Mesenchymal Transformation Research Articles

Comprehensive Bioinformatics Analysis Reveals the Potential Role of the hsa_circ_0001081/miR-26b-5p Axis in Regulating COL15A1 and TRIB3 within Hypoxia-Induced miRNA/mRNA Networks in Glioblastoma Cells.

Background/Objectives: The intrinsic molecular heterogeneity of glioblastoma (GBM) is one of the main reasons for its resistance to conventional treatment. Mesenchymal GBM niches are associated with hypoxic signatures and a negative influence on patients' prognosis. To date, competing endogenous RNA (ceRNA) networks have been shown to have a broad impact on the progression of various cancers. In this study, we decided to construct hypoxia-specific microRNA/ messengerRNA (miRNA/mRNA) networks with a putative circular RNA (circRNA) regulatory component using available bioinformatics tools. Methods: For ceRNA network construction, we combined publicly available data deposited in the Gene Expression Omnibus (GEO) and interaction pairs obtained from miRTarBase and circBank; a differential expression analysis of GBM cells was performed with limma and deseq2. For the gene ontology (GO) enrichment analysis, we utilized clusterProfiler; GBM molecular subtype analysis was performed in the Glioma Bio Discovery Portal (Glioma-BioDP). Results: We observed that miR-26b-5p, generally considered a tumor suppressor, was upregulated under hypoxic conditions in U-87 MG cells. Moreover, miR-26b-5p could potentially inhibit TRIB3, a gene associated with tumor proliferation. Protein-protein interaction (PPI) network and GO enrichment analyses identified a hypoxia-specific subcluster enriched in collagen-associated terms, with six genes highly expressed in the mesenchymal glioma group. This subcluster included hsa_circ_0001081/miR-26b-5p-affected COL15A1, a gene downregulated in hypoxic U-87 MG cells yet highly expressed in the mesenchymal GBM subtype. Conclusions: The interplay between miR-26b-5p, COL15A1, and TRIB3 suggests a complex regulatory mechanism that may influence the extracellular matrix composition and the mesenchymal transformation in GBM. However, the precise impact of the hsa_circ_0001081/miR-26b-5p axis on collagen-associated processes in hypoxia-induced GBM cells remains unclear and warrants further investigation.

TGFBR3 inhibits progression of papillary thyroid cancer by inhibiting PI3K/AKT pathway and EMT.

Transforming growth factor beta receptor III (TGFBR3) has been shown to play a tumor suppressive role in a variety of cancers. However, its role in papillary thyroid cancer (PTC) remains unknown. TGFBR3 expression levels in PTC were analyzed utilizing TCGA and GEO database. Edu, wounding healing and Transwell assays were used to evaluate cell proliferation, migration and invasion. Transcriptome sequencing, qRT-PCR and Western blotting were used to detect the underlying mechanism of TGFBR3 in PTC progression. This study demonstrated that TGFBR3 expression was significantly down-regulated in PTC compared to normal thyroid tissues. Low expression of TGFBR3 was associated with poor prognosis of patients with PTC. Furthermore, TGFBR3 expression positively correlated with thyroid differentiation score. In investigating the biological impact of TGFBR3 over-expression in PTC cell lines, we found that the proliferation, migration and invasion of PTC cells were significantly inhibited in response to TGFBR3 overexpression. Moreover, we also demonstrated that overexpression of TGFBR3 inhibited PI3K/AKT pathway and epithelial mesenchymal transformation processes. Lastly, TGFBR3 expression was found to be involved in tumor immune infiltration, highlighting its potential influence on immune dynamics within the tumor microenvironment in PTC. TGFBR3 plays a tumor suppressive role in PTC progression by inhibiting PI3K/AKT pathway and EMT.

Clustering-independent estimation of cell abundances in bulk tissues using single-cell RNA-seq data.

