Transcription Factor Snail Research Articles

As shown hesperidin suppresses TGF-β2-induced proliferation and epithelial-mesenchymal transition of retinal pigment epithelial cells.

This study investigates the potential therapeutic effects and molecular mechanisms of hesperidin treatment on cell migration and epithelial-mesenchymal transition, key stages of proliferative vitreoretinopathy (PVR). Human retinal pigment epithelial cells (ARPE-19) were treated with 10ng/ml transforming growth factor-beta 2 (TGF-β2) alone or in combination with 1.56μM hesperidin for 48h. The impact of treatment on cell migration was evaluated using a wound healing assay. Apoptosis was assessed using DNA staining. mRNA and protein expression were evaluated using real-time PCR and Western blot, respectively. Hesperidin inhibits the proliferation and transformation of the cells by inducing apoptosis and reverses the cell morphology modified by TGF-β2. Hesperidin inhibits cell migration induced by TGF-β2. Upon treatment with hesperidin, the levels of mesenchymal markers upregulated by TGF-β2, such as MMP-1, -2, -9, fibronectin, α-SMA and the transcription factors Snail, Slug and ZEB-1, were downregulated. Conversely, the epithelial marker E-cadherin is upregulated with hesperidin treatment. Additionally, TIMP-1 and TIMP-2 expression levels, which are downregulated, increase with the treatment. These results suggest that hesperidin may inhibit the migration and EMT processes of RPE cells involved in the development of PVR, indicating its potential as a therapeutic agent for treating PVR.

Abstract A041: Deciphering the crosstalk between cancer cells and stroma that drives EMT- coupled fibrosis

Abstract In the tumor microenvironment (TME) cancer cells engage dynamically with the stromal component in promoting tumor progression and metastasis formation that are accompanied by intratumor fibrosis. Extensive extracellular matrix (ECM) remodeling and stiffness boost fibrosis which co-occurs with poor immune infiltration. Very little is known about the cross-interactions between tumor cells and stroma. A better understanding of this dynamic relationship may advance the development of new therapies. Transforming growth factor (TGF)-β signaling in both cancer epithelial cells and stroma is crucial for the establishment of intratumor fibrosis that enhances tumor progression. We previously discovered that in cancer cells, TGF-β signaling synergizes with RAS signal to regulate classical EMT transcription factors (Snail, Zeb1) and fibrogenic factors (Il11, Pdgf, Ctgfb, Wisp and others). This synergistic program is required for metastasis formation in pancreatic and lung adenocarcinoma (LUAD) models. However, how this dual program is affecting the stromal population in vivo remains unknown. Additionally, the role of each TGF-β dependent fibrogenic factor in regulating the stroma is still unclear. Using metastasis in vivo assays, we tested the metastatic ability of LUAD cells wild-type in comparison to Knock-Out cells targeting each one of the TGF-β dependent fibrogenic factors. We found that these factors are drivers of metastasis. More interestingly, these fibrogenic factors have a preferential effect depending on the metastatic site, suggesting that survival of the cancer cells in a certain tissue is dependent on the stromal niche within the site, which will act in favor or not of their growth. We also deeply characterize the stroma of micro-metastatic lesions by using high-throughput multiplex immunofluorescence to analyze the whole tissue TME at single cell resolution and found that lack of any of these fibrogenic factors compromise the establishment of a robust ECM at the very beginning of the metastatic colonization, to favor immune infiltration and consequently lead to cancer cell clearance. Together, these findings shed light on the potential role of these specific fibrogenic factors in metastasis, how their absence impacts the TME and how targeting these factors could be beneficial in metastatic disease. Citation Format: Elena Spina, Liangji Li, Junho Lee, Joan Massaguè. Deciphering the crosstalk between cancer cells and stroma that drives EMT- coupled fibrosis [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr A041.

Metformin inhibits migration and epithelial-to-mesenchymal transition in non-small cell lung cancer cells through AMPK-mediated GDF15 induction

The growth differentiation factor 15 (GDF15) may serve as a biomarker of metformin, which mediates the bodyweight lowering effect of metformin. However, whether GDF15 also serves as a molecular target of metformin to inhibit carcinogenesis remains largely unknown. This study examined the role and molecular mechanisms of GDF15 in the anticancer effects of metformin in non-small cell lung cancer (NSCLC) cells, which has never been reported before. We found that metformin significantly inhibited the migration of NSCLC A549 and NCI-H460 cells and reduced the expression of epithelial-to-mesenchymal transition (EMT)-related molecules, including neuro-cadherin (N-cadherin), matrix metalloproteinase 2 (MMP2), and the zinc finger transcription factor Snail, but increased epithelial cadherin (E-cadherin) expression. Furthermore, metformin increased GDF15 and its upstream transcription factors activated transcription factor 4 (ATF4) and C/EBP-homologous protein (CHOP) expressions and increased AMP-activated protein kinase (AMPK) phosphorylation in NSCLC cells. GDF15 siRNA partially reverses the inhibitory effect of metformin on NSCLC cell migration. Moreover, metformin-induced increases in GDF15, CHOP, and ATF4 expression and the inhibition of migration were partially reversed by treatment with Compound C, a specific AMPK inhibitor. Meanwhile, metformin significantly inhibited NCI-H460 xenograft tumor growth in nude mice, increased GDF15 expression, and regulated EMT- and migration-related protein expression in xenograft tumors. In conclusion, our results provide novel insights into revealing that GDF15 can serve as a potential molecular target of metformin owing to its anti-cancer effect in NSCLC, which is mediated by AMPK activation.

