Upregulation Of N-cadherin Research Articles

Co-Stimulation with TWEAK and TGF-β1 Induces Steroid-Insensitive TSLP and CCL5 Production in BEAS-2B Human Bronchial Epithelial Cells.

Steroid-resistant asthma is a common cause of refractory asthma. Type 2 inflammation is the main inflammatory response in asthma, and the mechanism underlying the steroid-resistance of type 2 inflammation has not been completely elucidated. Tumor-necrosis-factor-like apoptosis-inducing factor (TWEAK) and transforming growth factor (TGF)-β1 are involved in epithelial-mesenchymal transition (EMT) and the production of thymic stromal lymphopoietin (TSLP) and C-C motif chemokine ligand 5 (CCL5). We herein hypothesize that the combined exposure to TWEAK and TGF-β1 may result in the development of steroid resistance in bronchial epithelial cells. The bronchial epithelial cell line BEAS-2B was cultured with or without TGF-β1 or TWEAK, in the presence or absence of dexamethasone (DEX). The roles of Smad-independent pathways and MAP kinase phosphatase 1 (MKP-1) were also explored. Co-stimulation of TWEAK and TGF-β1 induced E-cadherin reduction, N-cadherin upregulation, and TSLP and CCL5 production, which were not suppressed by DEX. Inhibition of the nuclear factor kappa beta (NF-κB) and mitogen-activated protein kinase pathways downregulated steroid-unresponsive TSLP and CCL5 production, whereas knockdown of MKP-1 improved steroid-unresponsive TSLP production, induced by co-stimulation with TWEAK and TGF-β1. Therefore, co-stimulation with TWEAK and TGF-β1 can induce the steroid-insensitive production of TSLP and CCL5 in the bronchial epithelium and may contribute to airway inflammation.

Antiapoptotic and Prometastatic Roles of Cytokine FAM3B in Triple-Negative Breast Cancer

BackgroundTriple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer. FAM3B, a secreted protein, has been extensively studied in various types of tumors. However, its function in breast cancer remains poorly understood. MethodsWe analyzed FAM3B expression data from breast cancer patients available at TCGA database and overall survival was analyzed by using the Kaplan-Meier plotter. MDA-MB-231 TNBC tumor cell line and hormone-responsive MCF-7 cell lines were transfected to overexpress FAM3B. We assessed cell death, tumorigenicity, and invasiveness in vitro through MTT analysis, flow cytometry assays, anchorage-independent tumor growth, and wound healing assays, respectively. We performed in vivo evaluation by tumor xenograft in nude mice. ResultsIn silico analysis revealed that FAM3B expression was lower in all breast tumors. However, TNBC patients with high FAM3B expression had a poor prognosis. FAM3B overexpression protected MDA-MB-231 cells from cell death, with increased expression of Bcl-2 and Bcl-xL, and reduced caspase-3 activity. MDA-MB-231 cells overexpressing FAM3B also exhibited increased tumorigenicity and migration rates in vitro, displaying increased tumor growth and reduced survival rates in xenotransplanted nude mice. This phenotype is accompanied by the upregulation of EMT-related genes Slug, Snail, TGFBR2, vimentin, N-cadherin, MMP-2, MMP-9, and MMP-14. However, these effects were not observed in the MCF-7 cells overexpressing FAM3B. ConclusionFAM3B overexpression contributes to tumor growth, promotion of metastasis, and, consequently, leads to a poor prognosis in the most aggressive forms of breast cancer. Future clinical research is necessary to validate FAM3B as both a diagnostic and a therapeutic strategy for TNBC.

Inhibition of EGFR attenuates EGF-induced activation of retinal pigment epithelium cell via EGFR/AKT signaling pathway.