Single-cell RNA-sequencing has transformed the study of biological tissues by enabling transcriptomic characterizations of their constituent cell states. Computational methods for gene expression deconvolution use this information to infer the cell composition of related tissues profiled at the bulk level. However, current deconvolution methods are restricted to discrete cell types and have limited power to make inferences about continuous cellular processes like cell differentiation or immune cell activation. We present ConDecon, a clustering-independent method for inferring the likelihood for each cell in a single-cell dataset to be present in a bulk tissue. ConDecon represents an improvement in phenotypic resolution and functionality with respect to regression-based methods. Using ConDecon, we discover the implication of neurodegenerative microglia inflammatory pathways in the mesenchymal transformation of pediatric ependymoma and characterize their spatial trajectories of activation. The generality of this approach enables the deconvolution of other data modalities such as bulk ATAC-seq data.

Expression changes of miRNAs and EMT-related genes in human mesothelial cells induced by long-term exposure to asbestos

Objective: To investigate the effects of long-term exposure to chrysotile and crocidolite on miRNAs and epithelial mesenchymal transformation (EMT) -related gene expression in human pleural mesothelial cells. Methods: In November 2020, fluorescence quantitative polymerase chain reaction (RT-qPCR) was used to detect the expressions of EMT-related genes in human pleural mesothelioma cells (NCl-H2052 cells, NCl-H2452 cells) and human normal mesothelial cells (Met-5A cells). MiRNAs with abnormal expression in human pleural mesothelioma cells were screened out from the previous miRNA chip data of research group, and target genes of differentially expressed miRNAs were predicted using miRWalk database (http: //mirwalk.umm.uni-heidelberg.de). RT-qPCR was used to verify the abnormal expression of EMT-related miRNAs in cell lines. Met-5A cells were treated with 5μg/cm(2) chrysotile and crocidolite respectively for 48 h a time, once a week and a total of 10 times. Chrysotile group, crocidolite group and control group were set up. And the control group was added with the same volume of PBS. The expression changes of EMT-related genes and abnormal expression miRNAs in each group were detected by RT-qPCR. The differences among the groups were compared by one-way ANOVA, and the differences between the control group and the experimental group were compared by dunnet-t test. Results: Compared with Met-5A cells, the expression levels of Vimentin and Twist genes were increased, and the expression level of E-cadherin genes was decreased in NCl-H2052 cells and NCl-H2452 cells (P<0.001). Target genes of miRNAs with abnormal expression in miRNA chip were predicted, and the results showed four abnormally expressed miRNAs associated with EMT and verified the expression of these four miRNAs in the cell lines. Compared with Met-5A cells, the expression level of hsa-miR-155-5p was increased in NCl-H2052 cells and NCl-H2452 cells, the expression levels of hsa-miR-34b-5p, hsa-miR-34c-5p and hsa-miR-28-5p were decreased in NCl-H2052 cells and NCl-H2452 cells (P<0.001), which was consistent with the results of chip analysis. After exposure of Met-5A cells, it was found that compared with the control group, the expression levels of Vimentin and Twist genes, hsa-miR-155-5p, hsa-miR-34b-5p and hsa-miR-34c-5p in the crocidolite group were increased, while the expression level of E-cadherin gene was decreased (P<0.05). Compared with the control group, the expression levels of Vimentin, Twist and E-cadherin genes in chrysotile group were increased, while the expression levels of hsa-miR-34b-5p, hsa-miR-34c-5p and hsa-miR-28-5p were decreased (P<0.05) . Conclusion: Long-term exposure to chrysotile and crocidolite could cause Met-5A cells to produce miRNAs and EMT-related gene expression changes similar to mesothelioma cells.

The Role of TGF-β1 and Mutant SMAD4 on Epithelial-Mesenchymal Transition Features in Head and Neck Squamous Cell Carcinoma Cell Lines.

