ZEB1 Protein Expression Research Articles

Melatonin inhibits tongue squamous cell carcinoma: Interplay of ER stress-induced apoptosis and autophagy with cell migration

Tongue squamous cell carcinoma (TSCC) occupies a high proportion of oral squamous cell carcinoma. TSCC features high lymph node metastasis rates and chemotherapy resistance with a poor prognosis. Therefore, an effective therapy strategy is needed to improve patient prognosis. Melatonin (MT) is a natural indole compound shown to have anti-tumor effects in several cancers. This study focused on the role and mechanism of MT in TSCC cells. The results of the study suggest that MT could inhibit cell proliferation in CRL-1623 cells. Western blot analysis showed the down-regulate of cyclin B1 and the up-regulate P21 protein by MT. MT was also shown to down-regulate the expression of Zeb1, Wnt5A/B, and β-catenin protein and up-regulate E-cadherin to inhibit the migration of CRL-1623 cells. MT also promoted the expression of ATF4, ATF6, Bip, BAP31 and CHOP in CRL-1623 cells leading to endoplasmic reticulum stress, and induced autophagy and apoptosis in CRL-1623 cells. Western blots showed that MT could promote the expression of Bax, LC3, and Beclin1 proteins and inhibit the expression of p62. We screened differentially expressed long non-coding RNAs (lncRNAs) in MT-treated cells and found that the expression of MALAT1 and H19 decreased. Moreover, MT inhibited tumor growth in nude mice inoculated with CRL-1623 cells. These results suggest that MT could induce autophagy, promote apoptosis, and provide a potential natural compound for the treatment of TSCC.

CD73 mitigates ZEB1 expression in papillary thyroid carcinoma

BackgroundZEB1, a core transcription factor involved in epithelial-mesenchymal transition (EMT), is associated with aggressive cancer cell behavior, treatment resistance, and poor prognosis across various tumor types. Similarly, the expression and activity of CD73, an ectonucleotidase implicated in adenosine generation, is an important marker of tumor malignancy. Growing evidence suggests that EMT and the adenosinergic pathway are intricately linked and play a pivotal role in cancer development. Therefore, this study focuses on exploring the correlations between CD73 and ZEB1, considering their impact on tumor progression.MethodsWe employed CRISPR/Cas9 technology to silence CD73 expression in cell lines derived from papillary thyroid carcinoma. These same cells underwent lentiviral transduction of a reporter of ZEB1 non-coding RNA regulation. We conducted studies on cell migration using scratch assays and analyses of cellular speed and polarity. Additionally, we examined ZEB1 reporter expression through flow cytometry and immunocytochemistry, complemented by Western blot analysis for protein quantification. For further insights, we applied gene signatures representing different EMT states in an RNA-seq expression analysis of papillary thyroid carcinoma samples from The Cancer Genome Atlas.ResultsSilencing CD73 expression led to a reduction in ZEB1 non-coding RNA regulation reporter expression in a papillary thyroid carcinoma-derived cell line. Additionally, it also mitigated ZEB1 protein expression. Moreover, the expression of CD73 and ZEB1 was correlated with alterations in cell morphology characteristics crucial for cell migration, promoting an increase in cell polarity index and cell migration speed. RNA-seq analysis revealed higher expression of NT5E (CD73) in samples with BRAF mutations, accompanied by a prevalence of partial-EMT/hybrid state signature expression.ConclusionsCollectively, our findings suggest an association between CD73 expression and/or activity and the post-transcriptional regulation of ZEB1 by non-coding RNA, indicating a reduction in its absence. Further investigations are warranted to elucidate the relationship between CD73 and ZEB1, with the potential for targeting them as therapeutic alternatives for cancer treatment in the near future.

Mechanisms of LPS–induced epithelial mesenchymal transition in bEECs

High-concentrate diets cause subacute ruminal acidosis, resulting in increased blood lipopolysaccharide (LPS) levels in cows. We found that the peak LPS in cows fed with high-concentrate diets coincides the period of embryo implantation in a large-scale dairy farm. As epithelial-mesenchymal transition (EMT) should be tightly regulated during normal embryo implantation in cows, we speculated that increased LPS may cause abnormal EMT, thereby inhibiting embryo implantation in cows. To confirm that elevated LPS levels induce abnormal EMT in cows, we treated bovine endometrial epithelial cells (bEECs) with LPS for 48 h and analyzed the protein levels of ZEB1, a major EMT-related transcription factor, which is positively regulated by the TGFβ/SMAD3 pathway. In addition, we analyzed the changes in expression of three EMT-related genes (E-cadherin, N-cadherin, and Vimentin), and examined the morphology and migratory ability of the cells. The results showed that elevated LPS levels increased protein expression of ZEB1, vimentin, and N-cadherin, and reduced that of E-cadherin. Elevated LPS also increased bEECs migration rate, and induced the cells to acquire a mesenchymal phenotype. Furthermore, benzyl butyl phthalate (BBP)-induced ZEB1 overexpression significantly decreased E-cadherin levels and increased N-cadherin levels. As LPS treatment also decreased the expression of Bta-miR-200b, we further found that Bta-miR-200b targets to the 3′UTR of ZEB1 through the confirmation of dual-luciferase reporter system. And the increased level of Bta-miR-200b by mimic enhanced the expression of E-cadherin and yet inhibited the expression of N-cadherin in protein, which exactly opposite to the results induced by LPS. In conclusion, LPS induced EMT in bEECs by upregulating ZEB1, while Bta-miR-200b could inhibit the occurrence of EMT by binding ZEB1 3′UTR. These results provide a new insight for low reproductive rate of dairy cows under the background of high-concentrate diets.

