Snail Axis Research Articles

Circular RNA hsa_circ_0004689 (circSWT1) promotes NSCLC progression via the miR-370-3p/SNAIL axis by inducing cell epithelial-mesenchymal transition (EMT).

Previous studies have reported the role of circular RNAs (circRNAs) in the progression of non-small-cell lung cancer (NSCLC). SWT1-derived circRNAs were confirmed to affect the apoptosis of cardiomyocytes; however, the biological functions of SWT1-derived circRNAs in cancers are still unknown. Here, we investigated the potential role of SWT1-derived circRNAs in NSCLC. We used quantitative real-time polymerase chain reaction (qRT-PCR) to measure the expression of circSWT1 in NSCLC tissues and paired normal tissues. The potential functions of circSWT1 in tumor progression were assessed by CCK-8, colony formation, wound healing, and matrigel transwell assays in vitro and by xenograft tumor models in vivo. Next, epithelial-mesenchymal transition (EMT) was evaluated by western blotting, immunofluorescence, and immunohistochemistry (IHC). Moreover, circRIP, RNA pulldown assays, luciferase reporter gene assays, and FISH were conducted to illuminate the molecular mechanisms of circSWT1 via the miR-370-3p/SNAIL signal pathway. Then, we knocked out SNAIL in A549 and H1299 cells to identify the roles of circSWT1 in the progression and EMT of NSCLC through SNAIL. Finally, circSWT1 functions were confirmed in vivo using xenograft tumor models. CircSWT1 expression was significantly upregulated in NSCLC tissues, and high expression of circSWT1 predicted poor prognosis in NSCLC via survival analysis. In addition, overexpression of circSWT1 promoted the invasion and migration of NSCLC cells. Subsequently, we found that overexpression of circSWT1 induced EMT and that knockdown of circSWT1 inhibited EMT in NSCLC cells. Mechanistically, circSWT1 relieved the inhibition of downstream SNAIL by sponging miR-370-3p. Moreover, we found that these effects could be reversed by knocking out SNAIL. Finally, we verified that circSWT1 promoted NSCLC progression and EMT in xenograft tumor models. CircSWT1 promoted the invasion, migration, and EMT of NSCLC. CircSWT1 could serve as a potential biomarker and a potential therapeutic target for NSCLC.

Abstract 12879: Endothelial Cu Exporter Atp7a Deficiency Promotes Cu-Dependent Endothelial-to-Mesenchymal Transition and Atherosclerosis via Orchestrating Glycolysis and Ros Signaling

Introduction: Oxidative stress, inflammation and metabolic reprogramming contribute to endothelial cell (EC) dysfunction and atherosclerosis. Copper (Cu), an essential micro-nutrient, promotes atherosclerosis via unknown mechanism. Since excess Cu is toxic, bioavailability of intracellular Cu is tightly controlled by Cu exporter ATP7A. However, the role of ATP7A and its molecular linkage with inflammation, oxidative stress and metabolism in EC dysfunction and atherosclerosis are entirely unknown. Results: Here we found a dramatic reduction of ATP7A expression in human and mouse atherosclerotic aorta. Surprisingly, ATP7A knockdown (KD) in human aortic ECs using shRNA changed the cell morphology from EC to mesenchymal types. This was confirmed by RNAseq/qPCR showing reduced EC markers and increased mesenchymal markers, indicating endothelial to mesenchymal transition (EndMT). Mechanistically, loss of ATP7A in ECs increased [Cu]i and PFKFB3-dependent glycolysis (1.8- fold by ECAR in Seahorse assay). Overexpression of PFKFB3 or constitutively active PFK2, which enhances glycolysis, or direct Cu treatment induced EndMT. In parallel, ATP7A KD in ECs increased mitochondrial (mito)ROS (9.8 fold)-histone demethylase JMJD2B, which induced inflammatory EndMT inducer TGFβ2-EndMT transcription factor Snail (2 fold) axis. Of note, ATP7A KD-induced glycolysis as well as mitoROS- TGFβ2-Snail axis involved in EndMT were inhibited by Cu chelator TTM. In vivo, Cu transport dysfunctional ATP7A mutant/ApoE -/- mice showed enhanced atherosclerosis and CD31 + /αSMA + cells representing EndMT in aorta compared to control ApoE -/- mice, which was further confirmed by EC-specific ATP7A -/- /mTmG reporter mice. Conclusion: Our study demonstrates that ATP7A dysfunction in ECs drives EndMT by orchestrating mitoROS-JMJD2B-TGFβ as well as PFKFB3-glycolysis axis in a Cu dependent manner, leading to Snail induction to accelerates atherosclerosis. Thus, ATP7A is a potential therapeutic target for treatment of inflammatory vascular disease.

