E-cadherin Protein Expression Research Articles

MiR-129–2-3p binds SEMA4C to regulate HCC development and inhibit the EMT

BackgroundAmong primary liver cancers, HCC is the most prevalent. Small noncoding RNAs called miRNAs control the expression of downstream target genes to take part in a variety of physiological and pathological processes, including those related to cancer. MethodsmiR-129–2–3p and SEMA4C expression levels were assessed using RT-qPCR. The CCK-8, invasion, and wound healing assays were used to confirm the capacity of HCC cells for proliferation, invasion and migration respectively. Serum SEMA4C levels were detected via ELISA. The RIP and dual-luciferase reporter assays were used to confirm the existence of intergenic binding sites. Cell apoptosis assay and cell cycle assay were performed to detect the apoptosis rate and cycle distribution of cells, and WB was performed to detect the protein expression of SEMA4C, RhoA, ROCK1, E-cadherin, N-cadherin, and vimentin. Furthermore, cancer-inhibiting role of miR-129–2–3p were further confirmed by animal tests. ResultsmiR-129–2–3p expression was reduced in HCC tissues and cells. Overexpression of miR-129–2–3p decreased the proliferation, invasion, migration, and EMT in HCC cells, whereas inhibition of miR-129–2–3p had the opposite effects. Our research also showed that SEMA4C was increased in HCC tissues, serum and cells, and that SEMA4C knockdown prevented HCC cell invasion, migration, proliferation, and EMT. Overexpression of SEMA4C reversed the inhibitory effect of miR-129–2–3p on HCC. ConclusionsOverall, we discovered that through binding to SEMA4C, miR-129–2–3p regulates HCC cell proliferation, invasion, migration, and EMT.

Mechanism of Zhenwu Decoction modulating TLR4/NF-κB/HIF-1α loop through miR-451 to delay renal fibrosis in type 2 CRS

BackgroundType 2 cardiorenal syndrome (CRS) is a progressive renal insufficiency in patients with chronic heart failure, but its pathophysiology is still unclear. The Chinese medicine Zhenwu Decoction plays an important role in the prevention and treatment of 2-CRS, however, its mechanism of action remains unknown. PurposeThe aim of this study was to investigate whether the ameliorative effect of ZWD on 2-CRS renal fibrosis is related to the modulation of miR-451 expression and thus mediating the TLR4/NF-κB/HIF-1α loop. Study design and methodsA type 2 CRS rat model was constructed using ligation of the left anterior descending branch of the coronary artery + 3/4 nephrectomy, and randomly divided into Control, Sham, Model, Captopril, ZWD-L, ZWD-M and ZWD-H groups.After 4 weeks of ZWD intervention, its effects on cardiac and renal functions of type 2 CRS rats were observed by hematuria and cardiac ultrasonography. Changes in kidney tissue morphology were observed by HE, Masson and PASM staining. The protein and mRNA expression of TLR4, NF-κB, HIF-1α and IκBα in kidney tissues were detected by immunohistochemistry and qPCR. Immunofluorescence was used to detect the protein expression of NF-κB and HIF-1α in renal tissues. Western blot and qPCR were used to detect the protein expression of MCP-1, ICAM-1, IL-1β, IL-6, TGF-β, α-SMA, FN, Smad2, Smad3, and E-cadherin in renal tissues. PCR was used to detect the protein expression of miR-451mRNA expression level in kidney tissues. ResultsIn this study, we found that ZWD was able to reduce the expression of Scr, BUN, NT-proBNP, and 24-hour quantitative urine protein, elevate LVEF, FS, CO, and reduce the level of LVIDS in type 2 CRS rats, as well as attenuate renal interstitial fibrosis and improve tubular swelling. In addition, Zhenwu Decoction up-regulated the expression of miR-451 in renal tissues and inhibited the expression of TLR4, NF-κB, and HIF-1α proteins and genes, which in turn inhibited the expression of inflammatory factors and fibrosis-related factors. ConclusionZWD was able to up-regulate the expression of miR-451 in renal tissues, inhibit the TLR4/NF-κB/HIF-1α response loop, and then inhibit the expression of inflammatory factors and fibrosis-related factors, improve renal fibrosis, and delay the pathological process of type 2 CRS.

ARHGAP17 Inhibits Hepatocellular Carcinoma Progression by Inactivation of Wnt/β-Catenin Signaling Pathway

ARHGAP17 Inhibits Hepatocellular Carcinoma Progression by Inactivation of Wnt/β-Catenin Signaling Pathway

Abstract GS03-04: Novel Mechanisms of CDH1 Inactivation in Breast Invasive Lobular Carcinoma Unveiled by the Integration of Artificial Intelligence and Genomics