Elevated expression levels of the protein kinase DYRK1B induce mesenchymal features in A549 lung cancer cells

BackgroundThe protein kinase DYRK1B is a negative regulator of cell proliferation but has been found to be overexpressed in diverse human solid cancers. While DYRK1B is recognized to promote cell survival and adaption to stressful conditions, the consequences of elevated DYRK1B levels in cancer cells are largely uncharted.MethodsTo elucidate the role of DYRK1B in cancer cells, we established a A549 lung adenocarcinoma cell model featuring conditional overexpression of DYRK1B. This system was used to characterize the impact of heightened DYRK1B levels on gene expression and to monitor phenotypic and functional changes.ResultsA549 cells with induced overexpression of wild type DYRK1B acquired a mesenchymal cell morphology with diminished cell-cell contacts and a reorganization of the pericellular actin cytoskeleton into stress fibers. This transition was not observed in cells overexpressing a catalytically impaired DYRK1B variant. The phenotypic changes were associated with increased expression of the transcription factors SNAIL and SLUG, which are core regulators of epithelial mesenchymal transition (EMT). Further profiling of DYRK1B-overexpressing cells revealed transcriptional changes that are characteristic for the mesenchymal conversion of epithelial cells, including the upregulation of genes that are related to cancer cell invasion and metastasis. Functionally, DYRK1B overexpression enhanced the migratory capacity of A549 cells in a wound healing assay.ConclusionsThe present data identify DYRK1B as a regulator of phenotypic plasticity in A549 cells. Increased expression of DYRK1B induces mesenchymal traits in A549 lung adenocarcinoma cells.

LRP1 Repression by SNAIL Results in ECM Remodeling in Genetic Risk for Vascular Diseases.

Genome-wide association studies implicate common genetic variations in the LRP1 (low-density lipoprotein receptor-related protein 1 gene) locus at risk for multiple vascular diseases and traits. However, the underlying biological mechanisms are unknown. Fine mapping analyses included Bayesian colocalization to identify the most likely causal variant. Human induced pluripotent stem cells were genome-edited using CRISPR-Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR associated protein 9) to delete or modify candidate enhancer regions and generate LRP1 knockout cell lines. Cells were differentiated into smooth muscle cells through a mesodermal lineage. Transcription regulation was assessed using luciferase reporter assay, transcription factor knockdown, and chromatin immunoprecipitation. Phenotype changes in cells were conducted using cellular assays, bulk RNA sequencing, and mass spectrometry. Multitrait colocalization analyses pointed at rs11172113 as the most likely causal variant in LRP1 for fibromuscular dysplasia, migraine, pulse pressure, and spontaneous coronary artery dissection. We found the rs11172113-T allele to associate with higher LRP1 expression. Genomic deletion in induced pluripotent stem cell-derived smooth muscle cells supported rs11172113 to locate in an enhancer region regulating LRP1 expression. We found transcription factors MECP2 (methyl CpG binding protein 2) and SNAIL (Zinc Finger Protein SNAI1) to repress LRP1 expression through an allele-specific mechanism, involving SNAIL interaction with disease risk allele. LRP1 knockout decreased induced pluripotent stem cell-derived smooth muscle cell proliferation and migration. Differentially expressed genes were enriched for collagen-containing extracellular matrix and connective tissue development. LRP1 knockout and deletion of rs11172113 enhancer showed potentiated canonical TGF-β (transforming growth factor beta) signaling through enhanced phosphorylation of SMAD2/3 (Mothers against decapentaplegic homolog 2/3). Analyses of the protein content of decellularized extracts indicated partial extracellular matrix remodeling involving enhanced secretion of CYR61 (cystein rich angiogenic protein 61), a known LRP1 ligand involved in vascular integrity and TIMP3 (Metalloproteinase inhibitor 3), implicated in extracellular matrix maintenance and also known to interact with LRP1. Our findings support allele-specific LRP1 expression repression by the endothelial-to-mesenchymal transition regulator SNAIL. We propose decreased LRP1 expression in smooth muscle cells to remodel the extracellular matrix enhanced by TGF-β as a potential mechanism of this pleiotropic locus for vascular diseases.