Targeting N7-methylguanosine tRNA modification blocks hepatocellular carcinoma metastasis after insufficient radiofrequency ablation

Radiofrequency heat ablation is an ideal radical treatment for hepatocellular carcinoma (HCC). However, insufficient radiofrequency ablation (IRFA) could lead to high recurrence of HCC. N7-methylguanosine (m7G) on tRNAs, an evolutionally conservative modification in mammals and yeast, modulates heat stress responses and tumor progression, while its function in HCC recurrence after IRFA remains unknown. Here, we found that IRFA significantly upregulates the level of m7G tRNA modification and its methyltransferase complex components METTL1/WDR4 in multiple systems including HCC patient-derived xenograft (PDX) mouse, patients' HCC tissues, sublethal-heat-treated models of HCC cell lines, and organoids. Functionally, gain-/loss-of-function assays showed that METTL1-mediated m7G tRNA modification promotes HCC metastasis under sublethal heat exposure both invitro and invivo. Mechanistically, we found that METTL1 and m7G tRNA modification enhance the translation of SLUG/SNAIL in a codon frequency-dependent manner under sublethal heat stress. Overexpression of SLUG/SNAIL rescued the malignant potency of METTL1 knockdown HCC cells after sublethal heat exposure. Our study uncovers the key functions of m7G tRNA modification in heat stress responses and HCC recurrence after IRFA, providing molecular basis for targeting METTL1-m7G-SLUG/SNAIL axis to prevent HCC metastasis after radiofrequency heat ablation treatment.

Transforming growth factor-β induced protein regulates pulmonary fibrosis via the G-protein signaling modulator 2 /Snail axis

Pulmonary fibrosis, a severe condition that can progress to respiratory failure and death, is characterized by aberrant activation/proliferation of fibroblasts and excessive extracellular matrix (ECM) deposition and has limited therapeutic options. Identifying novel mediators of pulmonary fibrosis is currently needed to facilitate the development of more effective therapeutic strategies targeting pulmonary fibrosis. The present study was designed to investigate whether transforming growth factor-β (TGF-β) induced protein (TGFBI), an extracellular matrix protein, regulates pulmonary fibrosis in vitro and in vivo and the possible mechanism of actions. It was found that protein expressions of TGFBI were significantly upregulated and G-protein signaling modulator 2 (GPSM2) expression downregulated in fibrotic lung tissues from bleomycin (BLM)-induced rats and TGF-β1-stimulated human lung IMR-90 fibroblasts. Either silencing TGFBI with specific siRNA or treatment with the TGF-β signaling inhibitor SB431542 significantly inhibited TGF-β1-induced fibrotic effects and dysregulation of GPSM2 and Snail expressions in IMR-90 fibroblasts. Moreover, GPSM2 overexpression also inhibited TGF-β1-induced fibrotic effects and Snail upregulation in IMR-90 fibroblasts. Silencing Snail with specific siRNA attenuated TGF-β1-induced fibrotic effects. Therefore, our findings suggest that the extracellular matrix protein TGFBI mediates pulmonary fibrosis through regulation of the GPSM2/Snail axis, which identifies TGFBI as a novel mediator of pulmonary fibrosis and may be a potential therapeutic target for the treatment of pulmonary fibrosis.

Snail acetylation by autophagy-derived acetyl-coenzyme A promotes invasion and metastasis of KRAS-LKB1 co-mutated lung cancer cells.