Abstract Background: Invasive lobular carcinoma (ILC) of the breast is the second most common histologic subtype of breast cancer (BC), following invasive ductal carcinoma of no special type (IDC-NST). The hallmark histologic feature of ILC is cellular discohesiveness, the result of bi-allelic inactivation of CDH1, and represents an important genotypic-phenotypic correlation in BC. Although most ILCs harbor CDH1 loss-of function mutations associated to loss-of-heterozygosity (LOH) of the wild type-allele, a subset of ILCs lack these alterations despite displaying a typical lobular phenotype. Here, we sought to identify alternative molecular mechanisms converging on CDH1 inactivation by employing an integrative artificial intelligence (AI) and genomics approach. Materials and Methods: A genomics-driven AI-based algorithm using hematoxylin and eosin (H&E) whole slide images (WSIs) as input, previously developed to detect bi-allelic CDH1 mutations (inactivating mutation associated to LOH) in BC was employed. WSIs of 1,057 BCs including ILCs (n=187) and non-lobular BCs (n=870) previously subjected to FDA-cleared tumor/normal targeted sequencing were subjected to analysis with the AI-based algorithm. Cases predicted to harbor CDH1 bi-allelic mutations by the AI-model but lacking CDH1 bi-allelic mutations by targeted sequencing were assessed through targeted sequencing data re-analysis, CDH1 gene promoter methylation evaluation and/or whole genome sequencing analysis. Results: AI-based analysis WSIs corresponding to 1,057 BCs resulted in the identification of 34 cases found to lack CDH1 bi-allelic mutations by targeted sequencing but predicted to harbor these genetic alterations by the AI-based model. CDH1 gene promoter methylation assessment revealed CDH1 promoter methylation in 18 cases. Targeted sequencing data reanalysis revealed other genetic mechanisms of CDH1 inactivation including CDH1 homozygous deletions (n=3), intragenic deletion with LOH (n=1), and likely pathogenic non-coding CDH1 alterations associated with LOH (n=2). WGS analysis of an ILC revealed a novel deleterious CDH1 fusion stemming from translocation t(13;16), resulting in loss of the 5’UTR, transcription start site and exons 1 and 2 of CDH1, associated with complete loss of E-cadherin protein expression. Taken together, we identified alternative/novel mechanisms of bi-allelic CDH1inactivation in 74% (25/34) cases analyzed. Conclusions: By applying an AI-based algorithm trained to detect a genetic alteration (i.e., CDH1 bi-allelic mutations), we were able to identify alternative epigenetic and genetic molecular mechanisms of CDH1 inactivation in ILCs, including novel non-coding CDH1 genetic alterations and a new inactivating CDH1 fusion gene. These findings indicate that molecular mechanisms affecting a single gene or process converging on the same phenotype can be unveiled by the integration of AI and genomics, highlighting the robustness of this approach for the discovery of novel biology. Citation Format: Fresia Pareja, Higinio Dopeso, Yikan Wang, Andrea Gazzo, David Brown, Pier Selenica, Jan Bernhard, Fatemeh Derakhshan, Edaise M. da Silva, Lorraine Colon-Cartagena, Thais Basili, Antonio Marra, Jillian Sue, Qiqi Ye, Arnaud Da Cruz Paula, Selma Yeni, Xin Pei, Hunter Green, Kaitlyn Gill, Yingjie Zhu, Matthew Lee, Ran Godrich, Adam Casson, Britta Weigelt, Nadeem Riaz, Hanna Y Wen, Edi Brogi, Matthew Hanna, Diana Mandelker, Jeremy Kunz, Brandon Rothrock, Sarat Chandarlapaty, Christopher Kanan, Gerard Oakley III, David Klimstra, Thomas Fuchs, Jorge Reis-Filho. Novel Mechanisms of CDH1 Inactivation in Breast Invasive Lobular Carcinoma Unveiled by the Integration of Artificial Intelligence and Genomics [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr GS03-04.

Overexpression of SOCS2 Inhibits EMT and M2 Macrophage Polarization in Cervical Cancer via IL-6/JAK2/STAT3 Pathway.

SOCS2 is a member of the suppressor of cytokine signaling (SOCS) protein family associated with the occurrence and development of multiple cancers. This study revealed the expression and molecular mechanisms of SOCS2 in cervical cancer. In this study, RT-qPCR, Western Blot, and immunohistochemistry were used to detect the expression level of SOCS2 in cervical cancer tissues and tumor cells. We overexpressed SOCS2 in SiHa cells via lentivirus. In-vitro experiments were used to investigate the changes in cervical cancer cell proliferation, migration, and invasion ability before and after SOCS2 overexpression. Western Blot was used to detect the expression of IL-6/JAK2/STAT3 pathway and EMTrelated proteins. M0 macrophages were co-cultured with the tumor-conditioned medium. The effect of SOCS2 on macrophage polarization was examined by RT-qPCR. SOCS2 expression level was significantly downregulated in cervical cancer tissues. SOCS2 was negatively correlated with CD163+M2 macrophages. Overexpression of SOCS2 inhibited the proliferation, migration, and invasion of cervical cancer cells. The expressions of Twist- 2, N-cadherin, and Vimentin were decreased, while the expression of E-cadherin was increased. Moreover, the expression of IL-6, p-JAK2, and p-STAT3 were decreased. After the addition of RhIL-6, the expression of E-cadherin protein in the LV-SOCS2 group was reversed. CM in the LV-SOCS2 group inhibited the polarization of M2 macrophages. SOCS2 acts as a novel biological target and suppressor of cervical cancer through IL- 6/JAK2/STAT3 pathway.

Integrated Approaches Revealed the Therapeutic Mechanisms of Zuojin Pill Against Gastric Mucosa Injury in a Rat Model with Chronic Atrophic Gastritis.