E-Cadherin and Snail Immunohistochemical Expression in Multiple Ipsilateral Breast Carcinoma Disease

Abstract Background As compared to invasive breast cancer- no special type (IBC-NST), Invasive lobular cancer (ILC) traditionally shows loss of function of the E-cadherin- catenin membrane complex, frequent downregulation of E-cad expression and is significantly more associated with Multiple Ipsilateral Breast Carcinoma (MIBC). Nevertheless, E cadherin downregulation was also described in MIBC with IBC-NST phenotype. MIBC was suggested to have a more aggressive biologic nature than unifocal disease, Epithelial mesenchymal transition (EMT) has been suggested as an attribute to such poorer prognosis. The transcription factor Snail commonly acts as one of the earliest switches of the EMT process and E-cadherin downregulation is one of its major downstream effects. Causation and association links between EMT and MIBC in different types of breast cancer needs further elucidation. Objective to investigate whether the association betweenE-cadherin downregulation and MIBC is attributed to EMT and whether such association and expression of EMT markers is differentially presented in ILC and IBC-NST. Methods This study was conducted retrospectively on 77 breast cancer cases diagnosed at Ain Shams University Hospitals (2012-2022). We included all ILC cases meeting study criteria (20 unifocal, 14 MIBC) and randomly selected sets of IBC- NST cases (13 unifocal, 16 MIBC) and mixed ductolobular IBC (7 unifocal, 7 MIBC). Immunohistochemical expression of E-cadherin and Snail was evaluated and compared in different study groups with subsequent statistical analysis. Results E- cadherin expression showed 15/40 (37.5%) negative expression, 15/40 (37.5%) with low expression, and 10/40 (25%) with high expression in the unifocal group, compared to 14/37 (37.8%) negative result, 13/37 (35.1%) low E-cadherin expression, and 10/37 (27%) with high E-cadherin expression in the MIBC group. There was no significant difference in E-cadherin expression regarding focality neither in all cases (p-value: 0.97) nor in each of the histological subgroups (p-values: 0.34 for IBC- NST, 1.00 for mixed, 0.2 for ILC). On the other hand, Snail expression was extensive in 24/37 MIBC cases (64.86%) as compared to 17/40 unifocal cases (42.5%), this was statistically significant (p-value: 0.038). Conclusions E-cadherin downregulation doesn’t seem to contribute to MIBC whether ILC, mixed or IBC-NST, however snail overexpression is significantly associated with MIBC. This suggests that EMT may have a role in multiplicity of IBC.

Activation of the Snail transcription factor induces Mdm2 gene expression

Epithelial-like tumor cells can become metastatic by undergoing molecular and phenotypic reprogramming in a process referred to as epithelial-to-mesenchymal transition (EMT). In response to EMT genes that promote migration and condition the tumor microenvironment to permit intravasation into the bloodstream, dissemination, and extravasation into new organs are induced. While the mutant p53 has been implicated in extravasation, one negative regulator of p53, the oncogene Mdm2, is required in the early stages of metastasis and the driver of EMT. This activity is independent of Mdm2’s role in the p53-Mdm2 autoregulatory feedback loop. Herein, we examine the EMT transcription factor Snail as a downstream effector of kinase signaling pathways. We show that the activation of upstream receptors and KRas signaling increase Snail levels. Snail binds to Ebox DNA motifs, and Mdm2 has two Ebox DNA binding domains in the second promoter. Snail binds to the second Ebox and induces Mdm2 gene expression. Knockdown of endogenous Snail by shRNA shows a decrease in Mdm2 and is associated with reduced migration. The reintroduction of Mdm2 in shSnail cells restores cellular migration. These data integrate upstream pathways that induce Snail-Mdm2 to promote the metastasis of tumor cells.

Hippo pathway effectors YAP, TAZ and TEAD are associated with EMT master regulators ZEB, Snail and with aggressive phenotype in phyllodes breast tumors

BackgroundPhyllodes tumors (PTs) of the breast are uncommon fibroepithelial neoplasms that tend to recur locally and may have metastatic potential. Their pathogenesis is poorly understood. Hippo signaling pathway plays an essential role in organ size control, tumor suppression, tissue regeneration and stem cell self-renewal. Hippo signaling dysfunction has been implicated in cancer. Recent evidence suggests that there is cross-talk between the Hippo signaling key proteins YAP/TAZ and the epithelial-mesenchymal transition (EMT) master regulators Snail and ZEB. In this study we aimed to investigate the expression of Hippo signaling pathway components and EMT regulators in PTs, in relation to tumor grade. MethodsExpression of Hippo signaling effector proteins YAP, TAZ and their DNA binding partner TEAD was evaluated by immunohistochemistry in paraffin-embedded tissue specimens from 86 human phyllodes breast tumors (45 benign, 21 borderline, 20 malignant), in comparison with tumor grade and with the expression of EMT-related transcription factors ZEB and Snail. ResultsNuclear immunopositivity for YAP, TAZ and TEAD was detected in both stromal and epithelial cells in PTs and was significantly higher in high grade tumors. Interestingly, there was a significant correlation between the expression of YAP, TAZ, TEAD and the expression of ZEB and SNAIL. ConclusionsOur results originally implicate Hippo signaling pathway in PTs pathogenesis and suggest that an interaction between Hippo signaling key components and EMT regulators may promote the malignant features of PTs.

