNA-212-3p inhibits several human cancers but its effects on hepatocellular carcinoma (HCC) remain unclear. In this study, we show that miR-212-3p is down-regulated in HCC cell lines and tissues, and correlates with vascular invasion (p = 0.001), and the absence of capsule formation (p = 0.009). We found that miR-212-3p influenced the epithelial to mesenchymal transition (EMT) of HCCLM3 and Huh7 cells. Mechanistically, miR-212-3p repressed cell invasion through the suppression of connective tissue growth factor (CTGF). We therefore validate the anti-HCC effects of miR-212-3p through its ability to suppress CTGF and subsequent EMT.

Abstract 1045: CRISPR/cas9 mediated generation of an EMT reporter cell line for metastatic breast cancer drug discovery and development

Abstract Among women, breast cancer continues to be the most common cancer, with metastasis being the leading cause of mortality in cancer patients around the world (DIZON et al. 2016). Epithelial to mesenchymal transition (EMT) - the process by which epithelial cells shift to the mesenchymal state, has been implicated in many aspects of breast cancer tumorigenesis, metastasis and drug resistance ). The accumulation of a large body of data on the association of EMT with cancer over the years has not resulted in EMT being an active target for therapeutic development. This is due in part to the lack of appropriate in vitro models. Here we have exploited some of the basic biology of EMT, to create an advanced in vitro metastatic breast cancer reporter cell line model for use in both basic research and discovery of new EMT inhibitors. During EMT, E-cadherin protein expression is down regulated in cancer cells in association with the loss of cell-to-cell adhesion, apico-basal polarity and a change to a spindle-shaped morphology. By installing an emerald green fluorescent protein (EmGFP) tag on the C-terminus of the e-cadherin (ECAD) gene in the epithelial BT-474 breast cancer cell line via CRISPR/Cas9 genome editing, end-point or real-time EMT status of cells can be tracked under defined conditions. The EMT reporter cell line was verified at the nucleic acid (genomic and mRNA) and protein levels as well as in cell-based assays. Bio-functional evaluation of the BT-474 ECAD EmGFP cell line, shows that it responds to EMT induction. The subsequent EMT status of cells can be monitored in real time by observing and measuring ECAD EmGFP expression, in addition, the resulting mesenchymal cells have increased invasive capacities. Furthermore, this EMT reporter cell line shows sensitivity to the MEK1/2 inhibitor U0126 - thereby providing the basis for the use of this cell line in high-throughput screening (HTS) applications including the identification of new anti-EMT drugs for metastatic breast cancer. The BT-474 ECAD EmGFP reporter cell line is also a convenient and sensitive model for basic science research on the mechanisms of metastasis. References DIZON, D. S., L. KRILOV, E. COHEN, T. GANGADHAR, P. A. GANZ et al., 2016 Clinical Cancer Advances 2016: Annual Report on Progress Against Cancer From the American Society of Clinical Oncology. J Clin Oncol 34: 987-1011. HAY, E. D., 1995 An overview of epithelio-mesenchymal transformation. Acta Anat (Basel) 154: 8-20. Citation Format: Metewo S. Enuameh, Weiguo Shu, Robert Newman. CRISPR/cas9 mediated generation of an EMT reporter cell line for metastatic breast cancer drug discovery and development [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1045.