The Zuojin Pill (ZJP) is widely used for treating chronic atrophic gastritis (CAG) in clinical practice, effectively ameliorating symptoms such as vomiting, pain, and abdominal distension in patients. However, the underlying mechanisms of ZJP in treating CAG has not been fully elucidated. This study aimed to clarify the characteristic function of ZJP in the treatment of CAG and its potential mechanism. The CAG model was established by alternant administrations of ammonia solution and sodium deoxycholate, as well as an irregular diet. Therapeutic effects of ZJP on body weight, serum biochemical indexes and general condition were analyzed. HE staining and AB-PAS staining were analyzed to characterize the mucosal injury and the thickness of gastric mucosa. Furthermore, network pharmacology and molecular docking were used to predict the regulatory mechanism and main active components of ZJP in CAG treatment. RT-PCR, immunohistochemistry, immunofluorescence and Western blotting were used to measure the expression levels of apoptosis-related proteins, gastric mucosal barrier-associated proteins and PI3K/Akt signaling pathway proteins. The results demonstrated that ZJP significantly improved the general state of CAG rats, alleviated weight loss and gastric histological damage and reduced the serum biochemical indicators. Network pharmacology and molecular docking found that ZJP in treating CAG by inhibiting inflammation, suppressing apoptosis, and protecting the gastric mucosal barrier via the PI3K/Akt signaling pathway. Further experiments confirmed that ZJP obviously modulated the expression of key proteins involved in gastric mucosal cell apoptosis, such as Bax, Bad, Apaf-1, cleaved-caspase-3, cleaved-caspase-9, Cytochrome C, Bcl-2, and Bcl-xl. Moreover, ZJP significantly reversed the protein expression of Occludin, ZO-1, Claudin-4 and E-cadherin. Our study revealed that ZJP treats CAG by inhibiting the PI3K/Akt signaling pathway. This research provided a scientific basis for the rational use of ZJP in clinical practice.

Angiotensin (1-7) Inhibits Transforming Growth Factor-Β1-Induced Epithelial-Mesenchymal Transition of Human Keratinocyte Hacat Cells in vitro.

Angiotensin (1-7) (Ang-(1-7)) is an emerging component of the renin-angiotensin system (RAS) with effective anti-fibrosis properties and has been shown to interfere with epithelial-mesenchymal transition (EMT) by numerous studies. In recent years, EMT has been proposed as a new therapeutic target for skin fibrotic diseases such as keloids. However, the effect of Ang-(1-7) on EMT in skin is still unclear. Hence, the purpose of this study was to explore the effect of Ang-(1-7) on Transforming growth factor-β1(TGF-β1)-induced EMT of human immortalized keratinocytes HaCaT in vitro. The study involved the use of the human immortalized keratinocyte cell line (HaCaT). The cells were cultured in high-glucose DMEM medium with 10% fetal bovine serum and 1% penicillin-streptomycin. Four groups were created for experimentation: control group (Group C), TGF-β1-treated group (Group T), Ang-(1-7)-treated group (Group A), and a group treated with both TGF-β1 and Ang-(1-7) (Group A + T). Various assays were conducted, including a cell proliferation assay using CCK-8 solution, a scratch wound healing assay to evaluate cell migration, and Western blotting to detect protein expressions related to cell characteristics. Additionally, quantitative real-time polymerase chain reaction (PCR) was performed to analyze epithelial-mesenchymal transition (EMT) related gene expression levels. The study aimed to investigate the effects of TGF-β1 and Ang-(1-7) on HaCaT cells. We found that Ang-(1-7) not only reduced the migration of HaCaT cells induced by TGF-β1 in vitro but also reduced the expression of α-SMA and vimentin, and restored the protein expression of E-cadherin and claudin-1. Mechanistically, Ang-(1-7) inhibits the phosphorylation levels of Smad2 and Smad3 in the TGF-β1 canonical pathway, and suppresses the expression of EMT-related transcription factors (EMT-TFs) such as SNAI2, TWIST1, and ZEB1. Taken together, our findings suggest that Ang-(1-7) inhibits TGF-β1-induced EMT in HaCaT cells in vitro by disrupting the TGF-β1-Smad canonical signaling pathway. These results may be helpful in the treatment of EMT in skin fibrotic diseases such as keloids.

Biological functions of LncRNA SNHG14 in the development of thyroid cancer cells via targeting miR-206.

This study aimed to determine lncRNA SNHG14 and miR-206 in Thyroid Cancer ( TC) and to explore the related mechanisms. Sixty-four samples of thyroid tissue were collected from patients with TC. TC cell lines and a normal human thyroid cell line (HTori-3) were bought. lncRNA SNHG14-siRNA (si-lncRNA SNHG14), lncRNA SNHG14-shRNA (sh-lncRNA SNHG14), blank plasmid (siRNA-NC), miR-206-inhibitor, miR-206-mimics were transfected into BHT101 and Ocut-2C cells. qRT-PCR quantified the expression of lncRNA SNHG14 and miR-206, and the expression of vimentin, Snail, N-cadherin, Slug, E-cadherin and ZO-1 proteins were identified via WB. MTT assay, flow cytometry, and Transwell were employed to determine cellular proliferation, apoptosis, and invasion, separately. The high expression of lncRNA SNHG14 and low expression of miR-206 were exhibited in patients with TC. lncRNA SNHG14 and miR-206 were related to lymph node metastases, TNM staging, as well as differentiation of TC. Silencing lncRNA SNHG14 and over-expressing miR-206 inhibited cell EMT, proliferation, and invasion, but accelerated apoptosis. WB demonstrated that silencing lncRNA SNHG14 and over-expressing miR-206 suppressed the expression of Akt, p-ERK1/2, p-p38, p-4EBP1, p-Akt, PI3K, vimentin, Snail, N-cadherin, and Slugn, as well as up-regulated the expression of E-cadherin and ZO-1. Rescue experiment showed that after BHT101 and Ocut-2C cells were transfected with either sh-lncRNA SNHG14+miR-206-mimics or si-lncRNA SNHG14+miR-206-inhibitor, the cellular proliferative, invasive, and apoptotic activities weren't different from those transfected with siRNA-NC. Suppression of lncRNA SNHG14 up-regulates miR-206 and affects EMT, as well as proliferative, invasive, and apoptotic activities of cells, which may become an underlying treatment target for TC.