Mitochondrial Ca2+ controls pancreatic cancer growth and metastasis by regulating epithelial cell plasticity.

Endoplasmic reticulum to mitochondria Ca2+ transfer is important for cancer cell survival, but the role of mitochondrial Ca2+ uptake through the mitochondrial Ca2+ uniporter (MCU) in pancreatic adenocarcinoma (PDAC) is poorly understood. Here, we show that increased MCU expression is associated with malignancy and poorer outcomes in PDAC patients. In isogenic murine PDAC models, Mcu deletion (Mcu KO) ablated mitochondrial Ca2+ uptake, which reduced proliferation and inhibited self-renewal. Orthotopic implantation of MCU-null tumor cells reduced primary tumor growth and metastasis. Mcu deletion reduced the cellular plasticity of tumor cells by inhibiting epithelial-to-mesenchymal transition (EMT), which contributes to metastatic competency in PDAC. Mechanistically, the loss of mitochondrial Ca2+ uptake reduced expression of the key EMT transcription factor Snail and secretion of the EMT-inducing ligand TGFβ. Snail re-expression and TGFβ treatment rescued deficits in Mcu KO cells and restored their metastatic ability. Thus, MCU may present a therapeutic target in PDAC to limit cancer-cell-induced EMT and metastasis.

LncRNA MEG3 Inhibits the Epithelial-mesenchymal Transition of Bladder Cancer Cells through the Snail/E-cadherin Axis.

This study aimed to investigate the role of the long noncoding RNA (lncRNA) maternally expressed gene 3 (MEG3) in the epithelial-mesenchymal transition (EMT) of bladder cancer cells and the potential mechanisms. Cell invasion, migration, and wound healing assays were conducted to assess the effects of MEG3 on the invasive and migratory capabilities of bladder cancer cells. The expression levels of E-cadherin were measured using Western blotting, RT-qPCR, and dual luciferase reporter assays. RNA immunoprecipitation and pull-down assays were performed to investigate the interactions between MEG3 and its downstream targets. MEG3 suppressed the invasion and migration of bladder cancer cells and modulated the transcription of E-cadherin. The binding of MEG3 to the zinc finger region of the transcription factor Snail prevented its ability to transcriptionally repress E-cadherin. Additionally, MEG3 suppressed the phosphorylation of extracellular regulated protein kinase (ERK), c-Jun N-terminal kinase (JNK), and P38, thereby decreasing the expression of Snail and stimulating the expression of E-cadherin. MEG3 plays a vital role in suppressing the EMT in bladder cancer cells, indicating its potential as a promising therapeutic target for the treatment of bladder cancer.

Loss of RAB25 Cooperates with Oncogenes in the Transformation of Human Mammary Epithelial Cells (HMECs) to Give Rise to Claudin-Low Tumors.

The loss of RAB25 expression-RAS superfamily of GTPase characteristic of numerous breast cancers-corresponds with H-RAS point mutations, particularly in triple-negative breast cancers (TNBC), a subtype associated with a poor prognosis. To address the poorly understood factors dictating the progression of TNBC tumors, we examine the cooperative effects that loss of RAB25 expression in human mammary epithelial cell (HMEC) lines with H-RAS mutations confers in tumorigenesis. HMECs were immortalized by transduction with LXSN CDK4 R24C, a mutant form of cyclin-dependent kinase, followed by transduction with hTERT, a catalytic subunit of the telomerase enzyme. We found that with the loss of RAB25 and overexpression of mutant H-RAS61L, immortal HMECs transformed toward anchorage-independent growth and acquired an increased ability to migrate. Furthermore, cells express low CD24, high CD44, and low claudin levels, indicating stem-like properties upon transformation. Besides, loss of RAB25 and overexpression of H-RAS61L resulted in increased expression of transcription factors Snail and Slug that drive these cells to lose E-cadherin and undergo epithelial-mesenchymal transition (EMT). This study confirms that loss of RAB25 and overexpression of mutant H-RAS can drive HMECs toward a mesenchymal stem-like state. Our findings reveal that RAB25 functions as a tumor suppressor gene, and loss of RAB25 could serve as a novel biomarker of the claudin-low type of TNBC.

