Transcription Factor Snail Research Articles

A conjugated 2D covalent organic framework as a drug delivery vehicle towards triple negative breast cancer malignancy.

Cancer, one of the deadliest diseases for both sexes, has always demanded updated treatment strategies with time. Breast cancer is responsible for the highest mortality rate among females worldwide and requires treatment with advanced regimens due to the higher probability of breast cancer cells to develop drug cytotoxicity followed by resistance. Covalent organic framework (COF) materials with ordered nanoscale porosity can serve as drug delivery vehicles due to their biocompatible nature and large internal void spaces. In this research work, we have employed a novel biocompatible COF, TRIPTA, as a drug delivery carrier towards breast cancer cells. It served as a drug delivery vehicle for cisplatin in triple negative breast cancer (TNBC) cells. We have checked the potency of TRIPTA in combating the proliferation of metastatic TNBC cells. Our results revealed that cisplatin loaded over TRIPTA-COF exhibited a greater impact on the CD44+/CD24− cancer stem cell niche of breast cancer. Retarded migration of cancer cells has also been observed with the dual treatment of TRIPTA and cisplatin compared to that of cisplatin alone. Epithelial–mesenchymal transition (EMT) has also been minimized by the combinatorial treatment of cisplatin carried by the carrier material in comparison to cisplatin alone. The epithelial marker E-cadherin is significantly increased in cells treated with cisplatin together with the carrier COF, and the expression of mesenchymal markers such as N-cadherin is lower. The transcriptional factor Snail has been observed under the same treatment. The carrier material is also internalized by the cancer cells in a time-dependent manner, suggesting that the organic carrier can serve as a specific drug delivery vehicle. Our experimental results suggested that TRIPTA-COF can serve as a potent nanocarrier for cisplatin, showing higher detrimental effects on the proliferation and migration of TNBC cells by increasing the cytotoxicity of cisplatin.

Delayed Administration of Nintedanib Ameliorates Fibrosis Progression in CG-Induced Peritoneal Fibrosis Mouse Model

Background: A multiple-target tyrosine kinase inhibitor, nintedanib, which is approved for treatment of interstitial pulmonary disease, has been demonstrated to have anti-fibrotic activity outside of the lungs. We explored its therapeutic effect in a murine model of peritoneal fibrosis. Methods: Daily intraperitoneal injections of chlorhexidine gluconate (CG) induced peritoneal fibrosis in mice. The effects of delayed administration of nintedanib (given at day 21 after CG injection and then given daily for 14 days) were determined by immunohistochemical staining, ELISA, and immunoblot analysis. Results: Delayed administration of nintedanib significantly inhibited peritoneal fibrosis progression as indicated by decreasing deposition and expression of extracellular matrix (ECM) proteins (fibronectin and type I collagen). Treatment with nintedanib also upregulated MMP-2 and reciprocally downregulated TIMP-2, along with reducing expression of α-SMA, β-vimentin, and two transcription factors (Snail and Twist), and retaining E-cadherin expression. Nintedanib also inhibited co-expression of β-vimentin with Snail or Twist as shown by immunofluorescent staining. Moreover, nintedanib decreased the number of CD31-positive blood vessels and CD31 expression in the injured peritoneum. Moreover, delayed application of nintedanib inhibited the expression of several cytokines/chemokines, including monocyte chemoattractant protein-1, tumor necrosis factor-α, interleukin-1β (IL-1β), and IL-6, and infiltration of CD68⁺ macrophages to the injured peritoneum. Finally, nintedanib blocked phosphorylation of STAT3, NF-κB, and Smad3 during the development of peritoneal fibrosis. Conclusions: Delayed administration of nintedanib inhibits progression of peritoneal fibrosis and partially reverses established peritoneal fibrosis by attenuating epithelial-mesenchymal transition, inflammation, and angiogenesis, as well as promoting ECM degradation. We conclude that nintedanib has a therapeutic potential to treat peritoneal fibrosis.

Exosomes Released from Decidual Stromal Cells Incured Insufficient Migration and Invasion of Trophoblast Because of Disturbing of β-TrCP-Mediated Ubiquitination and Degradation of Transcription Factor Snail in Unexplained Recurrent Spontaneous Abortion

Exosomes Released from Decidual Stromal Cells Incured Insufficient Migration and Invasion of Trophoblast Because of Disturbing of β-TrCP-Mediated Ubiquitination and Degradation of Transcription Factor Snail in Unexplained Recurrent Spontaneous Abortion

The laminin-binding integrins regulate nuclear factor κB-dependent epithelial cell polarity and inflammation.