To explore the effect of epidermal growth factor receptor (EGFR) inhibition by erlotinib and EGFR siRNA on epidermal growth factor (EGF)-induced activation of retinal pigment epithelium (RPE) cells. Human RPE cell line (ARPE-19 cells) was activated by 100 ng/mL EGF. Erlotinib and EGFR siRNA were used to intervene EGF treatment. Cellular viability, proliferation, and migration were detected by methyl thiazolyl tetrazolium (MTT) assay, bromodeoxyuridine (BrdU) staining assay and wound healing assay, respectively. EGFR/protein kinase B (AKT) pathway proteins and N-cadherin, α-smooth muscle actin (α-SMA), and vimentin were tested by Western blot assay. EGFR was also determined by immunofluorescence staining. EGF treatment for 24h induced a significant increase of ARPE-19 cells' viability, proliferation and migration, phosphorylation of EGFR/AKT proteins, and decreased total EGFR expression. Erlotinib suppressed ARPE-19 cells' viability, proliferation and migration through down regulating total EGFR and AKT protein expressions. Erlotinib also inhibited EGF-induced an increase of proliferative and migrative ability in ARPE-19 cells and clearly suppressed EGF-induced EGFR/AKT proteins phosphorylation and decreased expression of N-cadherin, α-SMA, and vimentin proteins. Similarly, EGFR inhibition by EGFR siRNA significantly affected EGF-induced an increase of cell proliferation, viability, and migration, phosphorylation of EGFR/AKT proteins, and up-regulation of N-cadherin, α-SMA, and vimentin proteins. Erlotinib and EGFR-knockdown suppress EGF-induced cell viability, proliferation, and migration via EGFR/AKT pathway in RPE cells. EGFR inhibition may be a possible therapeutic approach for proliferative vitreoretinopathy (PVR).

IGF-1R, E-cadherin, N-cadherin, and occludin gene expression and resistance to EGFR tyrosine-kinase inhibitors in patients with lung adenocarcinoma in Serbia.

8524 Background: Mutations in epidermal growth factor-receptor ( EGFR) are found in approximately 10% of patients with lung adenocarcinoma in Serbia. EGFR tyrosine-kinase inhibitors ( EGFR-TKIs) is a safe and effective targeted therapy. However, most patients develop resistance. Acquired resistance to EGFR-TKI is defined as disease progression after achieving response or stable disease and there is a possible molecular association with epithelial-mesenchymal transition (EMT), particularly N-cadherin, E-cadherin, occludin and insulin-like growth factor-1 receptor (IGF-1R). We assessed the gene expression levels of IGF-1R, E-cadherin, N-cadherin, and occludin as predictive factors of resistance to EGFR-TKIs and survival outcomes in patients with advanced EGFR mutation-positive lung adenocarcinoma in Serbia. Methods: 101 patients with stage IIIB/IV EGFR mutation positive lung adenocarcinoma treated with EGFR-TKIs were included in the study. Quantitative RT-PCR was performed to determine mutations in EGFR, and expression of N-cadherin, E-cadherin, occludin and IGF-1R. Kaplan-Meier method was employed to estimate overall-survival (OS) and progression-free survival (PFS), with significance set at p<0.05. Results: Median age of patients was 61.7 (39 - 81) years, 65.3% were females and 91% were diagnosed with metastatic lung adenocarcinoma. EGFR mutations were more frequently detected in females (p <0.001). Median overall survival (OS) was 21 months (15.2–26.7 CI 95%) and progression-free survival (PFS) was 10 months (7.4-12.5 CI 95%). Patients whose tumors had lower levels of IGF-1R, and N-cadherin expression had statistically significantly longer PFS (p <0.001). Patients with lower levels of IGF-1R and higher levels of E-cadherin gene expression were more likely to have a complete or partial response to therapy (p = 0.04). There was a statistically significant association between elevated IGF-1R expression and the occurrence of EGFR-TKI resistance in patients (p <0.001). There was a statistically significant association between increased N-cadherin expression and reduced E-cadherin expression with EGFR-TKI resistance in patients. Conclusions: The hallmark of EMT is the upregulation of N-cadherin followed by the downregulation of E-cadherin. In this group of patients, higher levels of IGF-1R and N-cadherin gene expression were associated with shorter PFS and resistance to EGFR TKI, compared to higher levels of expression of E-cadherin, which may indicate potential value of expression of these genes in predicting acquired resistance to EGFR-TKI. Therefore, more research is needed to prove the potential predictive value of E-cadherin, N-cadherin and IFG-1R in acquired resistance to EGFR-TKI and developing novel therapeutic options with anti-EMT properties that can overcome this problem.

Epithelial-mesenchymal transition contrast in the amputated tail and limb of the northern house gecko, Hemidactylus flaviviridis.

