Snail Expression Research Articles

Synthesis and Antitumor Activity of Brominated-Ormeloxifene (Br-ORM) against Cervical Cancer.

Aberrant regulation of β-catenin signaling is strongly linked with cancer proliferation, invasion, migration, and metastasis, thus, small molecules that can inhibit this pathway might have great clinical significance. Our molecular modeling studies suggest that ormeloxifene (ORM), a triphenylethylene molecule that docks with β-catenin, and its brominated analogue (Br-ORM) bind more effectively with relatively less energy (-7.6 kcal/mol) to the active site of β-catenin as compared to parent ORM. Herein, we report the synthesis and characterization of a Br-ORM by NMR and FTIR, as well as its anticancer activity in cervical cancer models. Br-ORM treatment effectively inhibited tumorigenic features (cell proliferation and colony-forming ability, etc.) and induced apoptotic death, as evident by pronounced PARP cleavage. Furthermore, Br-ORM treatment caused cell cycle arrest at the G1-S phase. Mechanistic investigation revealed that Br-ORM targets the key proteins involved in promoting epithelial-mesenchymal transition (EMT), as demonstrated by upregulation of E-cadherin and repression of N-cadherin, Vimentin, Snail, MMP-2, and MMP-9 expression. Br-ORM also represses the expression and nuclear subcellular localization of β-catenin. Consequently, Br-ORM treatment effectively inhibited tumor growth in an orthotopic cervical cancer xenograft mouse model along with EMT associated changes as compared to vehicle control-treated mice. Altogether, experimental findings suggest that Br-ORM is a novel, promising β-catenin inhibitor and therefore can be harnessed as a potent anticancer small molecule for cervical cancer treatment.

Continuous ultraviolet irradiation induces the development of irreversible resistance of melanoma cells to anticancer drugs

Introduction. Melanoma belongs to the group of the most malignant tumors characterized by aggressive growth and active metastasis. At the same time, the effectiveness of therapy, primarily targeted therapy, is largely limited by the rapid development of drug resistance.Aim. To study the effect of chronic ultraviolet (UV) irradiation on the formation of a population of radiation-resistant melanoma cells; to study the features of cell signaling and the sensitivity of UV-resistant melanoma cells to the antitumor drugs.Materials and methods. The experiments were carried out on in vitro cultured A375 melanoma cells. Cells were cultured in a standard DMEM + 10 % FBS medium; cell growth rate was analyzed using the MTT assay; cell survival after irradiation was analyzed using a colony-forming test. Determination of the transcriptional activity of the estrogen receptor (ER) was performed by reporter analysis upon transfection into cells of a plasmid containing the luciferase reporter gene controlled by estrogen responsive element. The immunoblotting method was used to analyze the expression of cellular proteins; comparative analysis of ERα and ERβ expression was performed by immunofluorescent method.Results. Long-term UV irradiation leads to the formation of a UV-resistant subpopulation of A375 melanoma cells, which is characterized by decreased sensitivity to targeted (vemurafenib) and hormonal (tamoxifen) drugs, increased expression of Snail, an activator of the epithelial-mesenchymal transition, and in the absence of noticeable changes in the expression of PI3K / mTOR signaling. Metformin reduces Snail expression in both parental and UV-resistant A375 cells and enhances the cytostatic effect in combination with vemurafenib or tamoxifen.Conclusion. The data obtained demonstrate a decrease in the sensitivity of melanoma cells to targeted drugs under the long-term exposure to UV. The ability of metformin to potentiate the action of targeted drugs and inhibit Snail allows us to consider metformin not only as an antitumor agent, but also as a potential inhibitor of the epithelial-mesenchymal transition.

Design of hypoxia responsive CRISPR-Cas9 for target gene regulation

The CRISPR–Cas9 system is a widely used gene-editing tool, offering unprecedented opportunities for treating various diseases. Controlling Cas9/dCas9 activity at specific location and time to avoid undesirable effects is very important. Here, we report a conditionally active CRISPR–Cas9 system that regulates target gene expression upon sensing cellular environmental change. We conjugated the oxygen-sensing transcription activation domain (TAD) of hypoxia-inducing factor (HIF-1α) with the Cas9/dCas9 protein. The Cas9-TAD conjugate significantly increased endogenous target gene cleavage under hypoxic conditions compared with that under normoxic conditions, whereas the dCas9-TAD conjugate upregulated endogenous gene transcription. Furthermore, the conjugate system effectively downregulated the expression of SNAIL, an essential gene in cancer metastasis, and upregulated the expression of the tumour-related genes HNF4 and NEUROD1 under hypoxic conditions. Since hypoxia is closely associated with cancer, the hypoxia-dependent Cas9/dCas9 system is a novel addition to the molecular tool kit that functions in response to cellular signals and has potential application for gene therapeutics.