The main laminin-binding integrins α3β1, α6β1 and α6β4 are co-expressed in the developing kidney collecting duct system. We previously showed that deleting the integrin α3 or α6 subunit in the ureteric bud, which gives rise to the kidney collecting system, caused either a mild or no branching morphogenesis phenotype, respectively. To determine whether these two integrin subunits cooperate in kidney collecting duct development, we deleted α3 and α6 in the developing ureteric bud. The collecting system of the double knockout phenocopied the α3 integrin conditional knockout. However, with age, the mice developed severe inflammation and fibrosis around the collecting ducts, resulting in kidney failure. Integrin α3α6-null collecting duct epithelial cells showed increased secretion of pro-inflammatory cytokines and displayed mesenchymal characteristics, causing loss of barrier function. These features resulted from increased nuclear factor kappa-B (NF-κB) activity, which regulated the Snail and Slug (also known as Snai1 and Snai2, respectively) transcription factors and their downstream targets. These data suggest that laminin-binding integrins play a key role in the maintenance of kidney tubule epithelial cell polarity and decrease pro-inflammatory cytokine secretion by regulating NF-κB-dependent signaling.

TGF-β/Smad signaling pathway plays a crucial role in patulin-induced pro-fibrotic changes in rat kidney via modulation of slug and snail expression

Patulin (PAT) is a mycotoxin that contaminates a variety of food and foodstuffs. Earlier in vitro and in vivo findings have indicated that kidney is one of the target organs for PAT-induced toxicity. However, no study has evaluated the chronic effects of PAT exposure at environmentally relevant doses or elucidated the detailed mechanism(s) involved. Here, using in vitro and in vivo experimental approaches, we delineated the mechanism/s involved in pro-fibrotic changes in the kidney after low-dose chronic exposure to PAT. We found that non-toxic concentrations (50 nM and 100 nM) of PAT to normal rat kidney cells (NRK52E) caused a higher generation of reactive oxygen species (ROS) (mainly hydroxyl (•OH), peroxynitrite (ONOO−), and hypochlorite radical (ClO−). PAT exposure caused the activation of mitogen-activated protein kinases (MAPKs) and its downstream c-Jun/Fos signaling pathways. Moreover, our chromatin immunoprecipitation (ChIP) analysis suggested that c-Jun/Fos binds to the promoter region of Transforming growth factor beta (TGF-β1) and possibly induces its expression. Results showed that PAT-induced TGF-β1 further activates the TGF-β1/smad signaling pathways. Higher activation of slug and snail transcription factors further modulates the regulation of pro-fibrotic molecules. Similarly, in vivo results showed that PAT exposure to rats through gavage at 25 and 100 μg/kg b. wt had higher levels of kidney injury/toxicity markers namely vascular endothelial growth factor (VEGF), kidney Injury Molecule-1 (Kim-1), tissue inhibitor of metalloproteinase-1 (Timp-1), and clusterin (CLU). Additionally, histopathological analysis indicated significant alterations in renal tubules and glomeruli along with collagen deposition in PAT-treated rat kidneys. Overall, our data provide evidence of the involvement of ROS mediated MAPKs and TGF-β1/smad pathways in PAT-induced pro-fibrotic changes in the kidney via modulation of slug and snail expression.

CCR9 initiates epithelial\u2013mesenchymal transition by activating Wnt/\u03b2-catenin pathways to promote osteosarcoma metastasis