The aim of the present study was to investigate possible differences in the sensitivity of HNSCC cells to known EMT regulators. Three HNSCC cell lines (UM-SCC-1, -3, -22B) and the HaCaT control keratinocyte cell line were exposed to transforming growth factor beta 1 (TGF-β1), a known EMT master regulator, and the cellular response was evaluated by real-time cell analysis (RTCA), Western blot, quantitative PCR, flow cytometry, immunocytochemistry, and the wound closure (scratch) assay. Targeted sequencing on 50 cancer-related genes was performed using the Cancer Hotspot Panel v2. Mutant, and wild type SMAD4 cDNA was used to generate recombinant SMAD4 constructs for expression in mammalian cell lines. The most extensive response to TGF-β1, such as cell growth and migration, β-actin expression, or E-cadherin (CDH1) downregulation, was seen in cells with a more epithelial phenotype. Lower response correlated with higher basal p-TGFβ RII (Tyr424) levels, pointing to a possible autocrine pre-activation of these cell lines. Targeted sequencing revealed a homozygous SMAD4 mutation in the UM-SCC-22B cell line. Furthermore, PCR cloning of SMAD4 cDNA from the same cell line revealed an additional SMAD4 transcript with a 14 bp insertion mutation, which gives rise to a truncated SMAD4 protein. Overexpression of this mutant SMAD4 protein in the highly epithelial control cell line HaCaT resulted in upregulation of TGF-β1 and vimentin. Consistent with previous reports, the invasive and metastatic potential of HNSCC tumor cells appears associated with the level of autocrine secretion of EMT regulators such as TGF-β1, and it could be influenced by exogenous EMT cytokines such as those derived from immune cells of the tumor microenvironment. Furthermore, mutant SMAD4 appears to be a significant contributor to the mesenchymal transformation of HNSCC cells.

TIPE2 inhibits the migration and invasion of epithelial ovarian cancer cells by targeting Smad2 to reverse TGF-β1-induced EMT.

Epithelial ovarian cancer is the deadliest gynecologic malignancy, characterized by high metastasis. Transforming growth factor-β1 (TGF-β1) drives epithelial- mesenchymal transformation (EMT), a key process in tumor metastasis. Tumor necrosis factor-α-induced protein 8 (TNFAIP8)-like 2 (TIPE2) acts as a negative regulator of innate and adaptive immunity and involves in various cancers. However, its relationship with TGF-β1 in ovarian cancer and its role in reversing TGF-β1-induced EMT remain unclear. This study examined TIPE2 mRNA and protein expression using quantitative RT-PCR (qRT-PCR), western blot and immunohistochemistry. The effects of TIPE2 overexpression and knockdown on the proliferation, migration and invasion of epithelial ovarian cancer cells were assessed through 5-ethynyl-2-deoxyuridine, colony-forming, transwell migration and invasion assays. The relationship between TIPE2 and TGF-β1 was investigated using qRT-PCR and enzyme-linked immunosorbent assay, while the interaction between TIPE2 and Smad2 was identified via co-immunoprecipitation. The results revealed that TIPE2 protein was significantly down-regulated in epithelial ovarian cancer tissues and correlated with the pathological type of tumor, patients' age, tumor differentiation degree and FIGO stage. TIPE2 and TGF-β1 appeared to play an opposite role to each other during the progression of human ovarian cancer cells. Furthermore, TIPE2 inhibited the metastasis and EMT of ovarian cancer cells by combining with Smad2 invitro or in an intraperitoneal metastasis model. Consequently, these findings suggest that TIPE2 plays a crucial inhibitory role in ovarian cancer metastasis by modulating the TGF-β1/Smad2/EMT signaling pathway and may serve as a potential target for ovarian cancer, providing important direction for future diagnostic and therapeutic strategies.