BackgroundAutophagy is elevated in metastatic tumors and is often associated with active epithelial‐to‐mesenchymal transition (EMT). However, the extent to which EMT is dependent on autophagy is largely unknown. This study aimed to identify the mechanisms by which autophagy facilitates EMT.MethodsWe employed a liquid chromatography‐based metabolomic approach with kirsten rat sarcoma viral oncogene (KRAS) and liver kinase B1 (LKB1) gene co‐mutated (KL) cells that represent an autophagy/EMT‐coactivated invasive lung cancer subtype for the identification of metabolites linked to autophagy‐driven EMT activation. Molecular mechanisms of autophagy‐driven EMT activation were further investigated by quantitative real‐time polymerase chain reaction (qRT‐PCR), Western blotting analysis, immunoprecipitation, immunofluorescence staining, and metabolite assays. The effects of chemical and genetic perturbations on autophagic flux were assessed by two orthogonal approaches: microtubule‐associated protein 1A/1B‐light chain 3 (LC3) turnover analysis by Western blotting and monomeric red fluorescent protein‐green fluorescent protein (mRFP‐GFP)‐LC3 tandem fluorescent protein quenching assay. Transcription factor EB (TFEB) activity was measured by coordinated lysosomal expression and regulation (CLEAR) motif‐driven luciferase reporter assay. Experimental metastasis (tail vein injection) mouse models were used to evaluate the impact of calcium/calmodulin‐dependent protein kinase kinase 2 (CAMKK2) or ATP citrate lyase (ACLY) inhibitors on lung metastasis using IVIS luciferase imaging system.ResultsWe found that autophagy in KL cancer cells increased acetyl‐coenzyme A (acetyl‐CoA), which facilitated the acetylation and stabilization of the EMT‐inducing transcription factor Snail. The autophagy/acetyl‐CoA/acetyl‐Snail axis was further validated in tumor tissues and in autophagy‐activated pancreatic cancer cells. TFEB acetylation in KL cancer cells sustained pro‐metastatic autophagy in a mammalian target of rapamycin complex 1 (mTORC1)‐independent manner. Pharmacological inhibition of this axis via CAMKK2 inhibitors or ACLY inhibitors consistently reduced the metastatic capacity of KL cancer cells in vivo.ConclusionsThis study demonstrates that autophagy‐derived acetyl‐CoA promotes Snail acetylation and thereby facilitates invasion and metastasis of KRAS‐LKB1 co‐mutated lung cancer cells and that inhibition of the autophagy/acetyl‐CoA/acetyl‐Snail axis using CAMKK2 or ACLY inhibitors could be a potential therapeutic strategy to suppress metastasis of KL lung cancer.

14-3-3η Promotes Invadosome Formation via the FOXO3-Snail Axis in Rheumatoid Arthritis Fibroblast-like Synoviocytes.

Erosive destruction of joint structures is a critical event in the progression of rheumatoid arthritis (RA), in which fibroblast-like synoviocytes (FLS) are the primary effectors. We previously reported that the ability of RA FLS to degrade extracellular matrix (ECM) components depends on the formation of actin-rich membrane protrusions, called invadosomes, through processes that remain elusive. 14-3-3η belongs to a family of scaffolding proteins involved in a wide range of cellular functions, and its expression is closely related to joint damage and disease activity in RA patients. In this study, we sought to assess the role of 14-3-3η in joint damage by examining its contribution to the invadosome formation phenotype of FLS. Using human primary FLS, we show that 14-3-3η expression is closely associated with their ability to form invadosomes. Furthermore, knockdown of 14-3-3η using shRNAs decreases the level of invadosome formation in RA FLS, whereas addition of the recombinant protein to FLS from healthy individuals promotes their formation. Mechanistic studies suggest that 14-3-3η regulates invadosome formation by increasing Snail expression, a mechanism that involves nuclear exclusion of the transcription repressor FOXO3. Our results implicate the 14-3-3η–FOXO3–Snail axis in promoting the aggressive ECM-degrading phenotype of RA FLS, and suggest a role for this scaffolding protein in cartilage degradation.

A reciprocal feedback of miR-548ac/YB-1/Snail induces EndMT of HUVECs during acidity microenvironment