The northern house gecko Hemidactylus flaviviridis exhibits appendage-specific responses to injuries. The autotomized tail regenerates, whereas the severed limb fails to regrow. Many site-specific cellular processes influence tail regeneration. Herein, we analyzed the epithelial-mesenchymal transition contrast in the lizard's amputated appendages (tail and limb). Morphological observations in the healing frame indicated the formation of regeneration blastema in the tail and scar formation in limb. Histology of the tail showed that epithelial cells closer to mesenchyme appeared less columnar and loosely packed, with little intercellular matrix. Whereas in the limb, the columnar epithelial cells remained tightly packed. Collagen deposition was seen in the limb at the intersection of wound epithelium and mesenchyme, favoring scarring by blocking the epithelial-mesenchymal transition. Markers for epithelial-mesenchymal transition were assessed at transcript and protein levels. The regenerating tail showed upregulation of N-cadherin, vimentin, and PCNA, favoring epithelial-mesenchymal transition, cell migration, and proliferation, respectively. In contrast, the scarring limb showed persistently elevated levels of E-cadherin and EpCAM, indicating retention of epithelial characteristics. An attempt was made to screen the resident epithelial stem cell population in both appendages to check their potential role in the epithelial-mesenchymal transition (EMT), hence the differential wound healing. Upregulation in transcript and protein levels of Nanog and Sox2 was observed in the regenerating tail. Fluorescence-activated cell sorting (FACS) provided supporting evidence that the epithelial stem cell population in tail remained significantly higher than in limb. Thus, this study focuses on the mechanistic role of the epithelial-mesenchymal transition in wound healing, highlighting the molecular details of regeneration and scarring events.

FLRT2 mediates chondrogenesis of nasal septal cartilage and mandibular condyle cartilage.

Nasal septal cartilages (NSCs) and mandibular condyle cartilages (MCCs) are two important cartilages for craniomaxillofacial development. However, the role of FLRT2 in the formation of NSCs and MCCs remains undiscovered. NSCs and MCCs were used for immunocytochemistry staining of collagen II, toluidine blue staining, and alcian blue staining. Quantitative reverse transcription‑PCR and western blot were used to detect mRNA and protein expressions of FLRT2, N-cadherin, collagen II, aggrecan, and SOX9. Cell proliferation of MCCs and NSCs was tested by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and cell counting kit‑8 assay. Cell migration of MCCs and NSCs was examined by wound healing assay and Transwell. Chondrogenesis of MCCs and NSCs were similar in morphological characteristics, while different in cell proliferation, migration, and extracellular matrix. FLRT2 promotes the proliferation and migration of NSCs. There were up-regulation of N-cadherin and down-regulation of collagen II, aggrecan, and SOX9 in NSC with knock down FLRT2. The current study, as demonstrated by Xie et al., reveals that FLRT2 overexpression in Sprague-Dawley neonatal rats promotes the proliferation and migration of NSCs and MCCs, decreases N-cadherin while increases collagen II, aggrecan, and SOX9 in NSC and MCCs. Altogether, FLRT2 mediates chondrogenesis of NSCs and MCCs.

EMT Dynamics in Lymph Node Metastasis of Oral Squamous Cell Carcinoma.

Epithelial-mesenchymal transition (EMT) enables tumor cell invasion and metastasis. Many studies have demonstrated the critical role of EMT in lymph node metastasis in oral squamous cell carcinoma (OSCC). During EMT, epithelial cancer cells lose intercellular adhesion and apical-basal polarity and acquire mesenchymal properties such as motility and invasiveness. A significant feature of EMT is cadherin switching, involving the downregulation of E-cadherin and upregulation of N-cadherin. The TGF-β/SMAD pathway can also induce EMT. We aimed to evaluate EMT markers as predictors of lymph node metastasis in OSCC. We performed genetic profiling of 159 primary OSCCs from TCGA and analyzed the expression of EMT markers, including cadherin switch genes (CDH1, CDH2), and TGF-β/SMAD pathway genes. Samples were divided into advanced (stage III-IV) and early (stage I-II) stage groups. Differential expression analysis was performed, as well as an independent validation study containing fresh OSCC samples. TGF-β/SMAD pathway genes such as SMAD6 were upregulated in advanced stage tumors. N-cadherin and SNAIL2 were overexpressed in node-positive tumors. Keratins were downregulated in these groups. Our findings demonstrate that EMT marker expression correlates with lymph node metastasis in OSCC. Developing therapies targeting regulators such as N-cadherin may prevent metastasis and improve outcomes.

Decreased NMIIA heavy chain phosphorylation at S1943 promotes mitoxantrone resistance by upregulating BCRP and N-cadherin expression in breast cancer cells.