TGF-β1/miR-30d-5p axis regulating the expression of EMT key factors to induce apoptosis of lung cancer cells

PurposePrevious studies have reported that miR-30d-5p gene expression can inhibit tumor proliferation, but its mechanism has not been fully elucidated. MethodsBased on previous findings, TGF-β1 was used to induce epithelial mesenchymal transformation in A549 cells. The expression levels of DUSP-1, E-cadherin and Vimentin were detected by Western blot. mRNA expression levels of DUSP-1, E-cadherin and Vimentin were determined by RT-qPCR. The expression level of miR-30d-5p was determined by RT-qPCR. ResultsThe induction effect of TGF-β1 could be reversed by the intervention of miR-30d-5p in A549 cells. miRNA inhibition experiment showed that miR-30d-5p inhibitor could effectively inhibit the expression of miR-30d-5p in A549 cells. After miR-30d-5p intervention, TGF-β1 was reversed on EMT-related genes (Dusp-1, E-cadherin, Vimentin). ConclusionTGF-β1 can induce epithelial mesenchymal transformation of A549 cells, and miR-30d-5p may be a key regulatory mechanism in regulating gene and protein expression of TGF-β1-mediated DUSP-1, E-cadherin and Vimentin. This provides a new perspective for understanding the anti-tumor effect of miR-30d-5p gene.

Trimethylamine N-oxide promotes the proliferation and migration of hepatocellular carcinoma cell through the MAPK pathway

Trimethylamine-n-oxide (TMAO) is a metabolite of intestinal flora following the consumption of phosphatidylcholine-rich foods. Clinical cohort studies have shown that plasma TMAO may be a risk factor for cancer development, including hepatocellular carcinoma (HCC), but fundamental research data supporting this hypothesis are lacking. In this study, HCC cells were treated with TMAO in vivo and in vitro to evaluate the effect on some indicators related to the malignancy degree of HCC, and the relevant molecular mechanisms were explored. In vitro, TMAO promoted the proliferation and migration of HCC cells and significantly upregulated the expression of proteins related to epithelial–mesenchymal transformation (EMT). In vivo, after HCC cells were inoculated subcutaneously in nude mice given water containing TMAO, the tumors grew faster and larger than those in the mice given ordinary water. The immunohistochemistry analysis showed that proliferation, migration and EMT-related proteins in the tumor tissues were significantly upregulated by TMAO. Furthermore, TMAO obviously enhanced the phosphorylation of MAPK signaling molecules in vivo and in vitro. In conclusion, TMAO promotes the proliferation, migration and EMT of HCC cells by activating the MAPK pathway.

The mechanism of NRF2 regulating cell proliferation and mesenchymal transformation in pulmonary hypertension

Pulmonary hypertension (PH) is a fatal disease with no existing curative drugs. NF-E2-related factor 2 (NRF2) a pivotal molecular in cellular protection, was investigated in PH models to elucidate its role in regulating abnormal phenotypes in pulmonary artery cells. We examined the expression of NRF2 in PH models and explored the role of NRF2 in regulating abnormal phenotypes in pulmonary artery cells. We determined the expression level of NRF2 in lung tissues of PH model decreased significantly. We found that NRF2 was reduced in rat pulmonary artery endothelial cells (rPAEC) under hypoxia, while it was overexpressed in rat pulmonary artery smooth muscle cells (rPASMC) under hypoxia. Next, the results showed that knockdown NRF2 in rPAEC promoted endothelial-mesenchymal transformation and upregulated reactive oxygen species level. After the rPASMC was treated with siRNA or activator, we found that NRF2 could accelerate cell migration by affecting MMP2/3/7, and promote cell proliferation by regulating PDGFR/ERK1/2 and mTOR/P70S6K pathways. Therefore, the study has shown that the clinical application of NRF2 activator in the treatment of pulmonary hypertension may cause side effects of promoting the proliferation and migration of rPASMC. Attention should be paid to the combination of NRF2 activators.

Effect of SIRT7 on inhibiting the epithelial-mesenchymal transformation in pancreatic cancer cells and related mechanism