Reversine inhibits proliferation and induces apoptosis of human osteosarcoma cells through targeting MEK1

Reversine, or 2-(4-morpholinoanilino)-6-cyclohexylaminopurine, is a 2,6-disubstituted purine derivative. This small molecule shows anti-tumor potential by playing a central role in the inhibition of several kinases related to cell cycle regulation and cytokinesis. In this study, systematic review demonstrated the feasibility and pharmacological mechanism of anti-tumor effect of reversine. Firstly, we grafted MNNG/HOS, U-2 OS, MG-63 osteosarcoma cell aggregates onto chicken embryonic chorioallantoic membrane (CAM) to examine the tumor volume of these grafts after reversine treatment. Following culture, reversine inhibited the growth of osteosarcoma cell aggregates on CAM significantly. In vitro experiment, reversine suppressed osteosarcoma cell viability, colony formation, proliferation, and induced apoptosis and cell cycle arrest at G0-G1 phase. Scratch wound assay demonstrated that reversine restrained cell migration. Reversine increased the protein expression of E-cadherin. The mRNA expression of Rac1, RhoA, CDC42, PTK2, PXN, N-cadherin, Vimentin in MNNG/HOS, U-2 OS and MG-63 cells were suppressed and PTEN increased after reversine treatment. Network pharmacology prediction, molecular docking and systematic review revealed MEK1 can be used as an effective target for reversine to inhibit osteosarcoma. Western blot results show the regulation of MEK1 and ERK1/2 by reversine was not consistent in different osteosarcoma cell lines, but we found that reversine significantly inhibited the protein expression of MEK1 in MNNG/HOS, U-2 OS and MG-63. All these suggested that reversine can exert its anti-tumor effect by targeting the expression of MEK1.

Abstract LB211: Genetic complexity in OSCC metastasis: From transcription factors to novel therapeutic targets

Abstract Background: The survival of patients with oral cavity squamous cell carcinoma (OSCC) is largely influenced by the extent of locoregional recurrence. Recurrence after standard therapy occurs in about 30% of patients with late-stage OSCC, highlighting the challenge in managing this aggressive form of cancer. The transition of cells from an epithelial to mesenchymal phenotype is a critical event in OSCC invasion and metastasis and is governed by a complex interplay of genes and signaling pathways, with transcription factors such as ZEB1/2 and SNAI1/2 being pivotal players in this process. However, these transcription factors have proven challenging to target therapeutically, and it is likely that additional genes play crucial roles in OSCC pathology. Furthermore, the biology of locoregionally aggressive OSCC cancer cells is poorly understood, and this gap in knowledge is critical to predicting patient outcomes and improving treatment. This lack of understanding underscores the need for further research into identifying the specific molecular mechanisms and genetic factors that drive the locoregional aggressiveness of OSCC. Objective: To evaluate the impact of individual genes on OSCC cell migration, perform a comprehensive analysis of gene-specific effects on cell migration, and develop a valuable platform for the discovery of potential therapeutic targets. Methods: Our lab has created an in vitro model system of “mesenchymal-like” OSCC which exhibits increased migration and chemotherapeutic resistance. Using this model, we generated several cell lines expressing either Cas9 or an activating dCas9. We next transfected genome wide sgRNA libraries. Using a transwell assay platform we are working to identify several key genes necessary for successful cell migration and genes that amplify OSCC cell migratory potential. Results: Our results indicate that OSCC invasion and regional metastasis is a multifaceted process influenced by a network of genes beyond the well-known transcription factors. By subdividing gene expression changes based on E-cadherin protein expression, we are working to therapeutically isolate genes that may be critical during the fleeting transition from an epithelial to mesenchymal phenotype necessary for aggressive OSCC biology. Conclusions: We have identified several genes whose knockout or activation resulted in a significant impact on cell migration, shedding light on potential candidates for therapeutic intervention. The findings from this study will open new avenues for the development of targeted therapies to improve the prognosis of OSCC patients by focusing on previously unexplored genes involved in the invasion and metastatic cascade. Citation Format: Guillermo Flores, Slyn Uaroon, Marisa Buchakjian. Genetic complexity in OSCC metastasis: From transcription factors to novel therapeutic targets [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB211.

MicroRNA-630 alleviates inflammatory reactions in rats with diabetic kidney disease by targeting toll-like receptor 4.