BackgroundOsteosarcoma (OS) patients with lung metastasis have poor prognoses, and effective therapeutic strategies for delaying or inhibiting the spread of lung metastasis from the primary OS site are lacking. Hence, it is critical to elucidate the underlying mechanisms of OS metastasis and to identify additional new effective treatment strategies for patients.MethodsDifferential expression and functional analyses were performed to identify key genes and relevant signaling pathways associated with OS lung metastasis. The expression of CCR9 in OS cell lines and tissues was measured by RT-qPCR, western blotting and immunohistochemistry. Cell migration and invasion were assessed by wound healing and Transwell Matrigel invasion assays, respectively. The regulatory relationship between CCR9 and the Wnt/β-catenin signaling pathway was further evaluated by rescue experiments.ResultsThe expression of CCR9 was elevated in OS cell lines and patients with lung metastasis. CCR9 promoted MG63 and HOS cell migration and invasion by activating the Wnt/β-catenin signaling pathway. Furthermore, knockdown of CCR9 repressed epithelial–mesenchymal transition (EMT) by downregulating mesenchymal markers (N-cadherin and Vimentin) and EMT-associated transcription factors (twist and snail) and upregulating an epithelial marker (E-cadherin).ConclusionsOur findings suggest that CCR9 promotes EMT by activating Wnt/β-catenin pathways to promote OS metastasis. CCR9 may be a promising therapeutic target to inhibit lung metastasis and serve as a novel prognostic marker for OS.

SNAIL1: Linking Tumor Metastasis to Immune Evasion.

The transcription factor Snail1, a key inducer of epithelial-mesenchymal transition (EMT), plays a critical role in tumor metastasis. Its stability is strictly controlled by multiple intracellular signal transduction pathways and the ubiquitin-proteasome system (UPS). Increasing evidence indicates that methylation and acetylation of Snail1 also affects tumor metastasis. More importantly, Snail1 is involved in tumor immunosuppression by inducing chemokines and immunosuppressive cells into the tumor microenvironment (TME). In addition, some immune checkpoints potentiate Snail1 expression, such as programmed death ligand 1 (PD-L1) and T cell immunoglobulin 3 (TIM-3). This mini review highlights the pathways and molecules involved in maintenance of Snail1 level and the significance of Snail1 in tumor immune evasion. Due to the crucial role of EMT in tumor metastasis and tumor immunosuppression, comprehensive understanding of Snail1 function may contribute to the development of novel therapeutics for cancer.

Abstract PO-059: Epithelial/mesenchymal identity dictates pancreatic cancer cell metastasis

Abstract Metastatic pancreatic adenocarcinoma (PDAC) is the dominant clinical presentation and highly treatment-resistant. However, not all metastasis is equal with metastatic disease in the lung having improved outcomes over the liver. These clinical differences suggest a therapeutic opportunity and urge analysis of the molecular underpinnings of PDAC metastasis. The acquisition of mesenchymal features by epithelial cancer cells is commonly associated with solid tumor metastasis and has been linked to the pancreatic cancer basal subtype and its association with treatment resistance and poorer outcomes, but its impact on pancreatic cancer metastasis needs further understanding. We explored the impact on metastasis of stabilized epithelial, partial-mesenchymal and mesenchymal cancer cells by generating several genetic mouse models based on the lineage-traced KPC mouse model (KrasLSL-G12D;p53R172H; or p53LSL; PDX1-Cre;EYFPLSL). Using single-cell RNA-sequencing we confirmed the KPC mouse model recapitulates the spectrum of epithelial-mesenchymal phenotypes observed in patients and can be genetically engineered to stabilize specific phenotypes. The stabilization of epithelial phenotypes through the homozygous loss of the mesenchymal-driving transcription factors Snail and Twist (Snai1F/F;Twist1F/F) had no impact on primary tumor progression but increased liver colonization. This increase in liver colonization was supported by a second epithelial-stabilized mouse model based on the loss of Zeb1 (Zeb1F/F). The stabilization of mesenchymal features through the heterozygous or homozygous loss of the epithelial adherin junction E-cadherin (Cdh1F/+ or Cdh1F/F) promoted lung metastasis. Interestingly, epithelial plasticity was still required for efficient lung colonization, but not rare liver metastasis. Additionally, mesenchymal gene expression correlated with an improved patient survival as well as metastatic localization, supporting the clinical observations of improved survival in lung metastasis. Using gene expression analysis of sorted bulk cancer cells and single-cells, migration assays, and multiplexed-immunohistochemistry, we observed that the epithelial/mesenchymal status of the cancer cells dictated different mechanisms for motility and interaction with the immune system. Mesenchymal cancer cells migrate as single-cells and attract fewer T-cells where epithelial cancer cells migrate collectively and have increased immune regulation gene expression. These data suggest the epithelial/mesenchymal status of cancers cells dictate the where and how of metastatic disease and could inform therapeutic interventions. Citation Format: Julienne L. Carstens, Sujuan Yang, Pedro Correa de Sampaio, Xiaofeng Zheng, Souptik Barua, Kathleen M. McAndrews, Arvind Rao, Jared K. Burks, Andrew D. Rhim, Raghu Kalluri. Epithelial/mesenchymal identity dictates pancreatic cancer cell metastasis [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2021 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2021;81(22 Suppl):Abstract nr PO-059.