Objective: To investigate the effect and mechanism of SIRT7 in epithelial mesenchymal transformation (EMT) of pancreatic cancer cells. Methods: The pancreatic cancer cells were divided into siControl, siSIRT7, over-expression SIRT7, siSIRT7+siCOL4A1, and siSIRT7+siSLUG groups using siRNA or plasmid transfection. The proliferation, migration and invasion of pancreatic cancer cells were detected by EdU, wound healing assay and Transwell experiments, respectively. The expression of EMT and cancer stem cell (CSC) markers were detected by quantitative real-time reverse transcription polymerase chain reaction assay (qRT-PCR) and western blot. RNA sequencing (RNA-seq) in SIRT7 knockdown PANC-1 cells was performed to explore the signaling pathways and target genes regulated by SIRT7. Then the target genes directly regulated by SIRT7 were identified with quantitative chromatin immunoprecipitation experiment (q-ChIP) and chromatin immunoprecipitation polymerase chain reaction (ChIP-PCR). The expressions of SIRT7 and target genes were detected by immunohistochemical (IHC) in pancreatic cancer tissues, and the correlation between SIRT7 and target gene expression was analyzed using TCGA dataset. The correlation between expression of SIRT7 or target genes and survival was analyzed on KM-plotter website. Finally, GeneMANIA, STRING and ENCORI were used to predict SIRT7-related proteins and miRNAs. Results: EdU assay showed that the cell proliferation rates in SIRT7-overexpressed PANC-1 [(19.33±0.35)%] and BxPC-3 cells [(17.00±1.89)%] were lower than those in the control group [(31.60±1.37)% and (24.33±0.78)%, respectively, P＜0.05]. The proliferation rates of SIRT7-knockdown PANC-1 [(23.94±1.00)% and (27.08±0.97)%] and BxPC-3 cells [(22.00±1.86)% and (25.96±1.61)%] were higher than those of the siControl group [(11.80±1.86)% and (13.42±1.39)%, respectively, P＜0.05]. In PANC-1 cells, the wound healing assay showed that the relative migration rate of SIRT7-overexpression cells [(76.67±2.74)%] was lower than that of control cells [(100.00±2.13)%, P＜0.05]; the relative migration rate of cells with SIRT7 knockdown [(134.22±4.08)% and (199.82±9.20)%, respectively] was higher than that of siControl group [(102.24±3.13)%, P＜0.05]. Compared with the control group, SIRT7 overexpression decreased the number of migrated BxPC-3 cells (45.66±1.69 vs 28.33±2.62, P＜0.05); while SIRT7 knockdown increased these numbers (65.66±2.86 and 82.00±2.94 versus 33.00±0.81, P＜0.01). Transwell experiment revealed that the number of invaded cells in SIRT7 overexpression groups (16.33±2.05 and 34.66±1.69) was lower than that control groups (54.33±4.64 and 58.66±5.90, P＜0.05); with SIRT7 knockdown, the numbers of invaded PANC-1 (63.66±2.49 and 69.33±3.29) and BxPC-3 cells (134.33±3.09 and 181.66±4.02) were higher than those in control groups (35.33±2.49 and 42.00±0.81, P˂0.05). Also, SIRT7 knockdown decreased the expressions of epithelial markers and increased the expressions of mesenchymal and CSC markers. RNA-seq analysis showed that SIRT7 was involved in regulating a variety of cancer-related signaling pathways, including the pancreatic cancer pathway and the EMT pathway. Furthermore, SIRT7 could directly bind to the promoter regions of target genes, such as COL4A1 and SLUG. SIRT7 was negatively correlated with the expression and function of COL4A1 and SLUG in pancreatic cancer cells. The expressions of SIRT7, COL4A1, SLUG and SOX2 were verified in pancreatic cancer tissues by IHC. Finally, SIRT7 was predicted to be associated with many proteins and miRNAs based on GeneMANIA, STRING, and ENCORI online tools. Conclusions: SIRT7 can inhibit the EMT of pancreatic cancer cells through transcriptionally inhibiting the expression of target genes, such as COL4A1 and SLUG. Thus, SIRT7 may serve as a potential tumor suppressor gene in pancreatic cancer.