Eriocitrin inhibits epithelial-mesenchymal transformation (EMT) in lung adenocarcinoma cells via triggering ferroptosis.

Lung adenocarcinoma (LUAD) is the most prevalent pathological subtype of non-small cell lung cancer (NSCLC), characterized by a high propensity for relapse and metastasis due to epithelial-mesenchymal transition (EMT) of cancer cells. Ferroptosis, a newly discovered regulated cell death modality, is interconnected with the EMT process in certain cancers. Eriocitrin, a natural flavonoid compound, exerts anti-inflammatory and anticancer effects. The aim of this study is to investigate the potential inhibitory effect of eriocitrin on lung adenocarcinoma metastasis and explore whether its underlying mechanism involves ferroptosis induction in cancer cells. The CCK8 assay and wound healing assay and transwell were conducted to determine the cell viability and migration ability of A549 and H1299 cells, respectively. EMT process was assessed by western blot and RT-PCR to detect protein and mRNA levels of EMT markers. ROS and cell iron were measured to determine ferroptosis level. Eriocitrin treatment significantly inhibited cell viability and migration ability in a concentration-dependent manner. Furthermore, eriocitrin administration for 24 hours resulted in enhanced expression of E-cadherin, while downregulating vimentin, N-cadherin and snail expression, indicating marked repression of the EMT process. Additionally, eriocitrin significantly induced ferroptosis in A549 and H1299 cells, as evidenced by increased ROS levels, downregulation of Nrf-2, SLC7A11 and GPX4 expression, and enhanced cellular iron accumulation. Moreover, pretreatment with the ferroptosis inhibitor ferrostatin-1 effectively abrogated the inhibitory effects of eriocitrin on EMT. Our findings further support the anti-cancer properties of eriocitrin, as evidenced by its ability to inhibit the EMT process in LUAD cells, which is partially mediated through induction of ferroptosis in cancer cells.

Exogenous Ang-(1-7) inhibits autophagy via HIF-1α/THBS1/BECN1 axis to alleviate chronic intermittent hypoxia-enhanced airway remodelling of asthma

Obstructive sleep apnoea (OSA)-induced chronic intermittent hypoxia (CIH) has been considered a risk factor for severe asthma. Airway remodelling, which could be modulated by autophagy, plays a key role in severe asthma. However, the extent of autophagy’s involvement in CIH-potentiated airway remodelling remains largely unexplored. Furthermore, we had found that angiotensin-(1-7) [Ang-(1-7)] has therapeutic effects on airway remodelling in asthma, but the underlying mechanism is either unclear. This study aimed to explore how CIH aggravates asthma and mechanism of protective effects of Ang-(1-7) on airway remodelling, with a focus on autophagy. We observed that CIH promoted epithelial-to-mesenchymal transition (EMT), indicated by elevated EMT and fibrotic markers such as Snail and Collagen IV, both in vitro and in vivo. CIH intensified cell autophagy, evident from increased LC3B expression and reduced p62 levels. Ang-(1-7) reversed the CIH-enhanced expression of Snail, Collagen IV, and LC3B. To explore how CIH enhanced autophagy in cellular and animal model of asthma, overexpression of hypoxia-inducible factor 1-alpha (HIF-1α) and Thrombospondin 1 (THBS1) were identified in CIH-exposure mice lung compared with normal mice lung tissues from the GEO database. Finally, through chromatin immunoprecipitation and immunoprecipitation assays, we verified that Ang-(1-7) inhibits CIH-induced binding of HIF-1α to the promoter of THBS1, and also disrupts the protein-protein interaction between THBS1 and the autophagy-associated protein Beclin 1 (BECN1), ultimately leading to autophagy inhibition. Our findings suggest that exogenous Ang-(1-7) can inhibit autophagy via HIF-1α/THBS1/BECN1 axis, thereby alleviating CIH-enhanced airway remodelling in asthma. These findings imply the potential therapeutic effect of Ang-(1-7) in asthma with OSA.