ZIP4 promotes non-small cell lung cancer metastasis by activating snail-N-cadherin signaling axis.

Non-small cell lung cancer (NSCLC) is one of the most critical health problems worldwide, with high incidence and poor survival rate. A zinc importer ZIP4 has been implicated in the process of tumor growth and metastasis of many cancers. However, its exact role and the underlying mechanism in NSCLC remains to be elucidated. In the present study, we found that human ZIP4 was substantially overexpressed in NSCLC tissues and was correlated with poor overall survival (OS) and progression-free survival (PFS). Overexpression of ZIP4 promoted cell migration, invasion and metastasis both in vitro and in a mouse lung metastasis model. Silencing of ZIP4 attenuated migration, invasion and metastasis. Mechanistically, overexpression of ZIP4 increased the expression of Snail, Slug and N-cadherin while genetic inactivation of ZIP4 downregulated the expression of above-mentioned genes. Further analysis showed that transcriptional factor Snail which modulates N-cadherin was involved in the process of ZIP4-mediated NSCLC migration and invasion. We also demonstrated that ZIP4 positively correlates with the levels of Snail, Slug and N-cadherin in mice lung metastasis tumors. Together, these results suggest that ZIP4 acts as an important regulator of Snail-N-cadherin signaling axis in promoting NSCLC progression and may serve as a novel predictive marker and therapeutic target in NSCLC.

19-Hydroxybufalin inhibits non-small cell lung cancer cell proliferation and promotes cell apoptosis via the Wnt/\u03b2-catenin pathway

BackgroundBufadienolides derived from the skin of toads are often regarded as the main active components with antitumor effects. 19-Hydroxybufalin (19-HB) is a monomer of bufadienolides; however, its effects and underlying molecular mechanisms on tumor growth remain to be ascertained. In this report, we focused on the antitumor effects of 19-HB on non-small cell lung cancer to provide a scientific basis for its further development and utilization.MethodsThe antitumor effects of 19-HB on the human NSCLC cell lines NCI-H1299 and NCI-H838 were examined in vitro. The cells were treated with different concentrations of 19-HB, and the inhibition of cell growth was measured by CCK-8 and colony formation assays. Furthermore, cell apoptosis was analyzed by flow cytometry, TUNEL staining, JC-1 staining, and western blotting. The effects on migration and invasion were evaluated by wound-healing assay, transwell assay, and western blotting. Finally, the antitumor effects of 19-HB were evaluated in vivo using a xenograft mouse model.Results19-HB-treated NSCLC cells showed inhibited cell viability and increased apoptosis. The expression levels of cleaved caspase-3, cleaved-PARP, and Bax/Bcl-2 were upregulated, while the mitochondrial membrane potential decreased. In contrast, migration, invasion, as well as the expression of MMP2, MMP7, MMP9, the epithelial–mesenchymal transition-related proteins N-cadherin and Vimentin, and the transcription factors Snail and Slug were inhibited. Furthermore, the expression levels of the key molecules in the Wnt/β-catenin signaling pathway (CyclinD1, c-Myc, and β-catenin) were decreased. In vivo, the growth of xenograft tumors in nude mice was also significantly inhibited by 19-HB, and there were no significant changes in biochemical indicators of hepatic and renal function.Conclusions19-HB inhibited the proliferation, migration, and invasion, and promoted the apoptosis of NSCLC cells via the Wnt/β-catenin pathway. In addition, 19-HB inhibited the growth of xenograft tumors in nude mice with little toxicity to the liver and kidney. Thus, 19-HB may be a potential antitumor agent for treating NSCLC.

Lobeglitazone, A Peroxisome Proliferator-Activated Receptor-Gamma Agonist, Inhibits Papillary Thyroid Cancer Cell Migration and Invasion by Suppressing p38 MAPK Signaling Pathway.