Association of mitochondrial phosphoenolpyruvate carboxykinase with prognosis and immune regulation in hepatocellular carcinoma

Mitochondrial phosphoenolpyruvate carboxykinase (PCK2), a mitochondrial isoenzyme, supports the growth of cancer cells under glucose deficiency conditions in vitro. This study investigated the role and potential mechanism of PCK2 in the occurrence and development of Hepatocellular carcinoma (HCC). The Cancer Genome Atlas (TCGA), Gene Expression Omnibus (GEO), and other databases distinguish the expression of PCK2 and verified by qRT-PCR and Western blotting. Kaplan–Meier was conducted to assess PCK2 survival in HCC. The potential biological function of PCK2 was verified by enrichment analysis and gene set enrichment analysis (GSEA). The correlation between PCK2 expression and immune invasion and checkpoint was found by utilizing Tumor Immune Estimation Resource (TIMER). Lastly, the effects of PCK2 on the proliferation and metastasis of hepatocellular carcinoma cells were evaluated by cell tests, and the expressions of Epithelial mesenchymal transformation (EMT) and apoptosis related proteins were detected. PCK2 is down-regulated in HCC, indicating a poor prognosis. PCK2 gene mutation accounted for 1.3% of HCC. Functional enrichment analysis indicated the potential of PCK2 as a metabolism-related therapeutic target. Subsequently, we identified several signaling pathways related to the biological function of PCK2. The involvement of PCK2 in immune regulation was verified and key immune checkpoints were predicted. Ultimately, after PCK2 knockdown, cell proliferation and migration were significantly increased, and N-cadherin and vimentin expression were increased. PCK2 has been implicated in immune regulation, proliferation, and metastasis of hepatocellular carcinoma, and is emerging as a novel predictive biomarker and metabolic-related clinical target.

Astragalus polysaccharides augment BMSC homing via SDF-1/CXCR4 modulation: a novel approach to counteract peritoneal mesenchymal transformation and fibrosis

PurposeThis study aimed to evaluate the potential of astragalus polysaccharide (APS) pretreatment in enhancing the homing and anti-peritoneal fibrosis capabilities of bone marrow mesenchymal stromal cells (BMSCs) and to elucidate the underlying mechanisms.MethodsForty male Sprague-Dawley rats were allocated into four groups: control, peritoneal dialysis fluid (PDF), PDF + BMSCs, and PDF + APSBMSCs (APS-pre-treated BMSCs). A peritoneal fibrosis model was induced using PDF. Dil-labeled BMSCs were administered intravenously. Post-transplantation, BMSC homing to the peritoneum and pathological alterations were assessed. Stromal cell-derived factor-1 (SDF-1) levels were quantified via enzyme-linked immunosorbent assay (ELISA), while CXCR4 expression in BMSCs was determined using PCR and immunofluorescence. Additionally, a co-culture system involving BMSCs and peritoneal mesothelial cells (PMCs) was established using a Transwell setup to examine the in vitro effects of APS on BMSC migration and therapeutic efficacy, with the CXCR4 inhibitor AMD3100 deployed to dissect the role of the SDF-1/CXCR4 axis and its downstream impacts.ResultsIn vivo and in vitro experiments confirmed that APS pre-treatment notably facilitated the targeted homing of BMSCs to the peritoneal tissue of PDF-treated rats, thereby amplifying their therapeutic impact. PDF exposure markedly increased SDF-1 levels in peritoneal and serum samples, which encouraged the migration of CXCR4-positive BMSCs. Inhibition of the SDF-1/CXCR4 axis through AMD3100 application diminished BMSC migration, consequently attenuating their therapeutic response to peritoneal mesenchyme-to-mesothelial transition (MMT). Furthermore, APS upregulated CXCR4 expression in BMSCs, intensified the activation of the SDF-1/CXCR4 axis’s downstream pathways, and partially reversed the AMD3100-induced effects.ConclusionAPS augments the SDF-1/CXCR4 axis’s downstream pathway activation by increasing CXCR4 expression in BMSCs. This action bolsters the targeted homing of BMSCs to the peritoneal tissue and amplifies their suppressive influence on MMT, thereby improving peritoneal fibrosis.

Wogonin Inhibits Colorectal Cancer Proliferation and Epithelial Mesenchymal Transformation by Suppressing Phosphorylation in the AKT Pathway.