Diabetic kidney disease (DKD) is a major complication of diabetes mellitus. Renal tubular epithelial cell (TEC) damage, which is strongly associated with the inflammatory response and mesenchymal trans-differentiation, plays a significant role in DKD; However, the precise molecular mechanism is unknown. The recently identified microRNA-630 (miR-630) has been hypothesized to be closely associated with cell migration, apoptosis, and autophagy. However, the association between miR-630 and DKD and the underlying mechanism remain unknown. To investigate how miR-630 affects TEC injury and the inflammatory response in DKD rats. Streptozotocin was administered to six-week-old male rats to create a hyperglycemic diabetic model. In the second week of modeling, the rats were divided into control, DKD, negative control of lentivirus, and miR-630 overexpression groups. After 8 wk, urine and blood samples were collected for the kidney injury assays, and renal tissues were removed for further molecular assays. The target gene for miR-630 was predicted using bioinformatics, and the association between miR-630 and toll-like receptor 4 (TLR4) was confirmed using in vitro investigations and double luciferase reporter gene assays. Overexpression of miR-630 in DKD rats led to changes in body weight, renal weight index, basic blood parameters and histopathological changes. The expression level of miR-630 was reduced in the kidney tissue of rats with DKD (P < 0.05). The miR-630 and TLR4 expressions in rat renal TECs (NRK-52E) were measured using quantitative reverse transcription polymerase chain reaction. The mRNA expression level of miR-630 was significantly lower in the high-glucose (HG) and HG + mimic negative control (NC) groups than in the normal glucose (NG) group (P < 0.05). In contrast, the mRNA expression level of TLR4 was significantly higher in these groups (P < 0.05). However, miR-630 mRNA expression increased and TLR4 mRNA expression significantly decreased in the HG + miR-630 mimic group than in the HG + mimic NC group (P < 0.05). Furthermore, the levels of tumor necrosis factor-alpha (TNF-α), interleukin-1β (IL-1β), and IL-6 were significantly higher in the HG and HG + mimic NC groups than in NG group (P < 0.05). However, the levels of these cytokines were significantly lower in the HG + miR-630 mimic group than in the HG + mimic NC group (P < 0.05). Notably, changes in protein expression were observed. The HG and HG + mimic NC groups showed a significant decrease in E-cadherin protein expression, whereas TLR4, α-smooth muscle actin (SMA), and collagen IV protein expression increased (P < 0.05). Conversely, the HG + miR-630 mimic group exhibited a significant increase in E-cadherin protein expression and a notable decrease in TLR4, α-SMA, and collagen IV protein expression than in the HG + mimic NC group (P < 0.05). The miR-630 targets TLR4 gene expression. In vivo experiments demonstrated that DKD rats treated with miR-630 agomir exhibited significantly higher miR-630 mRNA expression than DKD rats injected with agomir NC. Additionally, rats treated with miR-630 agomir showed significant reductions in urinary albumin, blood glucose, TLR4, and proinflammatory markers (TNF-α, IL-1β, and IL-6) expression levels (P < 0.05). Moreover, these rats exhibited fewer kidney lesions and reduced infiltration of inflammatory cells. MiR-630 may inhibit the inflammatory reaction of DKD by targeting TLR4, and has a protective effect on DKD.

Nickel induces epithelial-mesenchymal transition in pulmonary fibrosis in mice via activation of the oxidative stress-mediated TGF-β1/Smad signaling pathway.

Nickel (Ni) is recognized as a carcinogenic metal, and its widespread use has led to severe environmental and health problems. Although the lung is among the main organs affected by Ni, the precise mechanisms behind this effect remain poorly understood. This study aimed to elucidate the physiological mechanisms underlying Ni-induced pulmonary fibrosis (PF), using various techniques including histopathological detection, biochemical analysis, immunohistochemistry, western blotting, and quantitative real-time PCR. Mice were treated with nickel chloride (NiCl2), which induced PF (detected by Masson staining), up-regulation of α-smooth muscle actin (α-SMA), and collagen-1 mRNA and protein expression. NiCl2 was found to induce PF by: activation of the epithelial-mesenchymal transition (EMT) and the transforming growth factor-β1 (TGF-β1)/Smad signaling pathway; up-regulation of protein and mRNA expression of TGF-β1, p-Smad2, p-Smad3, vimentin, and N-cadherin; and down-regulation of protein and mRNA expression of E-cadherin. In addition, NiCl2 treatment increased malondialdehyde content while inhibiting antioxidant activity, as indicated by decreased catalase, total antioxidant capacity, and superoxide dismutase activities, and glutathione content. Co-treatment with the effective antioxidant and free radical scavenger N-acetyl cysteine (NAC) plus NiCl2 was used to study the effects of oxidative stress in NiCl2-induced PF. The addition of NAC significantly mitigated NiCl2-induced PF, and reversed activation of the TGF-β1/Smad signaling pathway and EMT. NiCl2-induced PF was therefore shown to be due to EMT activation via the TGF-β1/Smad signaling pathway, mediated by oxidative stress.

Abstract B004: Investigating loss of ARID1A protein on endometrial cancer progression through the destabilization of adherens junctions

Abstract The purpose of this work is to test the hypothesis that loss of ARID1A promotes endometrial cancer progression by destabilizing tumor cell adherens junctions proteins. ARID1A, an important structural subunit of the SWI/SNF chromatin remodeling complex, is mutated in 26-39% of endometrioid tumors. In endometrial cancer loss of ARID1A has been classically linked to loss of ARID1A expression and has been shown to correlate with increasing tumor grade. However, little is known about the functional impact of ARID1A loss on endometrial cancer progression. A common hallmark of aggressive endometrial cancer is decreased membranous expression of adherens junction proteins, E-Cadherin and β-Catenin. In colorectal and gastric cancer loss of ARID1A expression has been shown to directly downregulate E-Cadherin expression. To determine how loss of ARID1A affects adherens junctions and its impact on endometrial cancer progression we utilized human endometrial cancers and human endometrial cancer cell lines. Using immunohistochemistry, we determined the expression and localization of ARID1A and the adherens junction protein β-Catenin in forty-six human endometrioid type endometrial cancers. Intratumoral heterogeneity of ARID1A expression allowed for quantification of membranous E-Cadherin in relation to presence or absence of ARID1A protein within the same human endometrial tumor. To quantify membranous E-Cadherin we performed immunofluorescence and used line intensity measurements in intratumoral areas of ARID1A presence and ARID1A absence. To assess effects of ARID1A loss on adherens junction in vitro we used short-hairpin RNA to acutely knockdown ARID1A in the endometrial cancer cell lines Ishikawa and ECC-1. Protein expression of ARID1A and E-Cadherin in ARID1A knockdown cells was assessed by Western Blot. Within our cohort of forty-six human endometrial tumors, 75% of tumors with wild-type ARID1A and 45% of tumors with mutated ARID1A had absence of ARID1A protein expression. Due to the discordance between presence of an ARID1A gene mutation and the expression of ARID1A protein, we hypothesize that there are alternative mechanisms of ARID1A protein regulation in endometrial cancer. Additionally, tumors with loss of ARID1A show a trend towards nuclear localization of β-Catenin and have a significant decrease in membranous E-Cadherin expression (P&amp;lt;0.001). However, acute knockdown of ARID1A in Ishikawa and ECC-1 human endometrial cancer cells does not affect total E-Cadherin protein. This suggests that loss of ARID1A may decrease adherens junction stability, as decreased membranous expression of E-Cadherin without total protein change is a common phenotype of destabilized adherens junctions. Thus, we hypothesize that loss of ARID1A destabilizes adherens junctions leading to increased endometrial tumor aggression. Citation Format: Savannah E. Wright, Isurika Weerasinghe, Russell R. Broaddus, Andrew B. Gladden. Investigating loss of ARID1A protein on endometrial cancer progression through the destabilization of adherens junctions [abstract]. In: Proceedings of the AACR Special Conference on Endometrial Cancer: Transforming Care through Science; 2023 Nov 16-18; Boston, Massachusetts. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(5_Suppl):Abstract nr B004.