BackgroundResearches indicated the process of Endothelial-Mesenchymal-Transition (EndMT) of vascular endothelial cells (ECs) was critically involved in the progression of tumor. ECs demonstrated functional and phenotypic heterogeneity when located under different microenvironments. The extracellular pH of tumor tissues was acidic compared to that of normal tissues. However, there was still unclear whether the acidic microenvironment affected the EndMT of vascular ECs.MethodsHuman Umbilical Vein Endothelial Cell (HUVECs) was cultured under the normal or acidic medium to evaluate the alteration of morphology, migration, permeability, and EndMT markers. Microarray assay was adopted to analyze the differential expression of miRNAs in the acidity-treated HUVECs. Gain- and loss- of function experiments were performed to evaluate the functional role of miRNA-548ac on acidity-induced EndMT of HUVECs. Luciferase reporter and Chromatin-immunoprecipitation assays were conducted to assess the downstream pathway of miRNA-548ac in acidity-induced EndMT of HUVECs.ResultsOur results showed that HUVECs demonstrated mesenchymal transition under acidic conditions with the increase of migration, permeability, and expression of α-SMA and Vimentin, but the expression of vascular endothelial cadherin (VE-cadherin) and CD31 were reduced. In addition, the acidity-treated HUVECs remarkably facilitated the transmigration of pancreatic cancer cells. The expression of miRNA-548ac was significantly decreased in the acidity-treated HUVECs. Moreover, overexpression of miR-548ac inhibited the EndMT of HUVECs and consequently impeded the transmigration of pancreatic cancer cells. The miR-548ac inhibited the expression of YB-1 by binding to the 3’UTR of its mRNA, and YB-1 promoted the translation of Snail which was a critical regulator of EndMT. What’s more, Snail transcriptionally inhibited the expression of miR-548ac through binding to the promoter of its host gene.ConclusionsOur data implicated that the acidic microenvironment promoted the EndMT of HUVECs by the miR-548ac/YB-1/Snail axis, which could contribute to the metastasis of pancreatic cancer.

Involvement of Müller Glial Autoinduction of TGF-β in Diabetic Fibrovascular Proliferation Via Glial-Mesenchymal Transition.

PurposeMüller glial–mesenchymal transition (GMT) is reported as the fibrogenic mechanism promoted by TGF-β–SNAIL axis in Müller cells transdifferentiated into myofibroblasts. Here we show the multifaceted involvement of TGF-β in diabetic fibrovascular proliferation via Müller GMT and VEGF-A production.MethodsSurgically excised fibrovascular tissues from the eyes of patients with proliferative diabetic retinopathy were processed for immunofluorescence analyses of TGF-β downstream molecules. Human Müller glial cells were used to evaluate changes in gene and protein expression with real-time quantitative PCR and ELISA, respectively. Immunoblot analyses were performed to detect TGF-β signal activation.ResultsMüller glial cells in patient fibrovascular tissues were immunopositive for GMT-related molecular markers, including SNAIL and smooth muscle protein 22, together with colocalization of VEGF-A and TGF-β receptors. In vitro administration of TGF-β1/2 upregulated TGFB1 and TGFB2, both of which were suppressed by inhibitors for nuclear factor-κB, glycogen synthase kinase-3, and p38 mitogen-activated protein kinase. Of the various profibrotic cytokines, TGF-β1/2 application exclusively induced Müller glial VEGFA mRNA expression, which was decreased by pretreatment with small interfering RNA for SMAD2 and inhibitors for p38 mitogen-activated protein kinase and phosphatidylinositol 3-kinase. Supporting these findings, TGF-β1/2 stimulation to Müller cells increased the phosphorylation of these intracellular signaling molecules, all of which were also activated in Müller glial cells in patient fibrovascular tissues.ConclusionsThis study underscored the significance of Müller glial autoinduction of TGF-β as a pathogenic cue to facilitate diabetic fibrovascular proliferation via TGF-β–driven GMT and VEGF-A–driven angiogenesis.

TOPK mediates hypoxia-induced epithelial-mesenchymal transition and the invasion of nonsmall-cell lung cancer cells via the HIF-1α/snail axis

Hypoxia has been suggested to induce epithelial-mesenchymal transition (EMT) in various cancer types via the transcription factor hypoxia-inducible factor-1 alpha (HIF-1α). Here, we demonstrated that TOPK upregulates EMT and the invasion of H460 nonsmall-cell lung cancer cells through the induction of the HIF-1α/Snail axis and hypoxic signaling. The expression of endogenous TOPK, phosphorylated TOPK, HIF-1α and Snail was significantly increased upon hypoxia exposure, but TOPK depletion markedly abrogated the induced mRNA and protein levels of HIF-1α and Snail. Interestingly, TOPK knockdown restored the hypoxia-induced suppression of E-cadherin and diminished hypoxia-induced N-cadherin expression. In addition, Snail depletion suppressed hypoxia-induced N-cadherin expression, which was attenuated by TOPK knockdown. Moreover, knockdown of Snail decreased hypoxia-induced nonsmall-cell lung cancer cell migration and invasion, which were suppressed by TOPK depletion. In summary, we conclude that TOPK positively regulates HIF-1α expression through hypoxia signaling and thereby promotes Snail expression, leading to EMT and the invasion of nonsmall-cell lung cancer cells. These findings suggest that TOPK plays a critical role as a novel mediator of hypoxia signaling that regulates nonsmall-cell lung cancer development.