LncRNA HOTAIR down-expression inhibits the invasion and tumorigenicity of epithelial ovarian cancer cells by suppressing TGF-β1 and ZEB1

BackgroundEpithelial ovarian cancer (EOC) is a pathological type with a higher mortality rate among gynecological cancers today. Long-chain noncoding RNAs (lncRNAs) can regulate the transcription and expression of cellular genes. However, the downstream molecules regulated by lncRNA HOTAIR have not been well studied. The effects of downregulated lncRNA HOTAIR on EOC invasiveness and tumorigenicity in nude mice, along with TGF- β1 and ZEB1 in epithelial ovarian cancer cells, need to be investigated in further research.ResultsRT-qPCR was used to detect lncRNA HOTAIR and TGF-β1 and ZEB1 mRNA expression in EOC SKOV3 cells. The expression of lncRNA HOTAIR in SKOV3 cells transfected with the recombinant shHOTAIR interference plasmid was significantly lower than that of the negative control. Compared with the negative control, the matrix gel invasion ability of shHOTAIR SKOV3 cells in vitro and their tumorigenicity in nude mice were significantly reduced. Moreover, compared with the control, the expression of ZEB1 protein in shHOTAIR-SKOV3 xenograft tumors was significantly reduced. Downregulation of lncRNA HOTAIR expression significantly reduced TGF-β1 and ZEB1 mRNA expression, but increased the expression of E-cadherin mRNA. In summary, downregulated lncRNA HOTAIR in EOC SKOV3 cells transfected with shHOTAIR can inhibit TGF-β1, reduce ZEB1, increase E-cadherin, and significantly reduce the invasiveness and tumorigenicity of ovarian epithelial cancer SKOV3 cells.ConclusionsThese results suggest that the lncRNA HOTAIR may be an effective target for the treatment of human EOC.

Inhibiting miR-33b-5p Enhances Chemoresistance in Lung Adenocarcinoma by Targeting YWHAH to Regulate Epithelial-mesenchymal Transition.

Lung adenocarcinoma (LUAD) is the most malignant type of lung cancer, whose clinical treatment is seriously hindered by chemoresistance. Numerous reports have demonstrated that miR-33b-5p plays an essential role in alleviating the chemoresistance of multiple cancers, but there are currently no reports about the effects of miR-33b-5p on the chemoresistance in LUAD. Our study aimed to investigate the impacts of miR-33b-5p on the chemoresistance in LUAD and the underlying mechanism. Bioinformatics analyses were employed to investigate the relation between miR-33b-5p and YWHAH. The MTT assay and flow cytometry were respectively adopted to determine cell viability and apoptosis. A transwell assay was employed to evaluate cellular invasion and migration. qRT-PCR and western blotting were respectively employed to detect the gene expression of miR-33b-5p and the protein expression of YWHAH, MMP2, Snail, and Zeb1. Three bioinformatics analysis approaches predicted that YWHAH was the underlying targeted gene of miR-33b-5p and revealed the associated mechanisms. The concentration of paclitaxel (TAX) and cisplatin (DDP) needed to induce chemoresistance of LUAD cells was determined as 100 μM. Migration and invasion, as well as protein expression of YWHAH, MMP2, MMP8, Snail and Zeb1 were increased, but the apoptosis and levels of miR-33b-5p were reduced in A549 cells with chemoresistance. Knockdown of miR-33b-5p exerted the same effects produced by chemoresistance, but additional knockdown of YWHAH reversed the effects generated by inhibiting miR-33b-5p. Our study confirmed that knockdown of miR-33b-5p aggravated chemoresistance in LUAD via targeting YWHAH to regulate EMT.

Atovaquone inhibits colorectal cancer metastasis by regulating PDGFRβ/NF-κB signaling pathway

BackgroundColorectal cancer is a common malignant tumour. Invasive growth and distant metastasis are the main characteristics of its malignant biological behaviour, and they are also the primary factors leading to death in colon cancer patients. Atovaquone is an antimalarial drug, and its anticancer effect has recently been demonstrated in several cancer models in vitro and in vivo, but it has not been examined in the treatment of colorectal cancer.MethodsTo elucidate the effect of atovaquone on colorectal cancer. We used RNA transcriptome sequencing, RT‒PCR and Western blot experiments to examine the expression of NF-κB (p-P65), EMT-related proteins and related inflammatory factors (IL1B, IL6, CCL20, CCL2, CXCL8, CXCL6, IL6ST, FAS, IL10 and IL1A). The effect of atovaquone on colorectal cancer metastasis was validated using an animal model of lung metastases. We further used transcriptome sequencing, the GCBI bioinformatics database and the STRING database to predict relevant target proteins. Furthermore, pathological sections were collected from relevant cases for immunohistochemical verification.ResultsThis study showed that atovaquone could inhibit colorectal cancer metastasis and invasion in vivo and in vitro, inhibit the expression of E-cadherin protein, and promote the protein expression of N-cadherin, vimentin, ZEB1, Snail and Slug. Atovaquone could inhibit EMT by inhibiting NF-κB (p-P65) and related inflammatory factors. Further bioinformatics analysis and verification showed that PDGFRβ was one of the targets of atovaquone.ConclusionIn summary, atovaquone can inhibit the expression of NF-κB (p-P65) and related inflammatory factors by inhibiting the protein expression of p-PDGFRβ, thereby inhibiting colorectal cancer metastasis. Atovaquone may be a promising drug for the treatment of colorectal cancer metastasis.