Colorectal cancer is the third leading cause of cancer-related death worldwide. Hence, there is a need to identify new therapeutic agents to improve the current repertoire of therapeutic drugs. Wogonin, a flavonoid from the herbal medicine Scutellaria baicalensis, has unique antitumor activity. Our study aimed to further explore the inhibitory effects of wogonin on colorectal cancer and its specific mechanism. The results showed that wogonin significantly inhibited the proliferation of colorectal cancer cells as well as their ability to invade and metastasize. We detected phosphorylation of tumor-associated signaling pathways using a phosphorylated protein microarray and found that wogonin intervention significantly inhibited the phosphorylation level of the AKT protein in colorectal cancer cells. Through in vitro and in vivo experiments, it was confirmed that wogonin exerted its antitumor effects against colorectal cancer by inhibiting phosphorylation in the AKT pathway. Our discovery of wogonin as an inhibitor of AKT phosphorylation provides new opportunities for the pharmacological treatment of colorectal cancer.

Simvastatin attenuates silica-induced pulmonary inflammation and fibrosis in rats via the AMPK-NOX pathway

BackgroundSimvastatin (Sim), a hydroxy-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor, has been widely used in prevention and treatment of cardiovascular diseases. Studies have suggested that Sim exerts anti-fibrotic effects by interfering fibroblast proliferation and collagen synthesis. This study was to determine whether Sim could alleviate silica-induced pulmonary fibrosis and explore the underlying mechanisms.MethodsThe rat model of silicosis was established by the tracheal perfusion method and treated with Sim (5 or 10 mg/kg), AICAR (an AMPK agonist), and apocynin (a NOX inhibitor) for 28 days. Lung tissues were collected for further analyses including pathological histology, inflammatory response, oxidative stress, epithelial mesenchymal transformation (EMT), and the AMPK-NOX pathway.ResultsSim significantly reduced silica-induced pulmonary inflammation and fibrosis at 28 days after administration. Sim could reduce the levels of interleukin (IL)-1β, IL-6, tumor necrosis factor-α and transforming growth factor-β1 in lung tissues. The expressions of hydroxyproline, α-SMA and vimentin were down-regulated, while E-cad was increased in Sim-treated rats. In addition, NOX4, p22pox, p40phox, p-p47phox/p47phox expressions and ROS levels were all increased, whereas p-AMPK/AMPK was decreased in silica-induced rats. Sim or AICAR treatment could notably reverse the decrease of AMPK activity and increase of NOX activity induced by silica. Apocynin treatment exhibited similar protective effects to Sim, including down-regulating of oxidative stress and inhibition of the EMT process and inflammatory reactions.ConclusionsSim attenuates silica-induced pulmonary inflammation and fibrosis by downregulating EMT and oxidative stress through the AMPK-NOX pathway.

Targeted lung therapy with rosmarinic acid encapsulated in PLGA microspheres for radiation-induced pulmonary fibrosis

Radiation-induced pulmonary fibrosis (RIPF) is a frequent complication among individuals affected by nuclear accidents and patients undergoing chest radiation therapy for tumors. However, the availability of effective drugs for prevention and treatment of RIPF remains limited. Rosmarinic acid (RA), which displays inhibitory properties against RIPF, suffers from poor targeting in previous drug studies. To overcome this issue and achieve optimal therapeutic effects, continuous administration over several days is usually employed. In this study, we developed PLGA microspheres with an average size of 17 μm and drug loading efficiency of 24.97 % (±0.97 %), loaded with RA (RA/PLGAMS) using the membrane emulsification method. By controlling the size of microspheres in the pulmonary capillaries, we achieved lung targeting of microspheres, and based on the release of drugs as PLGA degrades, the residence time of drugs in the body is prolonged. And it was confirmed by in vivo imaging and SEM imaging that PLGAMS could exist in the lungs for at least 7 days. Our results demonstrated successful targeting of the lungs in a mouse model and sustained release of RA encapsulated in the PLGA microspheres. Notably, under the same administration conditions, the therapeutic efficacy of PLGA-encapsulated RA surpassed that of ordinary RA. We observed decreased expression of inflammatory factors, reduced M2 macrophage polarization, and weakened epithelial mesenchymal transformation following our treatment, which might explain the radiation damage alleviating effect of RA. After treatment, it was shown that the deep inspiratory capacity of the mice improved by 57.8 %, and WB showed a decrease of 52.4 % and 28.3 % in the expression of epithelial mesenchymal transition related proteins (TGF-β1 and α-SMA). In summary, PLGA microspheres offer a promising drug delivery system and treatment strategy for radiation-induced pulmonary fibrosis.