Mitoxantrone (MX) is an effective treatment for breast cancer; however, high efflux of MX that is accomplished by breast cancer resistance protein (BCRP) leads to acquired multidrug resistance (MDR), reducing MX's therapeutic efficacy in breast cancer. Non-muscle myosin IIA (NMIIA) and its heavy phosphorylation at S1943 have been revealed to play key roles in tumor metastasis and progression, including in breast cancer; however, their molecular function in BCRP-mediated MDR in breast cancer remains unknown. In this study, we revealed that the expression of NMIIA heavy chain phosphorylation at S1943 was downregulated in BCRP-overexpressing breast cancer MCF-7/MX cells, and stable expression of NMIIA-S1943A mutant increased BCRP expression and promoted the resistance of MCF-7/MX cells to MX. Meanwhile, NMIIA S1943 phosphorylation induced by epidermal growth factor (EGF) was accompanied by the downregulation of BCRP in MCF-7/MX cells. Furthermore, stable expression of NMIIA-S1943A in MCF-7/MX cells resulted in upregulation of N-cadherin and the accumulation of β-catenin on the cell surface, which inhibited the nucleus translocation of β-catenin and Wnt/β-catenin-based proliferative signaling. EGF stimulation of MCF-7/MX cells showed the downregulation of N-cadherin and β-catenin. Our results suggest that decreased NMIIA heavy phosphorylation at S1943 increases BCRP expression and promotes MX resistance in breast cancer cells via upregulating N-cadherin expression.

Expression of circRNA-0036474 in esophageal cancer and its clinical significance.

Circular RNAs are considered to play important roles in the progression of different cancers such as esophageal squamous cell carcinoma. However, the functions of circular RNAs in esophageal squamous cell carcinoma are still not clear. This study aimed to investigate the role and mechanism of circRNA-0036474 in the progression of esophageal squamous cell carcinoma. The hsa_circ_0036474 expression levels were found to be elevated in both EC109 cells and esophageal squamous cell carcinoma tissue samples. Moreover, knockdown of circRNA-0036474 expression in the EC109 cells induced migration and invasion, characterized by the down-regulation of E-cadherin, and up-regulation of N-cadherin and vimentin. In addition, the over-expressed hsa_circ_0036474 significantly decreased the activity of EC109 cells, elevated E-cadherin expression but declined N-cadherin and vimentin expression. Moreover, over-expressed mir-223-3p levels and interfered RERG expression verified the role of hsa_circ_0036474 in inhibiting the invasion and migration of EC109 cells, reducing the expression of N-cadherin and vimentin, and promoting the expression of E-cadherin. In conclusion, circRNA-0036474 mitigated the progression of esophageal squamous cell carcinoma through regulating mir-223-3p/RERG axis, presenting a potential therapeutic target for the treatment.

Sunitinib Treatment of VHL C162F Cells Slows Down Proliferation and Healing Ability via Downregulation of ZHX2 and Confers a Mesenchymal Phenotype.

von Hippel-Lindau (VHL) disease, due to mutations of the tumor suppressor VHL gene, is a rare hereditary syndrome with a high risk of developing clear cell renal cell carcinoma (ccRCC). We asked whether the VHL-C162F mutation interferes with proliferation, migration, healing and forming colony ability by using wild-type VHL (WT VHL) and VHL-C162F reconstituted cells. We then analyzed the in vitro impact of the sunitinib treatment on VHL-C162F cells. We showed that VHL-C162F mutations have no impact on cell morphology, colony formation and migration ability but confer a significant higher healing ability than in WT VHL cells. RNA sequencing analysis revealed that VHL-C162F mutation upregulates genes involved in hypoxia and epithelial mesenchymal transition (EMT) pathways by comparison with VHL WT cells. We next showed a decrease in healing ability in VHL-C162F cells depleting on ZHX2, an oncogenic driver of ccRCC, highlighting the potential involvement of ZHX2 in aggressiveness of the VHL-C162F cells. Moreover, we found that sunitinib treatment inhibits ZHX2 expression and induces a reduced proliferation correlating with downregulation of P-ERK. Sunitinib treatment also conferred a more mesenchymal profile to VHL-C162F cells with significant downregulation of E-cadherin and upregulation of N-cadherin, Slug and AXL. Sunitinib therapy may therefore promote disease progression in VHL-C162F patients.

Altered Phenotypes of Breast Epithelial × Breast Cancer Hybrids after ZEB1 Knock-Out.