Human dermal fibroblast subpopulations and epithelial mesenchymal transition signals in hidradenitis suppurativa tunnels are normalized by spleen tyrosine kinase antagonism in vivo.

Hidradenitis Suppurativa is a chronic inflammatory disease of which the pathogenesis is incompletely understood. Dermal fibroblasts have been previously identified as a major source of inflammatory cytokines, however information pertaining to the characteristics of subpopulations of fibroblasts in HS remains unexplored. Using in silico-deconvolution of whole-tissue RNAseq, Nanostring gene expression panels and confirmatory immunohistochemistry we identified fibroblast subpopulations in HS tissue and their relationship to disease severity and lesion morphology. Gene signatures of SFRP2+ fibroblast subsets were increased in lesional tissue, with gene signatures of SFRP1+ fibroblast subsets decreased. SFRP2+ and CXCL12+ fibroblast numbers, measured by IHC, were increased in HS tissue, with greater numbers associated with epithelialized tunnels and Hurley Stage 3 disease. Pro-inflammatory CXCL12+ fibroblasts were also increased, with reductions in SFRP1+ fibroblasts compared to healthy controls. Evidence of Epithelial Mesenchymal Transition was seen via altered gene expression of SNAI2 and altered protein expression of ZEB1, TWIST1, Snail/Slug, E-Cadherin and N-Cadherin in HS lesional tissue. The greatest dysregulation of EMT associated proteins was seen in biopsies containing epithelialized tunnels. The use of the oral Spleen tyrosine Kinase inhibitor Fostamatinib significantly reduced expression of genes associated with chronic inflammation, fibroblast proliferation and migration suggesting a potential role for targeting fibroblast activity in HS.

Detection of zeb1 Gene in Granulosa Cells in Women Undergoing IVF Treatment.

ZEB1 plays a role in epithelial-to-mesenchymal transition and acts as a repressor of E-cadherin, TGF-β, and Wnt/β-catenin. Since ZEB1 protein is expressed in estrogen-responsive tissues, and expression of the gene in the normal ovary and endometrium is positively correlated with high estrogen levels, we performed a direct analysis of granulosa cell samples to determine whether there are any significant changes in zeb1 expression during folliculogenesis. ZEB1 expression levels were measured in the granulosa cells of 56 infertile women undergoing IVF treatment. RNA extraction from granulosa cells was performed along with reverse transcription quantitative polymerase chain reaction (RT-qPCR) with SYBR Green I to determine zeb1 gene expression levels. Statistical analysis was performed by using t-test, while possible correlations of the expression of ZEB1 protein with body mass index (BMI), age, number of oocytes, and oocyte maturation were investigated. Zeb1 gene expression levels correlate significantly with body mass index (BMI) and age, but not with oocyte number and oocyte maturation stage. Obese women demonstrate a higher expression level of zeb1 gene compared to normal and overweight women. Moreover, zeb1 gene is overexpressed in women aged 35-40 years old and is under-expressed in women >40 years old. ZEB1 expression should be further investigated as it may unveil new potential findings of the zeb1 gene's role in female fertility and its use as a biomarker in fertility workups.

METTL3 promotes the malignancy of non-small cell lung cancer by N6-methyladenosine modifying SFRP2.

This study aimed to investigate the roles of METTL3, a regulator of m6A, in NSCLC. RT-qPCR was applied to determine mRNA of m6A-associated genes and SFRP2, and western blot were used for ZEB1 and MMP9 protein expression. Total m6A level was measured using methylated RNA immunoprecipitation (MeRIP) assay, and RIP was used to access m6A level of SFRP2. Cellular behaviors were detected using CCK-8 and tranwell assays. Xenograft assays were conducted to further verify the roles of METTL3 and SFRP2 in NSCLC. The expression level of METTL3 was higher in NSCLC than normal controls. However, downregulation of METTL3 restrained the proliferation, migration and invasion of NSCLC cells. Enhanced expression of METTL3 caused the inverse consequences. Moreover, SFRP2 was found to be negatively regulated by METTL3. Intriguingly, the anti-tumor functions of METTL3 knockdown in the phenotype of NSCLC cells and xenograft mice were overturned by inhibition of SFRP2. Silencing METTL3 resulted in the enhanced stability of SFRP2. Finally, downregulation of SFRP2 induced by METTL3 activated the Wnt/β-catenin signaling pathway in NSCLC. METTL3 acted as an oncogene in the pathogenesis of NSCLC via suppressing SFRP2 to activate Wnt/β-catenin signaling pathway, indicating that METTL3 might be a promising predictor in NSCLC.