Jiawei Danxuan Koukang Alleviates Arecoline Induced Oral Mucosal Lesions: Network Pharmacology and the Combined Ultra-High Performance Liquid Chromatography (UPLC) and Mass Spectrometry (MS).

Arecoline is one of the main toxic components of arecoline to cause oral mucosal lesions or canceration, which seriously affects the survival and life quality of patients. This study analyzed the mechanism of Jiawei Danxuan Koukang (JDK) in alleviating arecoline induced oral mucosal lesions, to provide new insights for the treatment of oral submucosal fibrosis (OSF) or cancerosis. Metabolomics was applied to analyze the composition of JDK and serum metabolites. The active ingredients of JDK were analyzed by the combined ultra-high performance liquid chromatography and mass spectrometry. The target network of JDK, metabolites and OSF was analyzed by network pharmacology, and molecular docking. Oral mucosal lesions and fibrosis were analyzed by HE and Masson staining. Cell differentiation, proliferation and apoptosis were detected. The expressions of α-SMA, Collagen I, Vimentin, Snail, E-cadherin, AR and NOTCH1 were detected by Western blot. Arecoline induced the gradual atrophy and thinning of rat oral mucosal, collagen accumulation, the increase expressions of fibrosis-related proteins and Th17/Treg ratio. JDK inhibited arecoline-induced oral mucosal lesions and inflammatory infiltration. Arecoline induced changes of serum metabolites in Aminoacyl-tRNA biosynthesis, Alanine, aspartate and glutamate metabolism and Arginine biosynthesis pathways, which were reversed by M-JDK. Quercetin and AR were the active ingredients and key targets of JDK, metabolites and OSF interaction. Arecoline promoted the expression of AR protein, and the proliferation of oral fibroblasts. Quercetin inhibited the effect of arecoline on oral fibroblasts, but was reversed by AR overexpression. Arecoline induced NOTCH1 expression in CAL27 and SCC-25 cells, and promoted cell proliferation, but was reversed by M-JDK or quercetin. JDK improved the arecoline-induced OSF and serum metabolite functional pathway. Quercetin targeted AR protein to improve arecoline-induced OSF. JDK and quercetin inhibited arecoline-induced NOTCH1 protein expression in CAL27 and SCC-25 cells to play an anti-oral cancer role.

Extracellular pyruvate kinase M2 induces cell migration through p-Tyr42 RhoA-mediated superoxide generation and epithelial-mesenchymal transition

In the tumor microenvironment (TME), communication between cancer cells and tumor-associated macrophages (TAMs) through secreted extracellular proteins promotes cancer progression. Here, we observed that co-culturing cancer cells (4T1) and macrophage cells (Raw264.7) significantly enhanced superoxide production in both cell types. Using MALDI-TOF, we identified PKM2 as a highly secreted protein by Raw264.7 cells and bone marrow-derived monocytes. The extracellular recombinant PKM2 protein not only enhanced cancer cell migration and invasion but also increased superoxide production. Additionally, PKM2 was found to associate with the cell surface, and its binding to integrin α5/β1 receptor was inhibited by antibodies specifically targeting it.Furthermore, we investigated downstream signaling pathways involved in PKM2-induced superoxide production. We found that knock-down of RhoA and p47phox using siRNAs effectively abolished superoxide generation in response to extracellular PKM2. Notably, extracellular PKM2 triggered the phosphorylation of p47phox at Ser345 residue and RhoA at Tyr42 residue (p-Tyr42 RhoA). Moreover, extracellular PKM2 exerted regulatory control over the expression of key epithelial-mesenchymal transition (EMT) markers, including ZEB1, Snail1, vimentin, and E-cadherin. Interestingly, p-Tyr42 RhoA translocated to the nucleus, where it bound to the ZEB1 promoter region. In light of these findings, we propose that extracellular PKM2 within the TME plays a critical role in tumorigenesis by promoting cancer cell migration and invasion through RhoA/p47phox signaling pathway.