ZEB1 plays a pivotal role in epithelial-to-mesenchymal transition (EMT), (cancer) cell stemness and cancer therapy resistance. The M13HS tumor hybrids, which were derived from spontaneous fusion events between the M13SV1-EGFP-Neo breast epithelial cells and HS578T-Hyg breast cancer cells, express ZEB1 and exhibit prospective cancer stem cell properties. To explore a possible correlation between the ZEB1 and stemness/ EMT-related properties in M13HS tumor hybrids, ZEB1 was knocked-out by CRISPR/Cas9. Colony formation, mammosphere formation, cell migration, invasion assays, flow cytometry and Western blot analyses were performed for the characterization of ZEB1 knock-out cells. The ZEB1 knock-out in M13HS tumor cells was not correlated with the down-regulation of the EMT-related markers N-CADHERIN (CDH2) and VIMENTIN and up-regulation of miR-200c-3p. Nonetheless, both the colony formation and mammosphere formation capacities of the M13HS ZEB1 knock-out cells were markedly reduced. Interestingly, the M13HS-2 ZEB1-KO cells harbored a markedly higher fraction of ALDH1-positive cells. The Transwell/ Boyden chamber migration assay data indicated a reduced migratory activity of the M13HS ZEB1-knock-out tumor hybrids, whereas in scratch/ wound-healing assays only the M13SH-8 ZEB1-knock-out cells possessed a reduced locomotory activity. Similarly, only the M13HS-8 ZEB1-knock-out tumor hybrids showed a reduced invasion capacity. Although the ZEB1 knock-out resulted in only moderate phenotypic changes, our data support the role of ZEB1 in EMT and stemness.

Perfluorooctanesulfonic Acid and Perfluorooctanoic Acid Promote Migration of Three-Dimensional Colorectal Cancer Spheroids.

Perfluorooctanesulfonic acid (PFOS) and perfluorooctanoic acid (PFOA) are persistent environmental contaminants that are of increasing public concern worldwide. However, their relationship with colorectal cancer (CRC) is poorly understood. This study aims to comprehensively investigate the effect of PFOS and PFOA on the development and progression of CRC in vitro using a series of biological techniques and metabolic profiling. Herein, the migration of three-dimensional (3D) spheroids of two CRC cell lines, SW48 KRAS wide-type (WT) and SW48 KRAS G12A, were observed after exposure to PFOS and PFOA at 2 μM and 10 μM for 7 days. The time and dose-dependent migration phenotype induced by 10 μM PFOS and PFOA was further confirmed by wound healing and trans-well migration assays. To investigate the mechanism of action, derivatization-mass spectrometry-based metabolic profiles were examined from 3D spheroids of SW48 cell lines exposed to PFOS and PFOA (2 μM and 10 μM). Our findings revealed this exposure altered epithelial-mesenchymal transition related metabolic pathways, including fatty acid β-oxidation and synthesis of proteins, nucleotides, and lipids. Furthermore, this phenotype was confirmed by the downregulation of E-cadherin and upregulation of N-cadherin and vimentin. These findings show novel insight into the relationship between PFOS, PFOA, and CRC.

Engineering alginate/carboxymethylcellulose scaffolds to establish liver cancer spheroids: Evaluation of molecular variances between 2D and 3D models

Natural polymeric hydrogels represent an optimal framework for 3D culture development. This study demonstrates a freeze-thaw-based ionic crosslinking technique for fabricating alginate/carboxymethylcellulose scaffold for culturing human hepatocellular carcinoma, Huh-7 cells to generate 3D spheroids. Consolidating morphological and biomechanical characterization of Alg/CMC scaffolds shows the formation of uniform hydrogels with significant crosslinking (ATR-FTIR), multiscale pores (FE-SEM), swelling/water absorbance, softer texture, viscoelasticity (rheology), spreading nature (contact angle), and degradation rate optimal for 3D culture establishment. The influence of cell seeding density and time with spheroid formation reveals a maximal size of 250–300 μm on day 7. Calcein AM and Propidium iodide staining confirm that a culmination of viable and dead cells generates spheroidal heterogeneity. RT-qPCR in 3D culture against RPL-13 and 2D culture controls indicate an upregulation of E-cadherin, N-cadherin, fibronectin, and integrin α9/β6. Further, western blotting and immunofluorescence confirm the collective display of cellular interactions in 3D spheroids. Thus, the expression profile signifies the role of key genes during the assembly and formation of 3D spheroids in 1%Alg/1%CMC scaffolds with a profound epithelial characteristic. In the future, this study will bring a 3D spheroid model in a platter for elucidating epithelial to mesenchymal transition of cells during in vitro disease modeling.

CLDN6 Suppresses Migration and Invasion of MCF-7 and SKBR-3 Breast Cancer Cells by Blocking the SMAD/Snail/MMP-2/9 Axis.