Two sequential gene expression programs bridged by cell division support long-distance collective cell migration.

The precise assembly of tissues and organs relies on spatiotemporal regulation of gene expression to coordinate the collective behavior of cells. In Drosophila embryos, the midgut musculature is formed through collective migration of caudal visceral mesoderm (CVM) cells, but how gene expression changes as cells migrate is not well understood. Here, we have focused on ten genes expressed in the CVM and the cis-regulatory sequences controlling their expression. Although some genes are continuously expressed, others are expressed only early or late during migration. Late expression relates to cell cycle progression, as driving string/Cdc25 causes earlier division of CVM cells and accelerates the transition to late gene expression. In particular, we found that the cell cycle effector transcription factor E2F1 is a required input for the late gene CG5080. Furthermore, whereas late genes are broadly expressed in all CVM cells, early gene transcripts are polarized to the anterior or posterior ends of the migrating collective. We show this polarization requires transcription factors Snail, Zfh1 and Dorsocross. Collectively, these results identify two sequential gene expression programs bridged by cell division that support long-distance directional migration of CVM cells.

Molecular alterations in claudin 18 suppressed and non-suppressed gastric adenocarcinomas to guide targeted therapies

ABSTRACT Background Gastric adenocarcinoma represents an aggressive type of cancer and an important cause of cancer mortality. Progress in gastric cancer therapeutics has resulted from a better understanding of the molecular pathogenesis of the disease and introduction of targeted therapies, but most gastric cancer patients still rely on non-targeted chemotherapy as the mainstay of treatment for advanced disease. Methods An analysis of publicly available series from The Cancer Genome Atlas (TCGA) gastric cancer cohort was undertaken to delineate the clinical and genomic landscape of gastric cancers with suppressed expression of claudin 18 compared with cancers with non-suppressed claudin 18. Claudin 18 suppressed cancers were defined as having an mRNA expression z-score relative to normal samples (log RNA Seq V2) of less than −1. Claudin 18 non-suppressed cancers were defined as having an mRNA expression z-score relative to normal samples (log RNA Seq V2) above 0.5. Results Gastric cancers with claudin 18 mRNA suppression represented 7.7% of the gastric adenocarcinomas of TCGA cohort, while non-suppressed cancers represented 46.6% of the cases. The two groups did not differ in clinical and genomic characteristics, such as mean age, histology, grade, and stage. The mutation landscape of claudin 18 suppressed cases included high mutation rates of TP53, of genes of the WNT/β-catenin pathway and of ubiquitin ligase FBXW7. Moreover, a subset of both claudin 18 suppressed and non-suppressed cancers displayed mutations in Mismatch Repair (MMR) associated genes or a high tumor mutation burden (TMB). At the mRNA expression level, claudin 18 suppressed gastric cancers showed up-regulation of EMT core transcription factor Snail 2 and down-regulation of genes of HLA cluster. The survival of gastric cancer patients with claudin 18 mRNA suppression was not significantly different compared with patients with non-suppressed claudin 18. Conclusion Sub-sets of gastric cancers with claudin 18 mRNA suppression displayed characteristics of potential therapeutic interest, such as mutations in WNT and PI3K pathways and MMR defects. These may guide the development of alternative targeted therapies, in this sub-set of gastric cancers which are not candidates for claudin 18 targeting therapies.

RETRACTED ARTICLE: The analysis of boric acid effect on epithelial-mesenchymal transition of CD133 + CD117 + lung cancer stem cells

Targeting lung cancer stem cells (LC-SCs) for metastasis may be an effective strategy against lung cancer. This study is the first on epithelial-mesenchymal transition (EMT) properties of boric acid (BA) in LC-SCs. LC-SCs were isolated using the magnetic cell sorting (MACS) method. Tumor-sphere formation and flow cytometry confirmed CSC phenotype. The cytotoxic effect of BA was measured by MTT analysis, and the effect of BA on EMT was examined by migration analysis. The expression levels of ZEB1, SNAIL1, ITGA5, CDH1, ITGB1, VIM, COL1A1, and LAMA5 genes were analyzed by RT-qPCR. E-cadherin, Collagen-1, MMP-3, and Vimentin expressions were analyzed immunohistochemically. Boric acid slightly reduced the migration of cancer cells. Increased expression of transcription factor SNAIL (p < 0.001), but not ZEB1, was observed in LC-SCs. mRNA expression levels of ITGB1 (p < 0.01), ITGA5 (p < 0.001), COL1A1 (p < 0.001), and LAMA5 (p < 0.001) increased; CDH1 and VIM decreased in LC-SCs. Moreover, while E-cadherin (p < 0.001) and Collagen-1 (p < 0.01) immunoreactivities significantly increased, MMP-3 (p < 0.001) and Vimentin (p < 0.01) immunoreactivities decreased in BA-treated LC-SCs. To conclude, the current study provided insights into the efficacy and effects of BA against LC-SCs regarding proliferation, EMT, and cell death for future studies.Graphical abstract