Novel polysaccharide identified from Cortinarius purpurascens demonstrated anti-fibrosis effects in cardiac fibroblasts

BackgroundMyocardial fibrosis is the primary pathological characteristic of cardiovascular disease, which can cause impaired cardiac function, severe heart failure, arrhythmia, and even sudden cardiac death. Cardiovascular diseases in Inner Mongolia can be prevented by using Cortinarius purpurascens. C. purpurascens polysaccharide (CPP) is the main active component of C. purpurascens. However, the action mechanism of CPP on myocardial fibrosis remain unclear. ObjectivesWe aimed isolate CPPs from purpurascens and to explore the mechanism of CPPs in myocardial fibrosis treatment so as to obtain a new experimental basis for the use of CPPs in the treatment of CVDs. MethodsCPPs were isolated and purified through water extraction, ethanol precipitation, deproteinization, and column chromatography and identified by gel permeation chromatography and Fourier-transformed infrared spectroscopy (FT-IR). The cell model of myocardial fibrosis induced by TGF-β1 was further treated with CPPs of different molecular weights. The expression levels of fibrosis proteins Col I, Col III and α-Smooth muscle actin (α-SMA) were detected by Western blotting, immunofluorescence assay or enzyme-linked immunosorbent assay (ELISA), and the expressions of Snail 1, Snail 2, and Slug were determined qRT-PCR. Western blotting was also performed to evaluate the expression level of TGF-β/Smad signaling-related protein. ResultsWe extracted and purified three CPPs, that TGF-β1 treatment increased the expression of Col I, Col III, α-SMA, and TGF-β/Smad signaling pathway-related proteins, while CPPs treatment significantly decreased the expression of Col I, Col III, and α-SMA proteins. Both the activation of the TGF-β/Smad signaling pathway and the mRNA expression of fibrosis transcriptional regulators such as Snail 1, Snail 2, and Slug were inhibited. ConclusionsCPPs can effectively inhibit the abnormal proliferation of CFs and myocardial fibrosis induced by TGF-β1 in mice. Therefore, CPPs may be worthy of further analyses as a novel therapeutic agent for cardiovascular diseases.

LRRC59 serves as a novel biomarker for predicting the progression and prognosis of bladder cancer.

Leucine-rich repeat-containing protein 59 (LRRC59) is an endoplasmic reticulum membrane protein involved in various cancers, but its role in bladder cancer (BC) has not been reported. The aim of the present study was to investigate the role of LRRC59 protein in BC progression and prognosis. The expression profile and clinical significance were retrieved from BC patients in the Cancer Genome Atlas database. The methylation status of LRRC59 was analyzed by UALCAN and MethSurv databases. Potential signaling pathways and biological functions were explored by functional enrichment analysis. Immunocyte infiltration was evaluated by CIBERSORT analysis. The prognostic value of LRRC59 was evaluated by Kaplan-Meier and Cox regression analyses. Overall survival (OS) was predicted by the nomogram plot established in this study. LRRC59 expression in 10 pairs BC and adjacent noncancerous tissues were analyzed by immunohistochemistry (IHC). Cell proliferation, migration, and invasion were detected by CCK8, colony formation assay, transwell assay, and cell scratch assay, respectively. Proteins related to epithelial-mesenchymal transition and apoptosis were detected by western blot. LRRC59 overexpression significantly decreased OS, disease-specific survival, and progress-free interval of BC patients. LRRC59 was a prognostic marker for OS and its hypomethylation status signified a poor prognosis. LRRC59 overexpression was correlated with infiltration of resting memory CD4 T cells, memory activated CD4 T cells, resting NK cells, macrophages M0, M1, M2, and neutrophils. IHC showed that the LRRC59 expression in BC tissue was significantly higher than that in adjacent noncancerous tissue. Knockdown of LRRC59 expression inhibited the proliferation of BC cells and reduced their migratory ability. Western blot showed that Snail and vimentin protein expressions decreased, while E-cadherin expressions increased. LRRC59 expression can predict the outcome of BC independently and serve as a new biomarker for diagnosis.

High Salt Promotes Inflammatory and Fibrotic Response in Peritoneal Cells.