The study examined the mechanisms of action of signal protein claudin 6 (CLDN6) on migration and invasion of breast cancer cell lines MCF-7 and SKBR-3. To this end, the signal proteins SMAD were blocked with their inhibitor SB431542, the genes CLDN6 and SNAIL were knocked down with short hairpin RNAs, and MMP2 and MMP9 were inhibited with TIMP-1. Expressions of MMP2 and MMP9 mRNAs were evaluated by reverse transcription PCR, Expressions of MMP-2, MMP-9, E-cadherin, N-cadherin, and vimentin were examined by Western blotting. Migration and invasion were analyzed by scratch test and Matrigel invasion assay. SB431542 inhibited expression of MMP2 and MMP9 in both cell lines. Single use of SB431542 inhibited expression of MMP-2/MMP-9 and corresponding mRNAs, but subsequent silencing of CLDN6 gene reversed this effect. TIMP-1 reversed down-regulation of E-cadherin, upregulation of N-cadherin and vimentin, facilitation of migration and invasion evoked by CLDN6 knocking down. Silencing of SNAIL gene inhibited migration and invasion, upregulated the expression of E-cadherin, and down-regulated expression of MMP2, MMP 9, N-cadherin, and vimentin. Thus, CLDN6 suppresses the epithelial-mesenchymal transition, migration, and invasion via blocking SMAD/Snail/MMP-2/9 signaling pathway in MCF-7 and SKBR-3 cancer cell lines.

CRP‑1 promotes the malignant behavior of hepatocellular carcinoma cells via activating epithelial‑mesenchymal transition and Wnt/β‑catenin signaling.

Hepatocellular carcinoma (HCC) is one of the most common malignancies worldwide. It has been reported that cysteine rich protein 1 (CRP-1) is dysregulated in several types of human cancer; however, its role in HCC is poorly understood. Therefore, the current study aimed to investigate the role of CRP-1 in HCC. Western blotting and reverse transcription-quantitative PCR results showed that CRP-1 was upregulated in HCC cell lines. Furthermore, for in vitro experiments, CRP-1 was knocked down and overexpressed in the HCC cell lines Hep 3B2.1-7 and BEL-7405, respectively. c-Myc and proliferating cell nuclear antigen upregulation, and cleaved caspase 3 and poly(ADP-ribose) polymerase downregulation suggested that CRP-1 silencing could inhibit the proliferation and colony-forming ability of HCC cells, and induce apoptosis. In addition, CRP-1 overexpression promoted the malignant behavior of HCC cells and induced epithelial-mesenchymal transition (EMT), as verified by E-cadherin downregulation, and N-cadherin and vimentin upregulation. Additionally, CRP-1 overexpression promoted the nuclear translocation of β-catenin, and activated the expression of cyclin D1 and matrix metalloproteinase-7. Furthermore, inhibition of Wnt/β-catenin signaling, following cell treatment with XAV-939, an inhibitor of the Wnt/β-catenin signaling pathway, abrogated the effects of CRP-1 on enhancing the proliferation and migration of HCC cells. These findings indicated that the regulatory effect of CRP-1 on HCC cells could be mediated by the Wnt/β-catenin signaling pathway. Overall, CRP-1 could promote the proliferation and migration of HCC cell lines, partially via promoting EMT and activating the Wnt/β-catenin signaling pathway.

Immunohistochemical expression of E-cadherin and N-cadherin in endometrioid endometrial carcinoma and its precursor lesions

ObjectivesEndometrial carcinoma (EC), majority being endometroid endometrial carcinoma (EEC), is the most common invasive gynecological cancer world-wide. Epithelial mesenchymal transition (EMT) is the process that cells undergo to switch from a polarized epithelial phenotype to a motile mesenchymal phenotype. Loss of E-cadherin and expression of N-cadherin are a critical step for development and progression of malignant tumors. In present study we evaluated the immunohistochemical (IHC) expression of E-cadherin and N-cadherin with clinicopathological parameters of EEC and its precursors lesions. MethodsIt was a prospective nested case-control study involving women aged 35–70 years, whose endometrial biopsy were obtained for histological diagnosis. Signed informed consent was obtained before enrolling the women in the study, and the approval to conduct the study was obtained from the institutional ethics committee. The IHC was done to measure the E-cadherin and N-cadherin. ResultsReduced expression of E-cadherin was observed in 69% of cancer and 65% of precancer, however only 32% of normal proliferative endometrium showed reduced E-cadherin expression. N-cadherin expression was observed in 82% of EEC and 81% of precancer cases, while 11% of normal proliferative endometrium showed N-cadherin expression. Statistically significant downregulation of E-cadherin and upregulation of N-cadherin were observed in EEC and precancerous lesions as compared to normal control. ConclusionSince downregulation of E-cadherin was also associated with upregulation of N-cadherin, the integration of immuno-expression of both E-cadherin and N-cadherin might offer a potential therapeutic target for EEC.