BackgroundPeroxisome proliferator-activated receptor-gamma (PPAR-γ) ligands have been widely shown to correlate with epithelial-mesenchymal transition (EMT) and cancer progression. Lobeglitazone (LGZ) is a novel ligand of PPAR-γ; and its role in EMT and metastasis in papillary thyroid carcinoma (PTC) is poorly understood. We aimed to investigate the role of LGZ in metastatic behavior of PTC cells.MethodsHalf maximal inhibitory concentration (IC50) values of LGZ in BRAF-mutated PTC cell lines (BCPAP and K1) were determined using MTT assay. Rosiglitazone (RGZ), the PPAR-γ ligand was used as a positive control. The protein expression of PPAR-γ, cell-surface proteins (E-cadherin, N-cadherin), cytoskeletal protein (Vimentin), transcription factor (Snail), p38 mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinase (ERK) 1/2 pathway, and matrix metalloproteinase (MMP)-2 expression were measured using Western blotting. Changes in E-cadherin expression were also determined using immunocytochemistry. Cell migration and invasion were analyzed using wound healing and Matrigel invasion assays.ResultsTreatment with LGZ or RGZ significantly inhibited transforming growth factor-beta1 (TGF-β1)-induced EMT-associated processes such as fibroblast-like morphological changes, EMT-related protein expression, and increased cell migration and invasion in BCPAP and K1 cells. LGZ restored TGF-β1-induced loss of E-cadherin, as observed using immunocytochemistry. Furthermore, LGZ and RGZ suppressed TGF-β1-induced MMP-2 expression and phosphorylation of p38 MAPK, but not ERK1/2. Although there was no change in PPAR-γ expression after treatment with LGZ or RGZ, the effect of downstream processes mediated by LGZ was hampered by GW9662, a PPAR-γ antagonist.ConclusionLGZ inhibits TGF-β1-induced EMT, migration, and invasion through the p38 MAPK signaling pathway in a PPAR-γ-dependent manner in PTC cells.

Extracellular RNA transfer from non-malignant human cholangiocytes can promote cholangiocarcinoma growth.

Extracellular vesicles (EV) within the cellular secretome are emerging as modulators of pathological processes involved in tumor growth through their ability to transfer donor‐derived RNA into recipient cells. While the effects of tumor and stromal cell EVs within the tumor microenvironment have been studied, less is known about the contributions of normal, nontransformed cells. We examined the impact of EVs within the cellular secretome from nonmalignant cells on transformed cell growth and behavior in cholangiocarcinoma cells. These effects were enhanced in the presence of the pro‐fibrogenic mediator TGF‐β. We identified miR‐195 as a TGF‐β responsive miRNA in normal cells that can be transferred via EV to tumor cells and regulate cell growth, invasion, and migration. The effects of miR‐195 involve modulation of the epithelial–mesenchymal transition through direct effects on the transcription factor Snail. These studies provide in vitro and in vivo evidence for the impact of normal cellular secretome on transformed cell growth, show the importance of EV RNA transfer, and identify mechanisms of EV‐mediated transfer of miRNA as a contributor to tumor development, which may provide new therapeutic opportunities for targeting human cholangiocarcinoma.

Requirement of Histone Deacetylase 6 for Interleukin-6 Induced Epithelial-Mesenchymal Transition, Proliferation, and Migration of Peritoneal Mesothelial Cells.

Aims: Influenced by microenvironment, human peritoneal mesothelial cells (HPMCs) acquired fibrotic phenotype, which was identified as the protagonist for peritoneal fibrosis. In this study, we examined the role of histone deacetylase 6 (HDAC6) for interleukin-6 (IL-6) induced epithelial-mesenchymal transition (EMT), proliferation, and migration of HPMCs.Methods: The role of HDAC6 in IL-6-elicited EMT of HPMCs was tested by morphological observation of light microscope, immunoblotting, and immune-fluorescence assay; and the function of HDAC6 in proliferation and migration of HPMCs was examined by CCK-8 assay, wound healing experiment, and immunoblotting.Results: IL-6 stimulation significantly increased the expression of HDAC6. Treatment with tubastatin A (TA), a highly selective HDAC6 inhibitor, or silencing of HDAC6 with siRNA decreased the expression of HDAC6. Moreover, TA or HDAC6 siRNA suppressed IL-6-induced EMT, as evidenced by decreased expressions of α-SMA, Fibronectin, and collagen I and the preserved expression of E-cadherin in cultured HPMCs. Mechanistically, HDAC6 inhibition suppressed the expression of transforming growth factor β (TGFβ) receptor I (TGFβRI), phosphorylation of Smad3, secretion of connective tissue growth factor (CTGF), and transcription factor Snail. On the other hand, the pharmacological inhibition or genetic target of HDAC6 suppressed HPMCs proliferation, as evidenced by the decreased optical density of CCK-8 and the expressions of PCNA and Cyclin E. The migratory rate of HPMCs also decreased. Mechanistically, HDAC6 inhibition blocked the activation of JAK2 and STAT3.Conclusion: Our study illustrated that IL-6-induced HDAC6 not only regulated IL-6 itself downstream JAK2/STAT3 signaling but also co-activated the TGF-β/Smad3 signaling, leading to the change of the phenotype and mobility of HPMCs. HDAC6 could be a potential therapeutic target for the prevention and treatment of peritoneal fibrosis.