Abstract 1383: Investigation of biological role and cytokine axis of cancer-associated fibroblast (CAF) in lung cancer brain metastasis

Abstract Background: Brain metastases (BM) are among the most lethal forms of cancer globally. Lung cancer BM are frequently encountered and are associated with poor prognosis, decreased survival rates, and therapeutic resistances. Cancer-associated fibroblasts (CAFs) have emerged as important players of the brain metastases tumor microenvironment (TME). Mechanistic roles of brain metastases CAFs (hBM-CAFs) in tumorigenesis and therapeutic resistances remain unclear. Material and Methods: In the present study, the patient's BM surgical sample was subjected to primary culture to isolate CAF. The hBM-CAF induced EMT and stemness phenotype was investigated by western blot. Biological role of hBM-CAF in tumorigenesis was evaluated using indirect co-culture method in vitro. FACS analysis was performed to investigate the changes in cisplatin cytotoxicity and radiation susceptibility of lung cancer cell lines when co-cultured with hBM-CAF. CX3CL1 and IL26 expression was confirmed by ELISA along with transcriptomic analysis of hBM-CAF. Molecular events were validated by using neutralizing CX3CL1 and IL26. The tumorigenicity and response to chemotoxicity of lung cancer cells were examined by co-injecting them with hBM-CAF in a mouse xenograft model. Results: Our experiments demonstrated that hBM-CAFs significantly enhanced EMT and stemness phenotype by upregulating transcription factor SNAIL1, TWIST1, Slug and CD133, CD44 respectively in lung cancer cells. We also observed that hBM-CAFs decreased cisplatin cytotoxicity and enhanced radiation resistance, as measured by FACS analysis of apoptotic cells. In vivo experiments using xenograft mouse models revealed that co-injection of lung cancer cells with hBM-CAFs led to faster tumor growth and increased cisplatin resistance. Transcriptomic analysis of hBM-CAFs revealed a distinct genomic signature compared to normal fibroblast (hNFs), with significant overexpression of IL26 and CX3CL1 genes in the hBM-CAFs secretome, as validated by ELISA. Moreover, we found that lung cancer cells expressed high levels of the IL26 receptor IL10Rβ and ILR20α, and CX3CL1 receptor CX3CR1. Our hBM-CAF significantly upregulated JAK-STAT3 and Akt-mTOR pathway. Eventually, we have confirmed that neutralizing IL26 and CX3CL1 inhibit phosphorylation of JAK-STAT3 and Akt-mTOR respectively and revoked the IL26-CX3CL1 induced EMT and stemness phenotype of lung cancer cells. Conclusion: Patient derived hBM-CAF promote aggressive phenotype of lung cancer cells by upregulating EMT and stemness transcriptome factors. Overexpression of IL26 and CX3CL1 cytokine led to the therapeutic resistance through JAK-STAT3 and Akt-mTOR signaling pathway respectively. Targeting IL26-CX3CL1 might be a novel therapeutic efficacy for lung cancer brain metastasis patients. Citation Format: Nah Ihm Kim, SM Abdus Salam, Eshrat Jahan, Eun-Jeong Ahn, Tae-Young Jung, Jae-Hyuk Lee, Joon Haeng Rhee, Sung Sun Kim, Kyung-Hwa Lee, Kyung-Sub Moon. Investigation of biological role and cytokine axis of cancer-associated fibroblast (CAF) in lung cancer brain metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1383.

Abstract 5390: Exploring signaling pathways of tumor-nerve interactions in prostate and breast cancer