Recent studies draw attention to how excessive salt (NaCl) intake induces fibrotic alterations in the peritoneum through sodium accumulation and osmotic events. The aim of our study was to better understand the underlying mechanisms. The effects of additional NaCl were investigated on human primary mesothelial cells (HPMC), human primary peritoneal fibroblasts (HPF), endothelial cells (HUVEC), immune cells (PBMC), as well as ex vivo on peritoneal tissue samples. Our results showed that a high-salt environment and the consequently increased osmolarity increase the production of inflammatory cytokines, profibrotic growth factors, and components of the renin-angiotensin-aldosterone system, including IL1B, IL6, MCP1, TGFB1, PDGFB, CTGF, Renin and Ace both in vitro and ex vivo. We also demonstrated that high salt induces mesenchymal transition by decreasing the expression of epithelial marker CDH1 and increasing the expression of mesenchymal marker ACTA2 and SNAIL1 in HPMCs, HUVECs and peritoneal samples. Furthermore, high salt increased extracellular matrix production in HPFs. We demonstrated that excess Na+ and the consequently increased osmolarity induce a comprehensive profibrotic response in the peritoneal cells, thereby facilitating the development of peritoneal fibrosis.

Retracted] MicroRNA‑124 acts as a tumor‑suppressive miRNA by inhibiting the expression of Snail2 in osteosarcoma.

[This retracts the article DOI: 10.3892/ol.2018.7994.].

Effect of acetylalkannin from Arnebia euchroma on proliferation, migration, and invasion of human melanoma A375 cells

This study aimed to explore the effect and mechanism of acetylalkannin from Arnebia euchroma on the proliferation, migration, and invasion of human melanoma A375 cells. A375 cells were divided into a blank group, and low-, medium-, and high-dose acetylalkannin groups(0.5, 1.0, and 2.0 μmol·L~(-1)). The MTT assay was used to detect cell proliferation. Cell scratch and transwell migration assays were used to detect cell migration ability, and the transwell invasion assay was used to detect cell invasion ability. Western blot was used to detect the protein expression of migration and invasion-related N-cadherin, vimentin, matrix metalloproteina-se-9(MMP-9), and Wnt/β-catenin pathway-related Wnt1, Axin2, glycogen synthase kinase-3β(GSK-3β), phosphorylated GSK-3β(p-GSK-3β), β-catenin, cell cycle protein D_1(cyclin D_1), and p21. Real-time fluorescence-based quantitative polymerase chain reaction(real-time PCR) was used to detect the mRNA expression of E-cadherin, matrix metalloproteinase-2(MMP-2), N-cadherin, vimentin, β-catenin, snail-1, and CD44. MTT results showed that the cell inhibition rates in the acetylalkannin groups significantly increased as compared with that in the blank group(P<0.01). The results of cell scratch and transwell assays showed that compared with the blank group, the acetylalkannin groups showed reduced cell migration and invasion, and migration and invasion rates(P<0.05, P<0.01) and weakened horizontal and vertical migration and invasion abilities. Western blot results showed that compared with the blank group, the high-dose acetylalkannin group showed increased expression of Axin2 protein(P<0.05), and decreased expression of N-cadherin, vimentin, MMP-9, Wnt1, p-GSK-3β, β-catenin, cyclin D_1, and p21 proteins(P<0.05, P<0.01). The expression of GSK-3β protein did not change significantly. PCR results showed that the overall trend of MMP-2, N-cadherin, vimentin, β-catenin, snail-1, and CD44 mRNA expression was down-regulated(P<0.01), and the expression of E-cadherin mRNA increased(P<0.01). Acetylalkannin can inhibit the proliferation, migration, and invasion of human melanoma A375 cells, and its mechanism of action may be related to the regulation of Wnt/β-catenin signaling pathway.

Protein kinase D activity is a risk biomarker in prostate cancer that drives cell invasion by a Snail/ERK dependent mechanism

Protein kinase D (PKD) family members play controversial roles in prostate cancer (PC). Thus, PKD1 is nearly absent in advanced tumours, where PKD2 and PKD3 are upregulated. Additionally, consequences of activation of these kinases on PC progression remain largely unclear. Here, we first investigated PKD function on PC cell motility, analysing the underlying molecular mechanisms. We find a striking decrease of Snail levels after PKD inhibition followed by cell migration and invasion impairment, demonstrating an unprecedented role of PKD activity on the regulation of this key transcription factor in PC progression. Specifically, we show that PKD2 activity mediates the effects of MEK/ERK pathway on Snail expression, establishing a joint function of ERK/PKD2/Snail cascade in PC cell invasion regulation. These results led us to address the clinical relevance of the correlation between PKD2 and ERK activities with Snail abundance in samples from PC patients at different stages, analysing its impact on tumour prognosis and patients´ survival. Importantly, this is the first study defining a direct correlation between active PKD2 and Snail levels, further linked to ERK activity. We also evidence that PKD2 activity is associated with important poor prognostic factors. Thus, PC patients with the expression pattern: active PKD2high/active ERKhigh/Snailhigh exhibit increased invasiveness and metastasis, and decreased survival. Our findings provide new insights for understanding the molecular mechanisms involved in PC progression, pinpointing the combination of active PKD2 and Snail levels, with the additional measurement of active ERK, as a confident biomarker to predict clinical outcome of patients with advanced PC.