Abstract 1234: Tumor cell-intrinsic PD-L1 promotes epithelial-to-mesenchymal transition in non-small cell lung cancer in interferon-g-independent and dependent way

Abstract The immune checkpoint molecule programmed cell death-ligand 1 (PD-L1, CD274) suppresses immunity when the cell surface PD-L1 is engaged by programmed death-1 (PD-1) on antitumor immune cells. Although PD-L1 has been established and targeted in cancer immunotherapy, the tumor-intrinsic role of PD-L1 is less appreciated in tumor biology and therapeutic development. In this study, we showed that PD-L1 facilitates epithelial-to-mesenchymal transition (EMT) in non-small cell lung cancer (NSCLC) cells by upregulating EMT transcription factors and mesenchymal markers, and downregulating epithelial marker. RNA-sequencing was performed on paired exogenously PD-L1-overexpressed A549 cells and wild-type cells, and PD-L1-depleted H460 cells and wild-type cells, and transcriptomic features associated with PD-L1 status were identified using Gene Set Enrichment Analysis (GSEA). In both the paired samples, PD-L1 expression was associated with EMT-related pathways. Subsequent in vitro experiment showed that exogenous overexpression of PD-L1 in A549 and H522 cells increased both mRNA and protein expression of Twist, Zeb1, Snail, Slug, Vimentin, Fibronectin, and N-Cadherin. Correspondingly, the transcription factors and mesenchymal markers were decreased in PD-L1-depleted H460 and H596 cells. Recombinant PD-1 treatment also increased the expression of EMT transcription factors and markers on A549 and H460 cells. Moreover, interferon-γ stimulation on tumor cells increased expression of EMT transcription factors and markers, which was suppressed by PD-L1-knockdown, suggesting that interferon-γ-induced PD-L1 contributes to EMT. Our findings suggest that PD-L1 facilitates NSCLC progression by promoting the EMT in interferon-γ-independent and dependent way, and this intrinsic PD-L1 function could be targeted for better clinical management of PD-L1-overexpressing NSCLCs. Citation Format: Hyein Jeong, Hyun Kyung Ahn, Sehui Kim, Yoon Kyung Jeon. Tumor cell-intrinsic PD-L1 promotes epithelial-to-mesenchymal transition in non-small cell lung cancer in interferon-g-independent and dependent way [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1234.

Constructing interactive networks of functional genes and metabolites to uncover the cellular events related to colorectal cancer cell migration induced by arsenite

Tumor cell migration induced by arsenite (iAsIII) is closely associated with cancer progression. However, transcriptomic and metabolic traits of migrative human cells exposed to iAsIII remain to be well characterized. Here, the combination of transcriptomics and metabolomics approaches were employed to construct interactive networks of functional genes and metabolites in human colorectal cancer (DLD-1) cells exposed to iAsIII. The number of DLD-1 cells passing through the Transwell membrane was at least 6 times greater in the iAsIII-treated groups than in controls. Following iAsIII treatment, the expression of ZEB1 and SLUG protein was significantly upregulated while the expression of CRB2 was downregulated (p < 0.05), indicating the onset of epithelial to mesenchymal transition (EMT). Meanwhile, integrin- and collagen-mediated biological adhesion were enhanced by SLUG under iAsIII treatment. The expression of matrix metallopeptidase (MMP) genes was fostered by iAsIII, which have the functions to degrade extracellular matrix. Glutamine metabolism could be considerably interfered by iAsIII, and in turn glutamine supplementation could effectively enhance DLD-1 cell movement. Overall, our results suggested that DLD-1 cell migration could be promoted by iAsIII via a series of cellular events, including EMT activation, altered cell adhesion, MMP-dependent matrix degradation, accompanying with a metabolic focus on glutamine.

The Transcription Factor Twist1 Has a Significant Role in Mycosis Fungoides (MF) Cell Biology: An RNA Sequencing Study of 40 MF Cases.

The purpose of this RNA sequencing study was to investigate the biological mechanism underlying how the transcription factors (TFs) Twist1 and Zeb1 influence the prognosis of mycosis fungoides (MF). We used laser-captured microdissection to dissect malignant T-cells obtained from 40 skin biopsies from 40 MF patients with stage I-IV disease. Immunohistochemistry (IHC) was used to determinate the protein expression levels of Twist1 and Zeb1. Based on RNA sequencing, principal component analysis (PCA), differential expression (DE) analysis, ingenuity pathway analysis (IPA), and hub gene analysis were performed between the high and low Twist1 IHC expression cases. The DNA from 28 samples was used to analyze the TWIST1 promoter methylation level. In the PCA, Twist1 IHC expression seemed to classify cases into different groups. The DE analysis yielded 321 significant genes. In the IPA, 228 significant upstream regulators and 177 significant master regulators/causal networks were identified. In the hub gene analysis, 28 hub genes were found. The methylation level of TWIST1 promoter regions did not correlate with Twist1 protein expression. Zeb1 protein expression did not show any major correlation with global RNA expression in the PCA. Many of the observed genes and pathways associated with high Twist1 expression are known to be involved in immunoregulation, lymphocyte differentiation, and aggressive tumor biology. In conclusion, Twist1 might be an important regulator in the disease progression of MF.