Abstract A significant portion of prostate cancer (PCa) and breast cancer (BCa) patients develop bone metastatic disease. In some cases, cancer spreads through the nervous system, a process known as perineural invasion (PNI). Neurite outgrowth is believed to be a precursor to PNI. Snail is an important gene which regulates the epithelial-mesenchymal transition (EMT) process in which tumor cells at the invasive front undergo this transition to promote invasion, migration, and subsequent metastasis. We recently published that Snail promotes neurite outgrowth in PCa cells. We hypothesize that Snail expression will stimulate neurite outgrowth in both PCa and BCa cells through extracellular vesicles from cancer cells interacting with neurons. To test this hypothesis, we first collected conditioned media from PCa and BCa cells expressing Snail and isolated exosomes. Exosomal markers were detected using western blot to confirm the isolated exosomes. Additionally, we used Transmission Electron Microscopy (TEM) to observe the secretion of exosomes. Proteomics was performed to analyze proteins expressed within exosomes of LNCaP Snail overexpressing or C4-2 Snail knockdown PCa cells. Our results showed that Snail-expressing cells secrete exosomes containing Talin1, specifically, the proteolyzed C-terminal rod domain, while full-length Talin1 is found in whole cell lysates from Snail-expressing cells. Talin1 has previously been associated with neurite outgrowth. Neurite outgrowth assays were then performed using conditioned medium collected from C4-2 PCa cells with Snail knockdown and MCF-7 BCa cells with Snail overexpression. Increased neurite outgrowth is observed in PC-12 cells and NPC (Induced Pluripotent Stem Cells differentiated neuronal progenitor cells) when cultured with conditioned medium collected from PCa and BCa cells expressing high levels of Snail. AKT activation was observed in the neuronal cells in response to conditioned media from Snail-expressing cells. Furthermore, we found that mH4, a proprietary small molecule inhibitor of Talin1, reduces Snail-mediated neurite outgrowth and AKT activation (p-AKT Thr308 and Ser473). Overall, we have uncovered a pathway whereby Snail transcription factor promotes secretion of exosomes containing Talin1 which promotes AKT signaling in neuronal cells and neurite outgrowth. Talin1 small molecule inhibitor shows promise for therapeutic targeting of tumor-nerve interactions. Acknowledgements: The authors acknowledge the use of core facilities supported by the National Institute on Minority Health and Health Disparities through grant number 5U54MD013376 and 1U54GM128729-01-DaCCoTA Scholars Program (PI: Rezvani) National Institute of General Medical Sciences (NIGMS). Citation Format: Bor-Jang Hwang, Gabrielle J. Edwards, Cole Davis Knoblich, Khosrow Rezvani, Valerie Odero-Marah. Exploring signaling pathways of tumor-nerve interactions in prostate and breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5390.

A New Benzo[6,7]oxepino[3,2-b] Pyridine Derivative Induces Apoptosis in Canine Mammary Cancer Cell Lines

CMC is the most frequently diagnosed cancer and one of the leading causes of death in non-spayed female dogs. Exploring novel therapeutic agents is necessary to increase the survival rate of dogs with CMC. MPOBA is a BZOP derivative that has a significant anticancer effect in a human cell line. The main goal of this study was to investigate the anticancer properties of MPOBA against two CMC cell lines (REM134 and CMGT071020) using a 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay, a wound healing assay, a transwell migration assay, an Annexin V-FITC apoptosis assay with a flow cytometry analysis, a mRNA expression analysis using quantitative real-time PCR (qRT-PCR), and an immunohistochemistry (IHC). According to the accumulated studies, MPOBA caused significant concentration- and time-dependent reductions in cell proliferation and cell migration and induced apoptosis in both CMC cell lines. In gene expression analysis, nine canine genes, including TP53, BCL-2, BAX, epidermal growth factor receptor (EGFR), snail transcription factor (SNAIL), snail-related zinc-finger transcription factor (SLUG), TWIST, E-cadherin, and N-cadherin, were investigated. The mRNA expression results revealed that MPOBA induced upregulation of TP53 and overexpression of the pro-apoptotic gene BAX, together with an inhibition of BCL-2. Moreover, MPOBA also suppressed the mRNA expression levels of SNAIL, EGFR, and N-cadherin and induced upregulation of E-cadherin, crucial genes related to the epithelial-to-mesenchymal transition (EMT). However, there was no significant difference in the IHC results of the expression patterns of vimentin (VT) and cytokeratin (CK) between MPOBA-treated and control CMC cells. In conclusion, the results of the present study suggested that MPOBA exhibited significant anticancer activity by inducing apoptosis in both CMCs via upregulation of TP53 and BAX and downregulation of BCL-2 relative mRNA expression. MPOBA may prove to be a potential candidate drug to be further investigated as a therapeutic agent for CMC.

EGFR core fucosylation, induced by hepatitis C virus, promotes TRIM40-mediated-RIG-I ubiquitination and suppresses interferon-I antiviral defenses

Aberrant N-glycosylation has been implicated in viral diseases. Alpha-(1,6)-fucosyltransferase (FUT8) is the sole enzyme responsible for core fucosylation of N-glycans during glycoprotein biosynthesis. Here we find that multiple viral envelope proteins, including Hepatitis C Virus (HCV)-E2, Vesicular stomatitis virus (VSV)-G, Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-Spike and human immunodeficiency virus (HIV)-gp120, enhance FUT8 expression and core fucosylation. HCV-E2 manipulates host transcription factor SNAIL to induce FUT8 expression through EGFR-AKT-SNAIL activation. The aberrant increased-FUT8 expression promotes TRIM40-mediated RIG-I K48-ubiquitination and suppresses the antiviral interferon (IFN)-I response through core fucosylated-EGFR-JAK1-STAT3-RIG-I signaling. FUT8 inhibitor 2FF, N-glycosylation site-specific mutation (Q352AT) of EGFR, and tissue-targeted Fut8 silencing significantly increase antiviral IFN-I responses and suppress RNA viral replication, suggesting that core fucosylation mediated by FUT8 is critical for antiviral innate immunity. These findings reveal an immune evasion mechanism in which virus-induced FUT8 suppresses endogenous RIG-I-mediated antiviral defenses by enhancing core fucosylated EGFR-mediated activation.