Effects of bromodomain and extra-terminal inhibitor JQ1 and interleukin-6 on breast cancer cells.

Bromodomain and extra-terminal (BET) proteins are recognized acetylated lysine of histone 4 and act as scaffolds to recruit many other proteins to promoters and enhancers of active genes, especially at the super-enhancers of key genes, driving the transcription process and have been identified as potential therapeutic targets in breast cancer. However, the efficacy of BET inhibitors such as JQ1 in breast cancer therapy is impeded by interleukin-6 (IL-6) through an as-yet-defined mechanism. We investigated the interplay between IL-6 and JQ1 in MCF-7 and MDA-MB-231 human breast cancer cells. The results demonstrate that the efficacy of JQ1 on the inhibition of cell growth and apoptosis was stronger in MDA-MB-231 cells than in MCF-7 cells. Further, MCF-7 cells, but not MDA-MB-231 cells, exhibited increased expression of CXCR4 following IL-6 treatment. JQ1 significantly reduced CXCR4 surface expression in both cell lines and diminished the effects of IL-6 pre-treatment on MCF-7 cells. While IL-6 suppressed the extension of breast cancer stem cells in MCF-7 cells, JQ1 impeded its inhibitory effect. In MCF-7 cells JQ1 increased the number of senescent cells in a time-dependent manner. Analysis of gene expression indicated that JQ1 and IL-6 synergistically increase SNAIL expression and decrease c-MYC expression in MCF-7 cells. So, the BET proteins are promising, novel therapeutic targets in late-stage breast cancers. BET inhibitors similar to JQ1 show promise as therapeutic candidates for breast cancers, especially when triple-negative breast cancer cells are increased and/or tumor-promoting factors like IL-6 exist in the tumor microenvironment.

Abstract B004: Serine promotes invasive phenotype through enhanced translation of SNAIL in ccRCC

Abstract Background: Metastatic clear cell renal cell carcinoma (ccRCC) is the most common type of renal carcinoma with a median survival rate of about 13 months and is refractory to chemotherapy. ccRCC is fundamentally a metabolic disease characterized by extensive reprogramming in amino acid metabolism. Serine is a nonessential amino acid (NEAA), and the serine biosynthetic pathway has been shown as an essential source of metabolic intermediates in many cancers. Analysis of the TCGA database reveals that ccRCC tumors have reduced levels of PHGDH, the first enzyme in the de novo serine biosynthesis pathway, and makes serine conditionally essential amino acid in these tumors. The role of serine in tumorigenesis is well established; however, its role in promoting invasive phenotype and metastasis is not explored. Methods: To gain insight into the role of nonessential amino acids in proinvaisive phenotypes, we performed wound healing and migration assays in the presence of individual NEAAs. The importance of serine synthesis for promoting invasive phenotype was also assessed by restoring PHGDH in low PHGDH ccRCC. Results: In an unbiased approach, we found that serine is required for the increased migratory and invasive phenotype in ccRCC. We also demonstrate that in low PHGDH ccRCC cells, exogenous serine is to maintain SNAIL expression and the enhanced migratory and invasive phenotype. We also show that serine depletion induces translational inhibition in low PHGDH ccRCC. Conclusion: The role of serine in tumorigenesis and cell proliferation is well established; however, its role in supporting invasive and metastatic phenotypes is previously unknown. Our data highlights a novel role for serine in promoting EMT through enhanced translational maintenance of SNAIL, which may be therapeutically targeted in ccRCC. Citation Format: Suman Karki. Serine promotes invasive phenotype through enhanced translation of SNAIL in ccRCC [abstract]. In: Proceedings of the AACR Special Conference: Advances in Kidney Cancer Research; 2023 Jun 24-27; Austin, Texas. Philadelphia (PA): AACR; Cancer Res 2023;83(16 Suppl):Abstract nr B004.