β-Mangostin inhibits the metastatic power of cervical cancer cells attributing to suppression of JNK2/AP-1/Snail cascade.

β-Mangostin is a natural mangostin with potent anticancer activity against various cancers. In this study, we further explored the anticancer activity of β-mangostin on cervical cancer cells. β-Mangostin did not affect cell viability and cell cycle distribution in HeLa and SiHa cells; however, it dose-dependently inhibited the migration and invasion of both the human cervical cancer cell lines. In addition, we observed that β-mangostin suppressed the expression of integrin αV and β3 and the downstream focal adhesion kinase/Src signaling. We also found that Snail was involved in the β-mangostin inhibited cell migration and invasion of HeLa cell. Then, our findings showed that β-mangostin reduced both nuclear translocation and messenger RNA expression of AP-1 and demonstrated that AP-1 could target to the Snail promoter and induce Snail expression. Kinase cascade analysis and reporter assay showed that JNK2 was involved in the inhibition of AP-1/Snail axis by β-mangostin in HeLa cells. These results indicate that β-mangostin can inhibit the mobility and invasiveness of cervical cancer cells, which may attribute to the suppression of both integrin/Src signaling and JNK2-mediated AP-1/Snail axis. This suggests that β-mangostin has potential antimetastatic potential against cervical cancer cells.

Propofol Reduces Epithelial to Mesenchymal Transition, Invasion and Migration of Gastric Cancer Cells through the MicroRNA-195-5p/Snail Axis.

BackgroundGastric cancer (GC) is a life-threating malignancy worldwide. Accumulating studies suggest propofol has anti-tumor functions in addition to the anesthetic effect. This study aimed to figure out the effects of propofol treatment in GC development.Material/MethodsHuman GC SGC-7901 and NCI-N87 cells were treated with different doses of propofol. Then the invasion and migration of GC cells was measured. SGC-7901 cells following 10 μM propofol treatment were applied in the following experiments. MicroRNAs (miRNAs) with differential expression in cells with or without propofol treatment were analyzed. Expression of miR-195-5p, Snail, vimentin and E-cadherin in SGC-7901 cells was measured, and then loss-of-function of miR-195-5p and gain-of-function of Snail were performed. Target relation between miR-195-5p and Snail was confirmed using luciferase assay. Xenograft tumor was induced in nude mice to identify the effect of propofol on GC in vivo.ResultsPropofol reduced epithelial to mesenchymal transition (EMT), invasion and migration of GC cells in a dose-dependent manner. Propofol elevated miR-195-5p expression but reduced Snail expression, and it reduced vimentin but increased E-cadherin expression in SGC-7901 cells. miR-195-5p directly bound to Snail. miR-195-5p inhibition or Snail promotion reversed propofol-inhibited malignant behaviors of SGC-7901 cells. In vitro results were reproduced in in vivo experiments.ConclusionsOur study found that propofol could inhibit EMT, invasion, and migration of GC cells by promoting miR-195-5p expression and suppressing Snail expression. This study may provide novel insights in GC treatment.

WISP-1 Promotes Epithelial-Mesenchymal Transition in Oral Squamous Cell Carcinoma Cells Via the miR-153-3p/Snail Axis.

Around half of all patients with oral squamous cell carcinoma (OSCC) present with lymphatic metastasis, a strong predictor of poor survival. Improving survival rates depends on preventing the first step in the “invasion-metastasis cascade,” epithelial-to-mesenchymal transition (EMT), and developing antilymphangiogenesis therapies that antagonize lymphatic metastasis. The extracellular matrix-related protein WISP-1 (WNT1-inducible signaling pathway protein-1) stimulates bone remodeling and tumor progression. We have previously reported that WISP-1 promotes OSCC cell migration and lymphangiogenesis induced by vascular endothelial growth factor C (VEGF-C). This investigation sought to determine the role of WISP-1 in regulating EMT in OSCC. Our analysis of oral cancer data from The Cancer Genome Atlas (TCGA) database revealed significant and positive associations between levels of WISP-1 expression and clinical disease stage, as well as regional lymph node metastasis. We also found higher levels of WISP-1 expression in serum samples obtained from patients with OSCC compared with samples from healthy controls. In a series of in vitro investigations, WISP-1 activated EMT signaling via the FAK/ILK/Akt and Snail signaling transduction pathways and downregulated miR-153-3p expression in OSCC cells. Our findings detail how WISP-1 promotes EMT via the miR-153-3p/Snail axis in OSCC cells.