An Ethanol Extract of Perilla frutescens Leaves Suppresses Adrenergic Agonist-Induced Metastatic Ability of Cancer Cells by Inhibiting Src-Mediated EMT.

Previous studies have indicated that the adrenergic receptor signaling pathway plays a fundamental role in chronic stress-induced cancer metastasis. In this study, we investigated whether an ethanol extract of Perilla frutescens leaves (EPF) traditionally used to treat stress-related symptoms by moving Qi could regulate the adrenergic agonist-induced metastatic ability of cancer cells. Our results show that adrenergic agonists including norepinephrine (NE), epinephrine (E), and isoproterenol (ISO) increased migration and invasion of MDA-MB-231 human breast cancer cells and Hep3B human hepatocellular carcinoma cells. However, such increases were completely abrogated by EPF treatment. E/NE induced downregulation of E-cadherin and upregulation of N-cadherin, Snail, and Slug. Such effects were clearly reversed by pretreatment with EPF, suggesting that the antimetastatic activity of EPF could be related to epithelial-mesenchymal transition (EMT) regulation. EPF suppressed E/NE-stimulated Src phosphorylation. Inhibition of Src kinase activity with dasatinib completely suppressed the E/NE-induced EMT process. Transfecting MDA-MB-231 cells with constitutively activated Src (SrcY527F) diminished the antimigration effect of EPF. Taken together, our results demonstrate that EPF can suppress the adrenergic agonist-promoted metastatic ability of cancer cells by inhibiting Src-mediated EMT. This study provides basic evidence supporting the probable use of EPF to prevent metastasis in cancer patients, especially those under chronic stress.

Abstract P2-26-19: Slc26a9 cooperates with HER2 to regulate the progression and development of HER2-positive breast cancer

Abstract Goals: Ion transporters play an important regulatory role in the progression and development of breast cancer (BC). Slc26a9 is a member of the Slc26a anion transporter family, which is mainly involved in regulating the secretion of chloride ions and bicarbonate, but the role of Slc26a9 in HER2-positive BC is still unclear. Methods: Tissue microarray and BC cell line were used to detect the expression level of Slc26a9 and its clinical relevance. By changing the expression level of Slc26a9 gene in BC cells, the effect of Slc26a9 gene on the biological behavior of BC cells and its related molecular mechanism were discussed. Results: We found that the expression of Slc26a9 was significantly upregulated in BC compared with adjacent tissues, and the upregulated Slc26a9 was associated with TNM staging and poor prognosis in BC patients. In addition, the expression of Slc26a9 was significantly upregulated in HER2-positive BC compared with HER2-negative BC, and similar results were obtained in BC cell lines, with Slc26a9 was the highest expression in HER2-enriched SKBR3 cells. Functionally, the proliferation, migration, invasion and anti-apoptotic abilities of SKBR3 cells were significantly inhibited after silencing Slc26a9, and tumorigenesis and metastasis were significantly inhibited in vivo. On the contrary, overexpression of Slc26a9 resulted in the opposite result. Mechanistically, overexpression of Slc26a9 activated the PI3K/AKT/mTOR signaling pathway, the key signaling pathway implicated in HER2-positive breast carcinogenesis, and promoted the expression of downstream proliferation related genes CCND1 (Cyclin D1) and c-Myc, and downregulated the expression of apoptosis related genes Caspase9, apoptosis-inducing factor (AIF) and endonuclease G (Endo G), indicating the simultaneous inhibition of caspase dependent and independent apoptosis pathway. At the same time, accompanied by changes in markers of epithelial-mesenchymal transition (EMT), including downregulation of E-cadherin and ZO-1, and upregulation of N-cadherin and Fibronectin, and SKBR3 cells changed from epithelioid morphology to mesenchymal morphology. In addition, immunofluorescence and protein nucleoplasm separation experiments showed that Slc26a9 upregulated the expression of HER2 and co-localized with HER2 in the nucleus. Co-immunoprecipitation experiments proved that Slc26a9 interacted with HER2. Furthermore, trastuzumab downregulated the expression of Slc26a9 by targeting HER2 in SKBR3 cells. Moreover, when Slc26a9 was overexpressed, the inhibitory effect of trastuzumab on HER2 was partially reversed, and we also verified the PI3K/AKT/mTOR signaling pathway. Not only that, we found that Slc26a9 was significantly upregulated in drug-resistant cell lines relative to parental cells by constructing SKBR3 drug-resistant cell lines, indicating that Slc26a9 expression was significantly correlated with chemotherapy resistance in HER2-positive BC. Conclusion(s): Slc26a9 may interact with HER2 in the form of molecular chaperones to activate PI3K/AKT/mTOR signaling pathway to promote the progression and development of HER2-positive BC and be associated with chemotherapy resistance, but the precise molecular mechanism needs further exploration. Conflict of Interest: No significant relationships. Citation Format: Zhengxing Zhou, Zhiyuan Ma, Xuemei Liu, Chengmin Zhang, Hu Wang, Renmin Mu, Xiaoming Cheng, Biguang Tuo, Taolang Li. Slc26a9 cooperates with HER2 to regulate the progression and development of HER2-positive breast cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-26-19.