Abstract PR007: Patient derived organoids as a model to study estrogen mediated endometrial cancer

Abstract The purpose of our study was to develop a more optimal in vitro model to study the mechanisms of hormone action on endometrial cancer. Endometrial cancer (EC) represents a major health disparity with a 21% increase in mortality in African American women. The cause for this excess mortality is unknown. Endometrial cancer is commonly characterized by histological subtype into Endometrioid or Non-Endometrioid with estrogen being a driver for many endometroid type tumors. Most EC cell lines do not have high expression of estrogen receptor or do not respond to physiological levels of hormones in vitro; this lack of functional experimental models has hampered progress in the study of this disease. We have developed Patient Derived Organoids (PDOs) from 9 patient-derived xenografts (PDX) that are from both African American and Non-Hispanic Caucasian women with various stages and subtypes of EC, as a model to study EC. This model system captures the characteristics of different histological and molecular subtypes in vitro, as they maintain not only EC tumor cells but also cells from the adjacent tumor stroma. Our developed PDOs were derived from multiple molecular subtypes of EC that have different characteristics and genomic features, allowing us to observe differences in the basal properties of each tumor in vitro. The use of this organoid model system is advantageous for studying EC because growth characteristics are similar to an in vivo model, with the organoids preserving the genotype and phenotype of the native EC, allowing us to observe cellular interactions. We have observed inter-organoid variation of protein expression of β-catenin, E-cadherin, and vimentin, proteins playing a pivotal role in cellular contact, signaling, and epithelial-mesenchymal transition (EMT). Using qPCR and Western blot analysis we have observed high expression levels of estrogen receptor and progesterone receptor in endometrioid type PDOs to begin the exploration of hormonal signaling in this model. Treatment of these same PDOs with β-estradiol resulted in an increase in proliferation of the stromal cells. Single cell RNA sequencing was performed on two PDO’s that had the highest expression of progesterone and estrogen receptors. This analysis demonstrated cell population heterogeneity with multiple cell clusters forming with differential gene expression. From this analysis we can see that the cells recapitulate the gene expression from the tumor of origin, with cells containing the same genetic mutation as the original patient. These results make this model potentially good for studying hormone signaling cascades seen in EC patients that have not been previously observed in established EC cell lines. These studies will also aid in the opportunity to explore the function of differential cell types in the pathogenesis of this disease. Citation Format: Breanna M.W. Jeffcoat, Andrew B. Galdden, Russell R. Broaddus. Patient derived organoids as a model to study estrogen mediated endometrial cancer [abstract]. In: Proceedings of the AACR Special Conference on Endometrial Cancer: Transforming Care through Science; 2023 Nov 16-18; Boston, Massachusetts. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(5_Suppl):Abstract nr PR007.

Long noncoding RNA GPRC5D-AS1 in renal cell carcinoma: a molecular mechanism study.

Clear cell renal cell carcinoma (RCC) is the most common subtype of RCC. Although targeted therapy can provide superior treatment outcomes, it is prone to drug resistance, and individual responses to immunotherapy vary greatly. Therefore, finding new diagnostic and therapeutic targets for RCC is of considerable importance. Long noncoding RNA (lncRNA) GPRC5D-AS1 can serve as a biomarker in clinical applications and the prognosis of lung squamous cell carcinoma. However, the specific mechanism of action of lncRNA GPRC5D-AS1 in RCC has not yet been clarified. Therefore, this paper explores the expression of lncRNA GPRC5D-AS1 in the renal cancer cell line 786-0, and conducts a preliminary study of its molecular mechanism. Selecting nude mice for tumor experiments is because of the high genomic and physiological similarity between mice and humans. Conducting tumor research on mice allows for better control of experimental conditions, aiding researchers in more accurately observing and analysing tumor characteristics and responses. Small interfering RNA (siRNA) and plasmid cloning DNA (pcDNA) 3.1 were used to transfect renal cancer cell line 786-0 to silence and overexpress the lncRNA GPRC5D-AS1 gene. Quantitative real-time fluorescence polymerase chain reaction was used to detect the difference in lncRNA GPRC5D-AS1 expression in blank control group, negative control group, siGPRC5D-AS1 group and oeGPRC5D-AS1 group. The effects of silence and overexpression of lncRNA GPRC5D-AS11 on the proliferation of 786-0 cells were detected in cell colony formation experiments; the changes in the migration and invasion of 786-0 cells were detected via cell scratch assay and transwell assay, respectively; the differences in tumor growth between groups were determined via tumorigenesis experiments in nude mice; and the expression of proliferation-related protein [β-catenin, Ki67 and proliferating cell nuclear antigen (PCNA)] and invasion-related protein (N-cadherin and E-cadherin) were detected via Western blotting. Compared with blank control group and negative control group, the siGPRC5D-AS1 group showed a significant decrease in the relative expression of lncRNA GPRC5D-AS1 (P<0.05), a significant increase in the number of proliferating cells and migrating cells (P<0.05), a significant increase in the tumor volume of nude mice (P<0.05), a significant increase in β-catenin, Ki67, PCNA and N-cadherin protein expression (P<0.05), and a significant decrease in E-cadherin protein expression (P<0.05); conversely, these results were opposite for the eGPRC5D-AS1 group. Silencing the expression of lncRNA GPRC5D-AS1 can enhance the proliferation, invasion, and migration ability of renal cancer cell line 786-0, which can be weakened by the overexpression of lncRNA GPRC5D-AS1.