New Functions of Long Noncoding RNAs during EMT and Tumor Progression.

Epithelial-to-mesenchymal transition (EMT) is orchestrated by aberrant activation of EMT transcription factors and expression of downstream target genes resulting in increased tumor initiation, metastatic potential, and therapeutic resistance. In this issue of Cancer Research, Wang and colleagues show that the long noncoding RNA SATB2-AS1 is dysregulated in colorectal carcinoma and correlates with poor survival and prognosis. They present a new functional mechanism illustrating how the SATB2-AS1-SATB2-Snail axis is involved in epigenetic modification that regulates colorectal carcinoma progression.See related article by Wang et al., p. 3542.

Reactive oxygen species induce epithelial‑mesenchymal transition, glycolytic switch, and mitochondrial repression through the Dlx‑2/Snail signaling pathways in MCF‑7 cells.

Reactive oxygen species (ROS) are important cellular second messengers involved in various aspects of cell signaling. ROS are elevated in multiple types of cancer cells, and this elevation is known to be involved in pathological processes of cancer. Although high levels of ROS exert cytotoxic effects on cancer cells, low levels of ROS stimulate cell proliferation and survival by inducing several pro‑survival signaling pathways. In addition, ROS have been shown to induce epithelial‑mesenchymal transition (EMT), which is essential for the initiation of metastasis. However, the precise mechanism of ROS‑induced EMT remains to be elucidated. In the present study, it was indicated that ROS induce EMT by activating Snail expression, which then represses E‑cadherin expression in MCF‑7 cells. It was further indicated that distal‑less homeobox‑2 (Dlx‑2), one of the human Dlx gene family proteins involved in embryonic development, acts as an upstream regulator of ROS‑induced Snail expression. It was also revealed that ROS treatment induces the glycolytic switch, a phenomenon whereby cancer cells primarily rely on glycolysis instead of mitochondrial oxidative phosphorylation for ATP production, even in the presence of oxygen. In addition, ROS inhibited oxidative phosphorylation and caused cytochromec oxidase inhibition via the Dlx‑2/Snail cascade. These results suggest that ROS induce EMT, the glycolytic switch and mitochondrial repression by activating the Dlx‑2/Snail axis, thereby playing crucial roles in MCF‑7 cancer cell progression.

Abstract 1036: WISP-1 promotes epithelial-mesenchymal transition in oral squamous cell carcinoma via the miR-153-3p/Snail axis

Abstract Oral squamous cell carcinoma (OSCC) is the most common oral malignancy and approximately 50% of patients with OSCC present with regional regional lymph node metastasis. The first step in the ‘invasion-metastasis cascade’ is the phenomenon of epithelial-to-mesenchymal transition (EMT), whereby epithelial cells of the primary tumor lose their apical-basal polarity and transition to a mesenchymal phenotype, gaining the ability to migrate and invade basement membrane and blood vessels. WNT1-inducible signaling pathway protein-1 (WISP-1/CCN4) is an extracellular matrix-related protein that belongs to the CCN family, capable of stimulating bone remodeling and tumor progression. Our previous research showed that WISP-1 promotes cell migration and VEGF-C-dependent lymphangiogenesis in OSCC cells. This investigation further examined the role of WISP-1 in regulating EMT. First, we analyzed data from the cancer genome atlas (TCGA) database, to determine the clinical importance of WISP-1 in oral cancer. We found that WISP-1 expression was significantly associated with clinical disease stage and regional lymph node metastasis. Moreover, we found higher levels of WISP-1 expression in serum samples obtained from 62 patients with OSCC compared with samples from healthy controls. Treatment of the OSCC cell line SCC4 with WISP-1 increased EMT by downregulating expression of the epithelial marker E-cadherin and upregulating the mesenchymal marker Snail. We also observed that WISP-1 regulated the expression of EMT markers via the FAK, ILK and AKT signal transduction pathways. Computational analysis confirmed that microRNA (miR)-29c directly targets the 3′ untranslated region (3′ UTR) of Snail. Treatment of SCC4 cells with WISP-1 inhibited miR-29c expression, while miR-29c mimic prevented WISP-1-induced enhancement of Snail expression, revealing an inverse correlation between miR-29c and Snail expression during WISP-1 treatment. Our findings provide insight into WISP-1-mediated regulation of EMT in OSCC. Citation Format: An-Chen Chang, Chih-Hsin Tang. WISP-1 promotes epithelial-mesenchymal transition in oral squamous cell carcinoma via the miR-153-3p/Snail axis [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 1036.