Control of gastruloid patterning and morphogenesis by the Erk and Akt signaling pathways.

Many developmental processes rely on the localized activation of receptor tyrosine kinases and their canonical downstream effectors Erk and Akt, yet the specific roles played by each of these signals is still poorly understood. Gastruloids, 3D cell culture models of mammalian gastrulation and axial elongation, enable quantitative dissection of signaling patterns and cell responses in a simplified, experimentally accessible context. We find that mouse gastruloids contain posterior-to-anterior gradients of Erk and Akt phosphorylation induced by distinct receptor tyrosine kinases, with features of the Erk pattern and expression of its downstream target Snail exhibiting hallmarks of size-invariant scaling. Both Erk and Akt signaling contribute to cell proliferation, whereas Erk activation is also sufficient to induce Snail expression and precipitate profound tissue shape changes. We further uncover that Erk signaling is sufficient to convert the entire gastruloid to one of two mesodermal fates depending on position along the anteroposterior axis. In all, these data demonstrate functional roles for two core signaling gradients in mammalian development and suggest how these modules might be harnessed to engineer user-defined tissues with predictable shapes and cell fates.

A novel long non-coding RNA XLOC_004787, is associated with migration and promotes cancer cell proliferation by downregulating mir-203a-3p in gastric cancer

BackgroundLong noncoding RNAs (lncRNAs) have been identified as important regulatory factors implicated in a wide array of diseases, including various forms of cancer. However, the roles of most lncRNAs in the progression of gastric cancer (GC) remain largely unexplored. This study investigates the biological function and underlying mechanism of a novel lncRNA, XLOC_004787 in GC.MethodsThe location of XLOC_004787 in GES-1 cells and HGC-27 cells were detected by fluorescence in situ hybridization (FISH) assay. The expression levels of XLOC_004787 were assessed using quantitative real-time fluorescence PCR (qRT-PCR) in various cell lines, including GES-1, MGC-803, MKN-45, BGC-823, SGC-7901, and HGC-27 cells. Functional assays such as Transwell migration, cell counting kit-8 (CCK-8), and colony formation experiments were employed to analyze the effects of XLOC_004787 and miR-203a-3p on cell migration and proliferation. Protein levels associated with GC in these cell lines were examined by Western blotting. The intracellular localization of β-catenin and P-Smad2/3 was assessed using immunofluorescence (IF) assay. Additionally, the interaction between XLOC_004787 and miR-203a-3p was investigated using a dual luciferase assay.ResultsXLOC_004787 was localized at both the cytoplasm and nucleus of GES-1 cells and HGC-27 cells. Compared to normal tissues and GES-1 cells, XLOC_004787 expression was significantly upregulated in GC tissues and cells, with the highest and lowest expression observed in SGC-7901 and HGC-27 cells, respectively. Furthermore, a reduced expression of XLOC_004787 was seen to inhibit migration and proliferation in SGC-7901 cells. Western blotting analysis revealed that a decrease in XLOC_004787 expression correspondingly decreased the expression of N-cadherin, mmp2, mmp9, Snail, Vimentin, β-catenin, C-myc, Cyclin D1, and TGF-β, while concurrently increasing E-cadherin expression. This was also associated with diminished expression of P-Smad2/3 in relation to Smad2/3, and reduced P-Gsk3β expression in comparison to Gsk3β. Additionally, the nuclear entry of P-Smad2/3 and β-catenin was reduced by lower XLOC_004787 expression. Amplifying XLOC_004787 expression via pcDNA_XLOC_004787 suggested a potential for cancer promotion. Notably, XLOC_004787 was found to negatively regulate mir-203a-3p expression, with potential binding sites identified between the two. Higher mir-203a-3p expression was observed to decrease migration and proliferation, and enhance E-cadherin expression. Conversely, suppression of mir-203a-3p expression suggested a potential promotion of proliferation and migration in GC cells.ConclusionsThese results suggest that XLOC_004787, found to be upregulated in GC tissues, potentially promotes proliferation and migration in GC cells. This occurs through the activation of TGF-β and Wnt/β-catenin signaling pathways and the expression of EMT-related proteins. Additionally, XLOC_004787 may influence cell migration and proliferation by modulating the signaling pathway via the adsorption and inhibition of mir-203a-3p.