Spatiotemporal gene targeting in the mouse corneal endothelium.

The inducible Cre-ERT2 recombinase system allows for temporal control of gene targeting, and it is useful to studying adult function of genes that have critical developmental roles. The Zeb1flox/flox: UBC-CreERT2 mouse was generated to conditionally target Zeb1 to investigate its role in mesenchymal transition in the mouse corneal endothelium in vivo. Hemizygous UBC-CreERT2 mice were crossed with homozygous mice harboring loxP-flanked Zeb1 alleles (Zeb1flox/flox) to generate the Zeb1flox/flox: UBC-CreERT2 mouse. 4-hydroxytamoxifen (4-OHT) exposure leads to excision of exon 6 of Zeb1, resulting in a loss function allele in the Zeb1flox/flox: UBC-CreERT2 mouse. Intracameral 4-OHT injection further isolates Zeb1 targeting to the anterior chamber. Mesenchymal transition and induction of Zeb1 expression in the corneal endothelium was achieved using FGF2 in ex vivo organ culture. Gene expression was analyzed by semi-quantitative reverse transcription-polymerase chain reaction and by immunoblotting in the mouse corneal endothelium in vivo. Following Cre-mediated targeting of Zeb1 by intracameral 4-OHT injection in Zeb1flox/flox: UBC-CreERT2 mice, FGF2 treatment in ex vivo organ culture resulted in abrogation of Zeb1 mRNA and protein expression in the corneal endothelium. The data show Zeb1, a critical mediator of fibrosis in corneal endothelial mesenchymal transition, can be targeted by intracameral injection of 4-OHT in the mouse corneal endothelium in vivo. These results suggest that genes with critical developmental roles can be targeted at a specific time in the corneal endothelium to study its role in adult disease using an inducible Cre-Lox strategy.

LINC00324 promotes cell proliferation and metastasis of esophageal squamous cell carcinoma through sponging miR-493-5p via MAPK signaling pathway

Long non-coding RNAs have been demonstrated to promote proliferation and metastasis via regulating the miRNA/mRNA regulatory axis in various malignancies. Based on our preliminary study, we investigated the mechanism of LINC00324 through miR-493-5p/MAPK1 in esophageal squamous cell carcinoma (ESCC) pathogenesis. Herein, we confirmed that LINC00324 is significantly upregulated in ESCC primary cells and esophageal squamous cell carcinoma cell line KYSE-70. Silencing of LINC00324 modulates cell proliferation markers, p21, p27, c-Myc, and Cyclin D1 and epithelial-to-mesenchymal transition markers, slug, snail, ZEB1, vimentin, ZO-1, and E-cadherin protein expression in ESCC. Through bioinformatics and dual luciferase reporter assays, we identified miR-493-5p as the direct target molecule of LINC00324. We further revealed that LINC00324 negatively regulates miR-493-5p expression in ESCC. Moreover, our multiple gain-and loss-of-functional experiments proved that a combination of miR-493-5p and LINC00324 significantly rescued ESCC cell proliferation and metastatic phenotypes. Mechanistically, LINC00324 promotes ESCC pathogenesis by acting as a competing endogenous RNA and sponges miR-493-5p activity thereby activating MAPK1 during ESCC progression. We believe that targeting LINC00324 /miR-493-5p/MAPK1 axis may provide new therapeutic avenues for ESCC.