MicroRNA-101 Targets CXCL12-Mediated Akt and Snail Signaling Pathways to Inhibit Cellular Proliferation and Invasion in Papillary Thyroid Carcinoma.

Escalating evidence suggests that microRNA-101 (miR-101) is implicated in the development and progression of various cancers, including papillary thyroid carcinoma (PTC). However, the biological function and molecular mechanisms of miR-101 in PTC are still unclear. In this study, we demonstrated that miR-101 expression was significantly decreased in PTC tissues and cell lines. Clinically, a low level of miR-101 was positively associated with advanced histological stages and lymph node and distant metastases. The expression of CXCL12 was negatively correlated with miR-101 level in PTC. CXCL12 was validated as a direct target of miR-101 in PTC cells. Functional experiments proved that miR-101 markedly reduced the proliferation, apoptosis escape, migration, and invasion of PTC cells. Moreover, CXCL12 restoration rescued the suppressive effects of miR-101 on PTC cells by activating Akt- and EMT-associated signaling pathways. Overall, miR-101 exerts oncostatic effects on PTC by downregulating CXCL12 and repressing its downstream Akt and Snail signaling pathways, suggesting that miR-101/CXCL12/Akt or Snail axis may serve as a potential therapeutic target for PTC.

NETO2 promotes invasion and metastasis of gastric cancer cells via activation of PI3K/Akt/NF-\u03baB/Snail axis and predicts outcome of the patients

Aberrant expression of neuropilin and tolloid-like 2 (NETO2) has been observed during the progression of some human carcinomas. However, the expression pattern and clinical relevance of NETO2 in gastric cancer (GC) remain to be elucidated. In this study, we found that NETO2 expression was higher in GC tissues compared with paired non-cancerous tissues. Moreover, the expression of NETO2 was positively correlated with clinical stage, invasion depth, lymph node metastasis, and tumor size, but inversely correlated with overall and disease-free survival rates. Cox regression analysis identified NETO2 as an independent prognostic indicator for GC patients. Overexpression of NETO2 facilitated migration and invasion of GC cells in vitro and metastasis in vivo in association with induction of epithelial-mesenchymal transition. Conversely, knockdown of NETO2 had the opposite effects. Mechanistically, silencing NETO2 reduced the phosphorylation of PI3K, AKT, and NF-κB p65 as well as the expression of Snail, whereas NETO2 overexpression achieved the opposite results. Furthermore, we identified TNFRSF12A as a mediator for NETO2 to activate PI3K/AKT/NF-κB/Snail axis. Collectively, our results demonstrate that NETO2 promotes invasion and metastasis of GC cells and represents a novel prognostic indicator as well as a potential therapeutic target in GC.

CircRNA_0084043 promote malignant melanoma progression via miR-153-3p/Snail axis

Circular RNAs (circRNAs) has been found to play an important role in the regulation of multiple diseases, and participate in cancer development. However, the role of circRNA in malignant melanoma has not been reported. In this study, human circRNA microarray was used to screen the dysregulated circRNA in melanoma. We found that circRNA_0084043 was significantly up-regulated in melanoma tissue, and the result was replicated in a larger sample size. High circRNA_0084043 expression was an independent risk factor of overall survival in melanoma patients. circRNA_0084043 promotes melanoma cell proliferation, invasion and migration. Bioinformatics and luciferase reporter assays confirmed that circRNA_0084043 directly binds to miR-153-3p, and Snail is directly targeted by miR-153-3p. We also demonstrated that circRNA_0084043 may act as the sponge of miR-153-3p to up-regulate Snail expression, and consequently function as an oncogene in melanoma. Overall, this result elucidates a new mechanism for circRNA_0084043 in melanoma development and provides a potential therapeutic target for melanoma patients.