HOXA13 promotes the proliferation, migration, and invasion of nasopharyngeal carcinoma HNE1 cells by upregulating the expression of Snail and MMP-2

Homeobox A13 (HOXA13) has been verified as an oncogen in some malignancies. However, its role in nasopharyngeal carcinoma (NPC) is still unclear. This study aims to explore the role of HOXA13 in NPC and its underlying mechanism. The mRNA expression of HOXA13 in NPC was obtained from the GSE53819 and GSE64634 datasets in the Gene Expression Omnibus (GEO) database. MTT, colony formation and transwell assays and xenograft tumour models were used to investigate the effects of HOXA13 on NPC HNE1 cells in vitro and in vivo. The expression of HOXA13, epithelial-mesenchymal transition-transcription factor (EMT-TF) Snail and matrix metalloproteinase 2 (MMP-2) was detected by immunohistochemistry, quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting. The results showed that HOXA13 was upregulated in NPC. Silencing HOXA13 suppressed the proliferation, migration, and invasion of HNE1 cells, which inhibited tumour growth, while overexpression of HOXA13 induced the opposite effects. In addition, the expression of Snail and MMP-2 at the transcriptional and protein levels was associated with the expression of HOXA13. In summary, our results suggest that HOXA13 plays a role as a cancer-promoting gene in NPC. The underlying mechanism may be related to the upregulation of Snail and MMP-2.

Regulation of Epithelial-Mesenchymal Transition by Cyrtopodion Scabrum: An in vitro Study against Colorectal Cancer Cells.

Natural treatment of cancer has received a lot of attention recently due to its advantages including low cost, and fewer side effects. In this study, we aimed to investigate the antimetastatic properties of Cyrtopodion scabrum, a common home gecko, through Epithelial-Mesenchymal Transition (EMT) process. Human colon cancer HCT116 cell line was selected and allocated into the following experimental groups: untreated control, vehicle control (DMSO), Retinoic acid (RA), and two treatment groups including aqueous C.scabrum Whole Extract (CWE) and C.scabrum Cell Extract (CCE) groups. The effects of the two different extracts on the viability, migration, and morphology of HCT116 cells were investigated using MTT, colony formation, and wound healing assay as well as microscopic evaluation. We also investigated the gene expression of E-cad, N-cad, and Snail genes using Real-Time PCR analysis. Our findings revealed that CWE and CCE were toxic to the HCT116 cell line with IC50 values of 590 and 680 µg/mL, respectively. Colony formation and migration ability of cancer cells were also inhibited by the two extracts, and the morphology of the cells were determined as epithelial phenotype. Moreover, the expression of N-cad and Snail were remarkably decreased in CWE and CCE, and RA groups, while E-cad didn't change significantly as compared to the control. The results suggest that C. scabrum extract (CsE) may induce its anti-cancer activity through the inhibition of cancer cell growth and the EMT process. CCE, as a valuable natural source, could be also suggested, to be used as an alternative/complementary medicine for the treatment of cancer, in clinical trials.

Aquaporin 1 is a prognostic marker and inhibits tumour progression through downregulation of Snail expression in intrahepatic cholangiocarcinoma.

Recently, some studies have suggested a link between AQP1 and cancer progression. The aim of the present study was to investigate the influence of AQP1 on the clinicopathology and prognosis of intrahepatic cholangiocarcinoma (ICC) patients. We retrospectively detected the expression of AQP1 protein in 307 patients with ICC who underwent partial hepatectomy. Western blot analysis was used to detect AQP1 protein levels in stable AQP1 overexpression and knockdown cell lines. The influence of AQP1 on the invasion and metastasis ability of ICC cells was assessed by wound-healing and Transwell assays in vitro as well as by a splenic liver metastasis model in vivo. Positive membranous AQP1 expression was identified in 34.2% (105/307) of the ICC specimens. Survival data revealed that positive AQP1 expression was significantly associated with favourable disease-free survival (DFS) and overall survival (OS) (p=0.0290 and p=0003, respectively). Moreover, high AQP1 expression inhibited the invasion and migration of ICC cells in vitro as well as inhibited liver metastasis in nude mice. Mechanistically, high AQP1 expression in ICC cells increased the levels of E-cadherin but decreased the levels of the Snail transcription factor. AQP1 expression is associated with a favourable prognosis in ICC patients. AQP1 inhibits ICC cell invasion, metastasis, and epithelial-mesenchymal transition (EMT) through downregulation of Snail expression.