BRD4-targeting PROTACs Synergize With Chemotherapeutics Against Osteosarcoma Cell Lines.

Osteosarcoma at an advanced stage has a poor outcome, and novel targeted therapies are needed, especially for metastatic disease. Bromodomain inhibitors (BETi) are epigenetic modulators that broadly impair the expression of oncogenic proteins and exert antitumor effects. BETi can be combined with chemotherapeutics to increase therapeutic responses with superior effects in the form of proteolysis targeting chimeras (PROTACs) that degrade proteins of interest (POI) in multiple cycles. This work aimed to investigate the efficacy of BETi, such as JQ1, dBET57, and MZ1 PROTACs in combination with cytotoxic drugs against osteosarcoma cell lines. Chemosensitivity of the osteosarcoma cell lines HOS, Saos-2, MG-63, and G292 were tested with BET-directed agents alone or in combination with cytotoxic drugs comprising cisplatin, doxorubicin, topotecan, and gemcitabine using cell viability assays. The BET degraders exhibited highest toxicity to HOS cells and showed synergistic activity in combination with the chemotherapeutics, except for the degrader - topotecan/gemcitabine combinations. Highest synergy between BET agents and chemotherapeutics were found for the more chemoresistant Saos-2 cells and potentiation of toxicity in MG-63 cells for the BET agents - doxorubicin combinations and the MZ1-topotecan pair. HOS and Saos-2 cell lines had reduced protein expression of AXL, BCL-X, e-cadherin, CAIX, EpCAM, ErbB2, and vimentin in response to JQ1, MZ1, and BET57. The study suggests that the application of novel BET PROTACs in combination with chemotherapeutics could represent a new therapeutic option to improve the therapy of osteosarcomas. First orally available PROTACs have reached clinical trials.

Exploring the Relationship between E-Cadherin and β-Catenin Cell Adhesion Proteins and Periacinar Retraction Clefting in Prostatic Adenocarcinoma.

Periacinar retraction clefts represent a histopathological criterion supporting the diagnosis of prostatic adenocarcinoma. The origin of these clefts in prostatic adenocarcinoma remains unclear. Exploring the established functions of E-cadherin and β-catenin as intercellular adhesion proteins, and aiming to elucidate the origin of periacinar retraction clefting, we conducted a correlation study between the immunohistochemical expression of E-cadherin and β-catenin and the presence of periacinar retraction clefts in prostatic adenocarcinoma. We examined 53 cases of morphologically diagnosed prostatic adenocarcinoma, assessing both the neoplastic and adjacent nonneoplastic prostatic tissues for the existence and degree of periacinar retraction clefts. Additionally, we analyzed the immunohistochemical expression of E-cadherin and β-catenin proteins in prostatic tissue and explored their correlation with periacinar retraction clefts, and Gleason score, Grade Group, preoperative serum prostate specific-antigen (sPSA) levels, surgical margin status, and Tumor, Node, Metastasis (TNM) stage in prostatic adenocarcinoma. Our study confirms that periacinar retraction clefting is significantly more extensive in prostatic adenocarcinoma than in nonneoplastic prostatic tissue (p < 0.001). We report a decreased expression of E-cadherin and β-catenin immunostaining in prostatic adenocarcinoma and a negative correlation with Gleason score and Grade Group. Periacinar retraction clefting positively correlated with E-cadherin and β-catenin ((rho = 0.350; p = 0.010) and (rho = 0.340; p = 0.012)) immunostaining in prostatic adenocarcinoma. Periacinar retraction clefts stand out as a dependable criterion in the diagnosis of prostatic adenocarcinoma. E-cadherin and β-catenin proteins are potential markers indicative of tumor progression and invasiveness in prostatic adenocarcinoma. Our discovery of a positive correlation between immunostaining of E-cadherin and β-catenin proteins and periacinar retraction clefts in prostatic adenocarcinoma aligns with the notion that periacinar retraction clefting is more characteristic of Gleason Grade3 pattern in prostatic adenocarcinomas, whereas the immunohistochemical expression of E-cadherin and β-catenin shows a decrease with increasing histopathological tumor grade.

CHL1 inhibits cell proliferation, migration and invasion by regulating the NF‑κB signaling pathway in colorectal cancer.