Abstract LB-274: Transcriptional factor Snail and MMP-9 signaling axis amplifies Neu1-MMP-9 crosstalk on EGF receptor to regulate tumor neovascularization, growth and metastasis in mouse model of human ovarian carcinoma

Abstract Snail is a transcriptional factor and repressor of E-cadherin. It is well known for its role in cellular invasion, epithelial to mesenchymal transition (EMT), tumor progression and metastases. shRNA lentiviral knockdown (KD) of Snail and its associated member Slug in human A2780 ovarian epithelial carcinoma cell line was investigated to identify its role in tumor neovascularization. Live cell sialidase, WST-1 and immunohistochemistry assays were used to evaluate sialidase activity, cell proliferation and the expression levels of tumor E-, N- and VE-cadherins, and host endothelial CD31+(PECAM-1) cells in A2780, A2780 Snail KD and A2780 Slug KD tumors grown in RAGxCγ double mutant mice. Oseltamivir phosphate (OP), anti-Neu1and MMP-9 specific inhibitor blocked Neu1 activity associated with epidermal growth factor (EGF) stimulated A2780 cells. Silencing Snail and Slug in A2780 cells abrogated the Neu1 activity following EGF stimulation of the cells compared to parental cells. OP treatment of A2780 and cisplatin-resistant A2780cis cells reproducibly and dose-dependently abated the cell viability with a LD50 of 7 and 4μM, respectively. Heterotopic xenografts of A2780 tumors developed abnormal bloody tumor vasculature in these mice. Anti-tumor activity of OP treatment at 50 mg/kg daily I.P. did not significantly impede A2780 tumor growth rate in a time-to-progression, but significantly cause a reduction of lung metastases compared with the untreated and OP 30 mg/kg cohorts. Silencing Snail in A2780 cells, but not the Slug KD member completely abrogated the tumor vascularization, growth and spread to the lungs in these mice. A2780 and A2780 Slug KD tumors expressed high levels of human N- and VE-cadherins, and host CD31+ endothelial cells. These findings uncover a novel organizational signaling platform connecting the Snail-MMP-9 signaling axis in amplifying the Neu1-MMP-9 cross-talk in regulating EGF receptors, tumor neovascularization, growth and invasiveness. Citation Format: Myron R. Szewczuk, Samar Abdulkhalek, Olivia Geen, Lacey Brodhagen, Fiona Haxho, Farah Alghamdi, Stephanie Allison, Duncan Simmons, Leah O'Shea, Ronald Neufeld. Transcriptional factor Snail and MMP-9 signaling axis amplifies Neu1-MMP-9 crosstalk on EGF receptor to regulate tumor neovascularization, growth and metastasis in mouse model of human ovarian carcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-274. doi:10.1158/1538-7445.AM2015-LB-274

Overexpression of c-myc induces epithelial mesenchymal transition in mammary epithelial cells

The c-myc gene is frequently overexpressed in human breast cancer and its target genes are involved in tumorigenesis. Epithelial mesenchymal transitions (EMT), where cells undergo a developmental switch from a polarized epithelial phenotype to a highly motile mesenchymal phenotype, are associated with invasion and motility of cancer cells. Basal E-cadherin expression was down-regulated in c-myc overexpressing MCF10A (c-myc-MCF10A) cells compared to GFP-overexpressing MCF10A (GFP-MCF10A) cells, while N-cadherin was distinctly increased in c-myc-MCF10A cells. Given that glycogen synthase kinase-3β (GSK-3β) and the snail axis have key roles in E-cadherin deregulation during EMT, we investigated the role of GSK-3β/snail signaling pathways in the induction of EMT by c-myc overexpression. In contrast to GFP-MCF10A cells, both the transcriptional activity and the ubiquitination-dependent protein stability of snail were enhanced in c-myc-MCF10A cells, and this was reversed by GSK-3β overexpression. We also found that c-myc overexpression inhibits GSK-3β activity through activation of extracellular signal-regulated kinase (ERK). Inhibition of ERK by dominant negative mutant transfection or chemical inhibitor significantly suppressed snail gene transcription. These results suggest that c-myc overexpression during transformation of mammary epithelial cells (MEC) is involved in EMTs via ERK-dependent GSK-3β inactivation and subsequent snail activation.