EMT-like Activation in CLL Provides Novel Therapeutic Target

Introduction: There is growing interest in the role of Epithelial to Mesenchymal Transition (EMT) regulators in immune cell development, but the functional role of EMT in B-cell biology and transformation remains obscured. The outcomes of the "classical" EMT activation of epithelial cells are known; committed migratory cells do not divide and become resistant to apoptotic stimuli. Importantly, EMT supports migratory cells' "stem cell-like" properties, allowing differentiation into more than one cell type and/or terminal differentiation and senescence. Chronic lymphocytic leukaemia (CLL) cells engage with antigen and microenvironment stimuli in tissue sites that lead to proliferation, followed by their egress from the tissue sites into the peripheral blood. This lifecycle implies functional changes altering CLL cell movement. Here we show EMT-like activation in CLL that correlates with disease progression and transformation. We identified a targetable candidate and show that it can be inhibited by small molecule ligands. Results: Our analysis of available expression datasets showed the expression of various EMT factors in healthy B cells. The single-cell analysis of lymph nodes confirms strong EMT-like activation in healthy B-cells (GSE159929), confirming the idea that genes involved in classical EMT would be expressed differently depending on the stage of B-cell differentiation. The following GENEVESTIGATOR analysis of B cell malignancies highlighted high EMT-like activation in CLL cases. However, differential expression (DE) analysis of peripheral blood (PB) samples from 44 CLL patients and 24 healthy B-cell donors identified six EMT factors aberrantly expressed in CLL cells compared to healthy B cells indicating significant changes in CLL EMT-like activation. At the transcription level, ZEB1 expression was higher and ZEB2 expression lower in PB CLL cells. The main stimulatory events in CLL cells appear restricted to affected tissue sites, primarily in lymph nodes (LN) where CLL cells migrate to reactivate and proliferate. Further, EMT factor protein expression was confirmed in CLL LN tissue microarrays. Strikingly, transformation into a more aggressive form (Richter syndrome) strongly correlates with an increase in ZEB2 but not ZEB1 protein expression. Interestingly, analysis of the published transcriptome data set of CLL cells co-cultured with nurse-like cells shows an increase in ZEB2 and a decrease in ZEB1 expression after 14 days (GSE13811). An apparent reliance of the EMT-like activation in CLL on the environmental stimuli has been demonstrated in CLL cells stimulated with BCR activation (anti-IgM) or mitogen (PMA), implying protection from cell death and activation of migration. Additionally, we observed higher S100A4 expression in CLL cells compared to mature B-cells from healthy individuals. Previously it has been shown that this calcium-dependent protein can control cell movement by direct interaction with non-muscle myosin and via regulation of stress fibre formation, thus facilitating cellular protrusions and increasing cell migration and invasion. Our analysis has shown the S100A4 positive correlation with CLL markers of poor prognosis, Zap70 and CD38. The S100A4 knockdown with siRNA dramatically impeded CLL migration in trans-well assay (Figure 1). Biochemical inhibitor screening for disruption of S100A4 interaction with myosin II identified several promising leads active in the low micromolar range in both biochemical and cell assays. Conclusions: Our findings improve the understanding of CLL pathobiology. Normally, healthy naïve B cells give rise to multiple B-cell subtypes with specific functions and locations. EMT-like activation likely contributes to B-cell development at different stages of differentiation and B-cell malignant transformation. Our investigation shows that the EMT-like activation is dysregulated in CLL cells compared to healthy B cells. The EMT expression signature changes upon B-cell receptor stimulation. These results suggest that targeting some of the aberrantly expressed EMT components such as S100A4 in CLL can be exploited, prolonging remission by, for example, impeding CLL returning to the tissue sites after an egress due to the treatment with Ibrutinib depriving these cells of vital microenvironmental support. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Abstract 2634: Vasoactive intestinal polypeptide (VIP) regulates ZEB1 expression in cancer

Abstract Background: In cancer immunology, VIP is a neuropeptide with pleiotropic effects that include inhibiting T cell activation, increasing Treg frequency, and augmenting the myeloid-derived suppressor cells function. VIP expression by cancer may represent a paracrine immune-checkpoint pathway while simultaneously having autocrine effects on cancer growth. We used an in silico model to identify genes whose over-expression in cancer was correlated with VIP expression and confirmed the role of VIP receptor (VIPR) signaling in vitro using VIPR inhibitors. Methods: The TCGA PanCan dataset (UCSC Xena) was used to compare mRNA expression data of VIP versus 760 genes involved in 48 cancer related pathways. Associations by cancer histology and germ layer were calculated. A Pearson’s Coefficient cutoff of R &amp;gt; [0.3] defined a significant gene association. VIP and target gene expression were confirmed and quantified by western blot analysis in cancer cell lines and VIPR signaling was inhibited using ANT008, a novel VIPR inhibitor. Results: Significant associations were observed between VIP expression and 10 genes (Table 1). Strong associations (R &amp;gt; [0.4]) were observed in cancers derived from ectodermal and mesodermal germ layers. The strongest gene association was for VIP and ZEB1 in stomach adenocarcinoma (STAD, R = 0.7643). In vitro experiments using Jurkat T cells confirmed expression of VIP and ZEB1 proteins. Treatment with the VIPR inhibitor ANT008 (10μM) reduced VIP and ZEB1 protein expression by 50%. A similar fold increase in ZEB1 expression was observed with exogenous VIP. PCR studies are currently underway. Conclusions: The results demonstrate autocrine signaling between VIP expression and cancer associated gene pathways. ZEB1 is a known EMT (epithelial mesenchymal transition) regulator, and our in vitro studies novelly link VIP to EMT and demonstrate that this pathway is therapeutically targetable using a VIPR inhibitor. Further studies in the pertinent tumor histologies are warranted. Table 1. Genes All Samples Ectoderm (All) Mesoderm (All) Endoderm (All) MAPK3 0.3120 0.3537 0.2252 0.3380 ZEB1 0.3621 0.4341 0.4901 0.3348 NOS2 0.3611 0.3186 0.4849 0.3428 TEK 0.3113 0.3336 0.5131 0.2600 PTCH1 0.3031 0.4040 -0.0061 0.2879 EI4G1 -0.3216 -0.4268 -0.3779 -0.2064 GMPS -0.3074 -0.4698 -0.3005 -0.0928 CDK2 -0.3186 -0.4687 -0.2345 -0.0563 RUVBL1 -0.3135 -0.3270 -0.4716 -0.1886 TIMELESS -0.3036 -0.4284 -0.3717 -0.1505 Citation Format: Ishani H. Rao, Rohan Dhamsania, Edmund K. Waller, Sanjay Chandrasekaran. Vasoactive intestinal polypeptide (VIP) regulates ZEB1 expression in cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2634.