Decidual stromal cells-derived exosomes incurred insufficient migration and invasion of trophoblast by disturbing of β-TrCP-mediated snail ubiquitination and degradation in unexplained recurrent spontaneous abortion

BackgroundExosomes released from decidual stromal cells (DSC-exos) play a crucial role in facilitating the epithelial–mesenchymal transition (EMT) of trophoblasts and insufficient trophoblasts EMT are associated with URSA (unexplained recurrent spontaneous abortion). However, the mechanisms underlying DSC-exos inducing EMT is not completely understood.MethodsDSC-exos of normal pregnant women (N-DSC-exos) and URSA patients (URSA-DSC-exos) were extracted and characterized. Characterization of the isolated DSC-exos was performed using with TEM (transmission electron microscopy), NTA (nanoparticle tracking analysis), and WB (western blot) techniques. Subsequently, these DSC-exos were co-cultured with trophoblasts cell lines (HTR-8/SVneo). The influence of both N-DSC-exos and URSA-DSC-exos on trophoblasts proliferation, invasion and migration, as well as on the expression of EMT-related proteins, was evaluated through a series of assays including CCK8 assays, wound healing assays, transwell assays, and western blot, respectively. Then rescue experiments were performed by β-TrCP knockdown or β-TrCP overexpressing trophoblasts with snail-siRNA transfection or β-TrCP overexpressing Lentivirus infection, respectively. Finally, animal experiments were employed to explore the effect of N-DSC-exos on embryo absorption in mice.ResultsWe found increased β-TrCP expression in the villus of URSA patients when compared to the normal pregnant women, alongside reduction in the levels of both snail and N-cadherin within URSA patients. N-DSC-exos can promote the EMT of the trophoblast by inhibiting β-TrCP-mediated ubiquitination and degradation of transcription factor snail. Moreover the capacity to promote EMT was found to be more potent in N-DSC-exos than URSA-DSC-exos. Down-regulation of snail or overexpression of β-TrCP can reverse the effects of N-DSC-exos on trophoblast. Finally, in vivo experiment suggested that N-DSC-exos significantly reduced the embryo resorption rate of spontaneous abortion mouse model.ConclusionsOur findings indicate that URSA-DSC-exos caused insufficient migration and invasion of trophoblast because of disturbing of β-TrCP-mediated ubiquitination and degradation of EMT transcription factor snail. Elucidating the underlying mechanism of this dysregulation may shed light on the novel pathways through which DSC-exos influence trophoblast function, thereby contributing to our understanding of their role in URSA.

Indomethacin and bromfenac may be effective in the prevention of posterior capsule opacification

Posterior capsule opacification (PCO) is the most common long‐term postoperative complication of cataract surgery, leading to secondary vision loss. Optimized intraocular lens (IOL) structure and appropriate pharmacological intervention can provide physical barriers and biological inhibition, respectively, to block the migration, proliferation, and epithelial‐mesenchymal transition (EMT) of lens epithelial cells (LECs) for PCO prophylaxis. We demonstrated that non‐steroidal anti‐inflammatory drugs (NSAIDs) are effective in preventing posterior capsule opacification. Indomethacin (INDOM) not only suppresses cell migration and down‐regulates the expression of cyclooxygenase‐2 (COX‐2) and EMT markers, including alpha‐smooth muscle actin (α‐SMA) and cyclin D1, but also promotes autophagy activation in LECs. Autophagy was also verified as a potential therapeutic target for down‐regulating EMT in LECs. Bromfenac effectively inhibits 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) signalling induced by transforming growth factor‐β2 (TGF‐β2) in vitro. Based on ultrasonic spray technology, we developed a drug‐eluting intraocular lens (IOL) using poly (lactic‐co‐glycolic acid) (PLGA) with sustained release ability of indomethacin/bromfenac for PCO prevention. The novel IOL, serving as a drug delivery platform, can carry an adjustable dose of drugs with sustained release ability for more than 28 days. In the rabbit PCO model, the novel IOL showed excellent anti‐inflammatory and anti‐PCO effects. In summary, indomethacin and bromfenac are effective pharmacological interventions for PCO prophylaxis, and the novel IOL we developed prevented PCO in vivo, providing a promising approach for PCO prophylaxis in clinical applications.