Blockade of Autophagy Prevents the Development and Progression of Peritoneal Fibrosis.

Peritoneal fibrosis (PF) is a major cause of ultrafiltration failure in long-term peritoneal dialysis (PD) patients. Nevertheless, limited measures have been shown to be effective for the prevention and treatment of PF. Some views reveal that activation of autophagy ameliorates PF but others demonstrate that autophagy promotes PF. It is obvious that the role of autophagy in PF is controversial and further studies are needed. Here, we investigated the role of autophagy in rat models of PF and damaged cultured human peritoneal mesothelial cells (HPMCs). Autophagy was highly activated in fibrotic peritoneum from two PF rat models induced by 4.25% peritoneal dialysate fluid (PDF) and 0.1% chlorhexidine gluconate (CG). Blockade of autophagy with 3-MA effectively prevented PF in both models and reversed epithelial to mesenchymal transition (EMT) by down-regulating TGF-β/Smad3 signaling pathway and downstream nuclear transcription factors Slug and Snail. Treatment with 3-MA also inhibited activation of EGFR/ERK1/2 signaling pathway during PF. Moreover, 3-MA prominently decreased STAT3/NF-κB-mediated inflammatory response and macrophage infiltration, and prevented peritoneal angiogenesis through downregulation of β-catenin signal. In addition, TGF-β1 stimulation up-regulated autophagic activity as evidenced by the increased autophagosome in vitro. Exposure of HPMCs to TGF-β1 resulted in the induction of EMT and activation of TGF-β/Smad3, EGFR/ERK1/2 signaling pathways. Treatment with 3-MA blocked all these responses. In addition, delayed administration of 3-MA was effective in reducing EMT induced by TGF-β1. Taken together, our study indicated that autophagy might promote PF and 3-MA had anti-fibrosis effect in vivo and in vitro. These results suggest that autophagy could be a potential target on PF therapy for clinical patients with long-term PD.

Retinoic Acid-Inducible Gene I-Like Receptors Activate Snail To Limit RNA Viral Infections.

Retinoic acid-inducible gene I-like receptors (RLRs) are important cytosolic pattern recognition receptors (PRRs) that sense viral RNA before mounting a response leading to the activation of type I IFNs. Several viral infections induce epithelial-mesenchymal transition (EMT), even as its significance remains unclear. Here, we show that EMT or an EMT-like process is a general response to viral infections. Our studies identify a previously unknown mechanism of regulation of an important EMT-transcription factor (EMT-TF) Snail during RNA viral infections and describe its possible implication. RNA viral infections, poly(I·C) transfection, and ectopic expression of RLR components induced Snail levels, indicating that RLR pathway could regulate its expression. Detailed examination using mitochondrial antiviral signaling protein knockout (MAVS-KO) cells established that MAVS is essential in this regulation. We identified two interferon-stimulated response elements (ISREs) in the SNAI1 promoter region and demonstrated that they are important in its transcriptional activation by phosphorylated IRF3. Increasing the levels of Snail activated RLR pathway and dramatically limited replication of the RNA viruses dengue virus, Japanese encephalitis virus (JEV), and vesicular stomatitis virus, pointing to their antiviral functions. Knockdown of Snail resulted in a considerable increase in the JEV titer, validating its antiviral functions. Finally, transforming growth factor β-mediated IFNB activation was dependent on Snail levels, confirming its important role in type I IFN activation. Thus, EMT-TF Snail is transcriptionally coregulated with type I IFN by RLRs and, in turn, promotes the RLR pathway, further strengthening the antiviral state in the cell. Our work identified an interesting mechanism of regulation of Snail that demonstrates potential coregulation of multiple innate antiviral pathways triggered by RLRs. Identification of antiviral functions of Snail also provides an opportunity to expand the sphere of RLR signaling. IMPORTANCE RLRs sense viral genomic RNA or the double-stranded RNA intermediates and trigger the activation of type I IFNs. Snail transcription factor, commonly associated with epithelial-mesenchymal transition (EMT), has been reported to facilitate EMT in several viral infections. Many of these reports are based on oncoviruses, leading to the speculation that EMT induced during infection is an important factor in the oncogenesis triggered by these infections. However, our studies reveal that EMT or EMT-like processes during viral infections have important functions in antiviral response. We have characterized a new mechanism of transcriptional regulation of Snail by IRF3 through interferon-stimulated response elements in their promoters, and this finding could have importance in nonviral contexts as well. We also identify that EMT-TF Snail promotes antiviral status of the infected cells through the RLR pathway. This study characterizes a new regulatory mechanism of activation of Snail and establishes its unidentified function in antiviral response.