Abstract 6098: Analysis of the impact of ZEB1 expression on pathway enrichment and immune cell abundance in prostate cancer

Abstract Prostate cancer (PCa) reactivates a latent embryonic program called epithelial-mesenchymal transition during disease progression. The mesenchymal phenotype contributes to invasion, metastasis and therapy response variation. In this study, we characterize the downstream effects of ZEB1 expression based on cohort dichotomization of quartiles of expression comparing the 25% of tumors with high ZEB1 expression to the 75% of tumors with lower ZEB1 expression. Differential gene expression (DGE), gene set enrichment analysis (GSEA) and immune cell abundance prediction were accessed using PCa expression data from TCGA (n=488). ZEB1 high status based on gene expression was stratified based on ZEB1 expression values higher than the 3rd expression quartile value (n=122). Contingency analysis showed that PTEN loss is more frequent in the ZEB1 high group (Chi-square, p=0.0005) which supports previously presented data from our group that showed an inverse correlation between ZEB1 and PTEN protein expression but showed no difference between the groups regarding presence of the TMPRSS2-ERG fusion. DGE showed that more than 13,000 genes are differentially expressed in the ZEB1 high group while GSEA showed that signaling pathways such as HALLMARK TGF BETA, HALLMARK IL2 STAT5, HALLMARK INFLAMMATORY RESPONSE and HALLMARK NOTCH were upregulated in the ZEB1 high group compared to the low group. Yet inflammatory response related pathways were observed enriched in the ZEB1 high group, CIBERSORT analysis showed that effector immune cells such as CD8+ T cells and T helper cells (Holm-Sidak, p=000003 and p=0.000613, respectively) were enriched in the ZEB1 low group in comparison to the ZEB1 high group, which supports previous studies that suggest that ZEB1 cooperates with PTEN loss and TMPRSS2-ERG fusion to promote an immune evasive phenotype in PCa. Collectively, these findings demonstrate that gene expression changes associated with increased ZEB1 expression may involve clinically relevant pathways such as modulation of the tumor immune microenvironment and NOTCH-dependent tumor progression. Citation Format: Luiz Paulo Chaves, André Luiz Caliari, William Lautert-Dutra, Francisco Cesar Souza Silva, Camila Morais Melo, Fabiano Pinto Saggioro, Rodolfo Borges dos Reis, Jeremy Andrew Squire. Analysis of the impact of ZEB1 expression on pathway enrichment and immune cell abundance in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6098.

Co‐expression of Zeb‐1 and K17 provides new insights into the role of EMT in pancreatic cancer progression

INTRODUCTIONDespite improvements in screening and therapeutic options, pancreatic ductal adenocarcinoma (PDAC) has the third‐worst mortality rate, highlighting the need for better diagnostic and therapeutic approaches. Epithelial‐mesenchymal transition (EMT) has been conferred with a greater likelihood of metastasis in multiple cancers, specifically identified through markers like Zeb‐1 and Twist‐1. Zeb‐1 (Zinc Finger E‐Box Binding Homeobox 1) was shown to repress E‐cadherin expression, consequently inducing EMT.Our lab has previously identified K17 (Keratin 17) as an independent prognostic biomarker of poor outcomes in PDAC patients. While increasing evidence has suggested that EMT drives pancreatic cancer migration and metastasis, there have not been any well‐established connections between K17 expression and EMT. This study aims to provide new insights into the role of K17 in EMT by evaluating the co‐expression of K17 and Zeb‐1 in human PDAC biopsies. We hypothesized that tumor cells co‐express K17 and Zeb‐1 as a hallmark of EMT in PDAC. In addition, our study aims to establish a novel method of scoring Zeb‐1 expression in stromal fibroblasts via immunofluorescence to remove variability in the stromal constituency.METHODSPDAC tissues obtained from patients who underwent surgical resection between 2013 and 2017 at Stony Brook University (n=20) were selected for serial sectioning. Double immunofluorescence and single immunohistochemical staining were achieved by incubation with Zeb‐1 and K17 antibodies followed by incubation of fluorescent or biotin conjugated secondary antibodies, respectively. Histologic examination was performed using immunofluorescence staining along with H&amp;E and immunohistochemical staining (IHC) to determine concordance.RESULTS● IHC for Zeb‐1 was detected predominantly in the nuclei of centroacinar cells, benign proliferative ducts, and stromal fibroblasts. Focal nuclear staining in tumor epithelial was also detected in some cases.● High levels of K17 and but decreased expression of Zeb‐1 were detected in diffusely infiltrative cells with morphologic features of EMT.● Both high Zeb‐1 and high K17 protein expression were associated with poor survival of PDAC patients.● Using confocal immunofluorescence microscopy, we detected co‐localization of Zeb‐1 and K17 in PDAC.● We quantified Zeb‐1 protein expression in PDAC samples from 20 patients and confirmed significantly higher expression in the tumor‐associated stroma of high‐grade tumors compared to stromal fibroblasts in areas devoid of tumor.CONCLUSIONOur results provide evidence that K17 is upregulated in tumor epithelial cells that show morphologic immunohistochemical properties of EMT. The loss of Zeb‐1 in diffusely infiltrative, K17‐positive tumor cells suggests that EMT upregulates K17, driving tumor aggression. Our results suggest that K17 may enhance PDAC aggression by mediating EMT. Hence, novel therapeutic agents targeting K17 may improve clinical care for this biologically distinct patient subgroup.