Cell adhesion molecule close homolog of L1 (CHL1) is implicated in tumorigenesis of various malignancies. However, its role and underlying molecular mechanisms in colorectal cancer (CRC) remain unclear. The present study aimed to evaluate the specific biological functions and mechanisms of CHL1, in order to provide a theoretical basis for the use of CHL1 as a biological target in CRC. CHL1 expression was originally determined in CRC cell lines. Subsequently, CHL1 overexpression was induced by plasmid transfection in HT29 and SW480 cells, and cell proliferation, migration and invasion were evaluated using the Cell Counting Kit-8, clone formation, organoids formation and Transwell assays. Immunofluorescence and western blotting were performed to assess the protein expression of E-cadherin or N-cadherin. Differentially expressed genes (DEGs) were further evaluated using RNA-sequencing (RNA-seq) in HT29 and SW480 cells following CHL1 overexpression and functional enrichment analysis. Western blotting was performed to validate the expression of proteins related to the nuclear factor κB (NF-κB) signaling pathway. The TNMplot online database revealed the significant downregulation of CHL1 in CRC tissues. The results indicated that exogenous CHL1 overexpression significantly inhibited the proliferative, organoid-forming, migratory and invasive abilities of HT29 and SW480 cells, and increased E-cadherin protein expression. Additionally, CHL1 overexpression reduced xenograft tumor growth in vivo. RNA-seq and functional analysis revealed that DEGs in CHL1 overexpressing cells were mainly enriched in the NF-κB signaling pathway. The expression of p-p65 and p-p65/p65 ratio were significantly reduced in HT29 and SW480 cells, following CHL1 overexpression. Additionally, the inhibitory effects of CHL1 overexpression on CRC cell proliferation, organoid formation, migration and invasion were partially counteracted following the overexpression of p65 expression. Overall, the present study demonstrates that CHL1 inhibits CRC cell growth, migration and invasion through the inactivation of the NF-κB signaling pathway.

2-Dodecyl-6-Methoxycyclohexa-2, 5-Diene-1, 4-Dione isolated from Averrhoa carambola L. root inhibits high glucose-induced EMT in HK-2 cells through targeting the regulation of miR-21–5p/Smad7 signaling pathway

Objective2-Dodecyl-6-Methoxycyclohexa-2, 5-Diene-1, 4-Dione (DMDD) isolated from Averrhoa carambola L. root, has been proven therapeutic effects on diabetic kidney disease (DKD). This research aims to assess DMDD’s effects on DKD and to investigate its underlying mechanisms, to establish DMDD as a novel pharmaceutical agent for DKD treatment. MethodsThe human renal tubular epithelial (HK-2) cells were induced by high glucose (HG) to mimic DKD and followed by DMDD treatment. The cytotoxicity of DMDD was assessed using the Cell Counting Kit-8 (CCK-8) assay. The migratory capacity of HK-2 cells was evaluated through transwell and scratch-wound assays. To investigate the effect of Smad7 and miR-21–5p, lentiviral transfection was employed in HK-2 cells. Additionally, the expression of proteins related to epithelial-mesenchymal transition (EMT) and TGFβ1/Smad2/3 pathway was checked by QRT-PCR, Western blot, and immunofluorescence techniques. ResultsThis study has shown that DMDD significantly suppresses cell migration and the expression of Vimentin, α-SMA, TGFβ1, and p-Smad2/3 in HK-2 cells under HG conditions. Concurrently, DMDD enhances the protein expression of E-cadherin and Smad7. Intriguingly, the therapeutic effect of DMDD was abrogated upon Smad7 silencing. Further investigations revealed that DMDD effectively inhibits miR-21–5p expression, which is upregulated by HG. Downregulation of miR-21–5p inhibits the activation of the TGFβ1/Smad2/3 pathway and EMT induced by HG. In contrast, overexpression of miR-21–5p negates DMDD’s therapeutic benefits. ConclusionDMDD mitigates EMT in HG-induced HK-2 cells by modulating the miR-21–5p/Smad7 pathway, thereby inhibiting renal fibrosis in DKD. These findings suggest that DMDD holds promise as a potential therapeutic agent for DKD.

Jaceosidin induces apoptosis and inhibits migration in AGS gastric cancer cells by regulating ROS-mediated signaling pathways

ABSTRACT Jaceosidin (JAC) is a natural flavonoid with anti-oxidant and other pharmacological activities; however, its anti-cancer mechanism remains unclear. We investigated the mechanism of action of JAC in gastric cancer cells. Cytotoxicity and apoptosis assays showed that JAC effectively killed multiple gastric cancer cells and induced apoptosis in human gastric adenocarcinoma AGS cells via the mitochondrial pathway. Network pharmacological analysis suggested that its activity was linked to reactive oxygen species (ROS), AKT, and MAPK signaling pathways. Furthermore, JAC accumulated ROS to up-regulate p-JNK, p-p38, and IκB-α protein expressions and down-regulate the p-ERK, p-STAT3, and NF-κB protein expressions. Cell cycle assay results showed that JAC accumulated ROS to up-regulate p21 and p27 protein expressions and down-regulate p-AKT, CDK2, CDK4, CDK6, Cyclin D1, and Cyclin E protein expressions to induce G0/G1 phase arrest. Cell migration assay results showed JAC accumulated ROS to down-regulate Wnt-3a, p-GSK-3β, N-cadherin, and β-catenin protein expressions and up-regulate E-cadherin protein expression to inhibit migration. Furthermore, N-acetyl cysteine pre-treatment prevented the change of these protein expressions. In summary, JAC induced apoptosis and G0/G1 phase arrest and inhibited migration through ROS-mediated signaling pathways in AGS cells.