Drug-eluting intraocular lens with sustained bromfenac release for conquering posterior capsular opacification

Cataract is the leading cause of visual impairment, and posterior capsular opacification (PCO) is the most common long-term complication of modern cataract surgery, which can cause severe visual impairment after surgery. The proliferation, migration, and epithelial-mesenchymal transition (EMT) of residual lens epithelial cells (LECs) stimulated by growth factors and cytokines, are the key pathological mechanisms involved in the development of PCO. This study demonstrated that non-steroidal anti-inflammatory drug (NSAID), bromfenac, was capable of effectively inhibiting cell migration, overexpression of EMT markers, such as fibronectin (FN), matrix metalloproteinase 2 (MMP2), α-smooth muscle actin (α-SMA), and transcription factor Snail, and extracellular signal-regulated kinase (ERK)/glycogen synthase kinase-3β (GSK-3β) signaling induced by transforming growth factor-β2 (TGF-β2) in vitro. The inhibitory effect of bromfenac on TGF-β2-induced EMT was also verified on a primary lens epithelial cell model using human anterior capsules. Furthermore, based on ultrasonic spray technology, we developed a drug-eluting intraocular lens (IOL) using poly (lactic-co-glycolic acid) (PLGA) with sustained bromfenac release ability for the prevention of PCO development. In the rabbit models of cataract surgery, bromfenac-eluting IOL exhibited remarkable PCO prevention and inflammation suppression effects with excellent biocompatibility. In conclusion, bromfenac can inhibit TGF-β2-induced cell migration and the EMT of LECs via ERK/GSK-3β/Snail signaling. The present study offers a novel approach for preventing PCO through PLGA-based drug sustained-release IOLs.

TGF-β1 inhibits microvascular-like formation by decreasing VCAM1 and ICAM1 via the upregulation of SNAIL in human granulosa cells

Three major endothelial cell junctional adhesion molecules (VCAM1, ICAM1 and E-SELECTIN) play important roles in the process of angiogenesis, a progression of extensive physiological vascularization that occurs during the formation of the corpus luteum. Our previous studies demonstrated that TGF-β1 is a negative regulator of luteinization and progesterone production in luteinized human granulosa (hGL) cells. Whether TGF-β1 can regulate the expression of these endothelial cell adhesion molecules and subsequent angiogenesis in hGL cells remains to be elucidated. Using dual inhibition approaches (small molecular inhibitors and siRNA-based knockdown), we provided the first data showing that TGF-β1 significantly upregulates the expression of the SNAIL transcription factor, which in turn suppresses the expression of VCAM1 and ICAM1 in hGL cells. Additionally, we demonstrate that the suppressive effects on the expression of VCAM1 and ICAM1 induced by TGF-β1 treatment were most likely via an ALK5-mediated SMAD-dependent signaling pathway. Furthermore, functional studies showed that hGL cells cultured on Matrigel exhibited two typical endothelial cell phenotypes, microvascular-like formation and a sprouting microvascular pattern. Notably, these phenotypes were significantly suppressed by either TGF-β1 treatment or knockdown of VCAM1 and ICAM1. Our findings suggest that TGF-β1 plays a potential role in the inhibition of granulosa cell angiogenesis by downregulating the expression of VCAM1 and ICAM1 during follicular development and corpus luteum formation.