Micro-RNA-215 and -375 regulate thymidylate synthase protein expression in pleural mesothelioma and mediate epithelial to mesenchymal transition

The standard front-line treatment for pleural mesothelioma (PM) is pemetrexed-based chemotherapy, whose major target is thymidylate synthase (TS). In several cancer models, miR-215 and miR-375 have been shown to target TS, while information on these miRNAs in PM are still limited although suggest their role in epithelial to mesenchymal transition. Seventy-one consecutive PM tissues (4 biphasic, 7 sarcomatoid, and 60 epithelioid types) and 16 commercial and patient-derived PM cell lines were screened for TS, miR-215, and miR-375 expression. REN and 570B cells were selected for miR-215 and miR-375 transient transfections to test TS modulation. ZEB1 protein expression in tumor samples was also tested. Moreover, genetic profile was investigated by means of BAP1 and p53 immunohistochemistry. Expression of both miR-215 and miR-375 was significantly higher in epithelioid histotype. Furthermore, inverse correlation between TS protein and both miR-215 and miR-375 expression was found. Efficiently transfected REN and 570B cell lines overexpressing miR-215 and miR-375 showed decreased TS protein levels. Epithelioid PM with a mesenchymal component highlighted by reticulin stain showed significantly higher TS and ZEB1 protein and lower miRNA expression. A better survival was recorded for BAP1 lost/TS low cases. Our data indicate that miR-215 and miR-375 are involved in TS regulation as well as in epithelial-to-mesenchymal transition in PM.

Abstract LB095: A domain-specific antibody to NRP2 down-regulated epithelial-mesenchymal transition genes and enhanced efficacy of standard-of-care therapeutics for aggressive breast cancer

Abstract INTRODUCTION: A growing body of evidence suggests that increased expression of the vascular endothelial growth factor (VEGF) co-receptor Neuropilin-2 (NRP2) is associated with aggressive breast cancers and that VEGF/NRP2 signaling contributes to clinical resistance to chemotherapy and tumor recurrence, making NRP2 a promising therapeutic target. A major limitation that has hampered the development of such a therapy, however, has been the lack of availability of high-quality anti-human NRP2 monoclonal antibodies (mAbs) that specifically block VEGF/NRP2 signaling. aTyr has generated a panel of high-quality, anti-human NRP2 mAbs that have the potential to be developed for the clinical management of diseases that involve NRP2 signaling. Among them, ATYR2810 has been characterized to bind to the b1 domain of NRP2 that encompasses the VEGF binding sites. It completely blocks the binding of VEGF to NRP2, and VEGF-induced NRP2/VEGFR dimerization. Importantly, ATYR2810 had no effect on Semaphorin 3F (Sema3F) induced NRP2/PlexinA1 dimerization, demonstrating its specificity for blocking the VEGF/NRP2 pathway. We have shown that ATYR2810, but not a Sema3F-blocking mAb has tumor-inhibitory effects on triple negative breast cancer (TNBC) cell lines or patient-derived organoids. RESULTS: We further evaluated the efficacy of ATYR2810 in combination with standard-of-care therapeutics including Cisplatin and Bevacizumab (anti-VEGF-A blocking antibody). In in vitro 3D methylcellulose colony formation assays, ATYR2810 sensitized TNBC cells to Cisplatin or Bevacizumab and considerably reduced colony formation in combination with these treatments. In an in vivo TNBC xenograft cancer model (MDA-MB-231), ATYR2810 augmented the anti-tumor effects of Cisplatin or Bevacizumab. To explore the underlying molecular mechanism, we performed gene expression profiling with samples treated by ATYR2810 alone and the combo therapy with Cisplatin or Bevacizumab. A number of gene markers of cancer stem cells (CSC) and/or epithelial-mesenchymal transition (EMT) were found to be down-regulated by ATYR2810 treatment in TNBC patient-derived organoids, including a key EMT transcription factor ZEB1. We also confirmed the reduction of ZEB1 protein expression by ATYR2810 treatment in TNBC cells. CONCLUSIONS: These results demonstrate the efficacy of ATYR2810 in combination with standard-of-care therapeutics in in vitro and in vivo TNBC models, and suggest its activity is mediated through inhibiting both EMT and cellular dedifferentiation that renders tumors more sensitive to the treatment regimes. The targeting of VEGF/NRP2 signaling by ATYR2810 may provide a new therapeutic option, and lead to the identification of new treatment biomarkers, which could offer improved efficacy and reduced toxicity in aggressive breast cancers. Citation Format: Zhiwen Xu, Christoph Burkart, Hira L. Goel, Justin Rahman, Clara Polizzi, Matt Seikkula, Luke Burman, Arthur M. Mercurio, Leslie A. Nangle. A domain-specific antibody to NRP2 down-regulated epithelial-mesenchymal transition genes and enhanced efficacy of standard-of-care therapeutics for aggressive breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB095.