MSX1 Regulates Goat Endometrial Function by Altering the Plasma Membrane Transformation of Endometrial Epithelium Cells during Early Pregnancy

MSX1 is an important member of the muscle segment homeobox gene (Msh) family and acts as a transcription factor to regulate tissue plasticity, yet its role in goat endometrium remodeling remains elusive. In this study, an immunohistochemical analysis showed that MSX1 was mainly expressed in the luminal and glandular epithelium of goat uterus, and the MSX1 expression was upregulated in pregnancy at days 15 and 18 compared with pregnancy at day 5. In order to explore its function, goat endometrial epithelial cells (gEECs) were treated with 17 β-estrogen (E2), progesterone (P4), and/or interferon-tau (IFNτ), which were used to mimic the physiological environment of early pregnancy. The results showed that MSX1 was significantly upregulated with E2- and P4-alone treatment, or their combined treatment, and IFNτ further enhanced its expression. The spheroid attachment and PGE2/PGF2α ratio were downregulated by the suppression of MSX1. The combination of E2, P4, and IFNτ treatment induced the plasma membrane transformation (PMT) of gEECs, which mainly showed the upregulation of N-cadherin (CDH2) and concomitant downregulation of the polarity-related genes (ZO-1, α-PKC, Par3, Lgl2, and SCRIB). The knockdown of MSX1 partly hindered the PMT induced by E2, P4, and IFNτ treatment, while the upregulation of CDH2 and the downregulation of the partly polarity-related genes were significantly enhanced when MSX1 was overexpressed. Moreover, MSX1 regulated the CDH2 expression by activating the endoplasmic reticulum (ER) stress-mediated unfolded protein response (UPR) pathway. Collectively, these results suggest that MSX1 was involved in the PMT of the gEECs through the ER stress-mediated UPR pathway, which affects endometrial adhesion and secretion function.

CD70 and PD-L1 (CD274) co-expression predicts poor clinical outcomes in patients with pleural mesothelioma.

Diffuse pleural mesothelioma (PM) is a highly aggressive tumour typically associated with short survival. Recently, the effectiveness of first-line immune checkpoint inhibitors in patients with unresectable PM was reported. CD70-CD27 signalling plays a co-stimulatory role in promoting T cell expansion and differentiation through the nuclear factor κB (NF-κB) pathway. Conversely, the PD-L1 (CD274)-PD-1 (PDCD1) pathway is crucial for the modulation of immune responses in normal conditions. Nevertheless, pathological activation of both the CD70-CD27 and PD-L1-PD-1 pathways by aberrantly expressed CD70 and PD-L1 participates in the immune evasion of tumour cells. In this study, 171 well-characterised PMs including epithelioid (n=144), biphasic (n=15), and sarcomatoid (n=12) histotypes were evaluated immunohistochemically for CD70, PD-L1, and immune cell markers such as CD3, CD4, CD8, CD56, PD-1, FOXP3, CD68, and CD163. Eight percent (14/171) of mesotheliomas simultaneously expressed CD70 and PD-L1 on the tumour cell membrane. PMs co-expressing CD70 and PD-L1 contained significantly higher numbers of CD8+ (p=0.0016), FOXP3+ (p=0.00075), and CD163+ (p=0.0011) immune cells within their microenvironments. Overall survival was significantly decreased in the cohort of patients with PM co-expressing CD70 and PD-L1 (p < 0.0001). In vitro experiments revealed that PD-L1 and CD70 additively enhanced the motility and invasiveness of PM cells. In contrast, PM cell proliferation was suppressed by PD-L1. PD-L1 enhanced mesenchymal phenotypes such as N-cadherin up-regulation. Collectively, these findings suggest that CD70 and PD-L1 both enhance the malignant phenotypes of PM and diminish anti-tumour immune responses. Based on our observations, combination therapy targeting these signalling pathways might be useful in patients with PM.