Caffeine prevents oxalate-induced epithelial-mesenchymal transition of renal tubular cells by its anti-oxidative property through activation of Nrf2 signaling and suppression of Snail1 transcription factor

Caffeine is an active ingredient found in coffee and energy beverages. Its hepatoprotective effects against liver fibrosis are well-documented. Nonetheless, its renoprotective effects against renal fibrogenesis and epithelial-mesenchymal transition (EMT) processes remain unclear and under-investigated. In this study, the protective effects of caffeine against oxalate-induced EMT in renal tubular cells were evaluated by various assays to measure expression levels of epithelial and mesenchymal markers, cell migrating activity, level of oxidized proteins, and expression of Nrf2 and Snail1. Oxalate at sublethal dose significantly suppressed cell proliferation but increased cell elongation, spindle index and migration. Oxalate also decreased expression of epithelial markers (zonula occludens-1 (ZO-1) and E-cadherin) but increased expression of mesenchymal markers (fibronectin, vimentin and α-smooth muscle actin (α-SMA)). All of these EMT-inducing effects of oxalate could be prevented by pretreatment with caffeine. While oxalate increased oxidized proteins and Snail1 levels, it decreased Nrf2 expression. Caffeine could preserve all these molecules to their basal (control) levels. Finally, silencing of Nrf2 expression by small interfering RNA (siRNA) could abolish such protective effects of caffeine on oxalate-induced EMT. Our data indicate that the renoprotective effects of caffeine against oxalate-induced EMT is mediated, at least in part, by its anti-oxidative property through activation of Nrf2 signaling and suppression of Snail1 transcription factor.

Epithelial cell plasticity drives endoderm formation during gastrulation

It is generally accepted that epiblast cells ingress into the primitive streak by epithelial-to-mesenchymal transition (EMT) to give rise to the mesoderm; however, it is less clear how the endoderm acquires an epithelial fate. Here, we used embryonic stem cell and mouse embryo knock‐in reporter systems to combine time-resolved lineage labelling with high-resolution single-cell transcriptomics. This allowed us to resolve the morphogenetic programs that segregate the mesoderm from the endoderm germ layer. Strikingly, while the mesoderm is formed by classical EMT, the endoderm is formed independent of the key EMT transcription factor Snail1 by mechanisms of epithelial cell plasticity. Importantly, forkhead box transcription factor A2 (Foxa2) acts as an epithelial gatekeeper and EMT suppressor to shield the endoderm from undergoing a mesenchymal transition. Altogether, these results not only establish the morphogenetic details of germ layer formation, but also have broader implications for stem cell differentiation and cancer metastasis.

Expression of the E5 Oncoprotein of HPV16 Impacts on the Molecular Profiles of EMT-Related and Differentiation Genes in Ectocervical Low-Grade Lesions.

Infection with human papillomavirus type 16 (HPV16) is one of the major risk factors for the development of cervical cancer. Our previous studies have demonstrated the involvement of the early oncoprotein E5 of HPV16 (16E5) in the altered isoform switch of fibroblast growth factor receptor 2 (FGFR2) and the consequent expression in human keratinocytes of the mesenchymal FGFR2c isoform, whose aberrant signaling leads to EMT, invasiveness, and dysregulated differentiation. Here, we aimed to establish the possible direct link between these pathological features or the appearance of FGFR2c and the expression of 16E5 in low-grade squamous intraepithelial lesions (LSILs). Molecular analysis showed that the FGFR2c expression displayed a statistically significant positive correlation with that of the viral oncoprotein, whereas the expression values of the epithelial FGR2b variant, as well as those of the differentiation markers keratin 10 (K10), loricrin (LOR) and involucrin (INV), were inversely linked to the 16E5 expression. In contrast, the expression of EMT-related transcription factors Snail1 and ZEB1 overlapped with that of 16E5, becoming a statistically significant positive correlation in the case of Snail2. Parallel analysis performed in human cervical LSIL-derived W12 cells, containing episomal HPV16, revealed that the depletion of 16E5 by siRNA was able to counteract these molecular events, proving to represent an effective strategy to identify the specific role of this viral oncoprotein in determining LSIL oncogenic and more aggressive profiles. Overall, coupling in vitro approaches to the molecular transcript analysis in ectocervical early lesions could significantly contribute to the characterization of specific gene expression profiles prognostic for those LSILs with a greater probability of direct neoplastic progression.