Role of COX-2/PGE2/EP4 Axis-induced Macrophage Functional Activation in NSCLC Development

Tumor microenvironment (TME) is one of the important factors in tumorigenesis and progression, in which tumor-associated macrophages (TAMs) play an important role in non-small cell lung cancer (NSCLC) progression. However, the mechanism of TAMs in NSCLC progression remains unclear, so this study aimed to investigate the role of TAMs in NSCLC progression and to find potential therapeutic targets. Gene Expression Profiling Interactive Analysis (GEPIA) database was used to analyze the expression of prostaglandin E2 receptor 4 (EP4) mRNA in NSCLC and normal lung tissues; the protein expression levels of cyclooxygenase-2 (COX-2), EP4, cluster of differentiation 86 (CD86), CD163 and CD31 were detected by immunohistochemistry (IHC) in 120 NSCLC tissues and 24 paracancerous tissues specimens. The nude mouse lung adenocarcinoma cell A549 and macrophage RAW264.7 co-transplanted tumor model was established. And the samples were collected by gavage with EP4 inhibitor E7046, and then stained with hematoxylin-eosin (HE), IHC, and immunofluorescence (IF), and then detected by Western blot for the epithelial mesenchymal transformation (EMT) of the tumor tissues of the nude mice in each group. Western blot was used to detect the expressions of EMT related protiens in each group of nude mice; full-length transcriptome sequencing was used to screen the key genes causing liver metastasis and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis was performed. EP4 mRNA expression level in NSCLC tissues was generally lower than that in normal lung tissues (P<0.05); COX-2, EP4, CD163, CD31 proteins were differentially expressed in NSCLC tissues and adjacent tissues, and differences were observed in many clinicopathological parameters of NSCLC patients; RAW264.7 shortened the latency period of tumorigenesis of A549 and promoted the proliferation of tumors and liver metastasis of tumors, and E7046 could reduce tumor cell proliferation activity, tumor tissue vascular density and M2-type macrophage infiltration in nude mice; IF staining showed that macrophages were mainly distributed around the metastatic foci of tumors; Western blot results showed that compared with A549 alone transplantation group, the relative expression of E-cadherin protein in tumor tissues of mice in A549 and RAW264.7 co-transplantation group was significantly decreased, and the difference was statistically significant (P<0.05), while the relative expression of N-cadherin protein was up-regulated, but the difference was not statistically significant (P>0.05); the main pathways enriched in the differential genes of the full-length transcriptome were the PI3K-AKT and MAPK signaling pathways. During NSCLC development, the COX-2/PGE2/EP4 axis may promote tumor progression by inducing macrophage functional activation, and EP4 may be a potential new target for tumor immunotherapy. This study provides new perspectives and ideas for in-depth exploration of the mechanisms of NSCLC development, as well as a theoretical basis for the development of new therapeutic strategies for NSCLC.

Abstract LB453: Distinct mechanism of CXCR7 activation in EGFR-mutated NSCLC by chemokines

Abstract Erlotinib and gefitinib, EGFR TKIs, have markedly improved outcomes for NSCLC patients with EGFR mutations. However, resistance eventually develops, with the T790M mutation evident in over 50% of resistant tumors. Other resistance pathways include MET amplification, SCLC transformation, and EMT. Osimertinib, a third-generation EGFR TKI, targets the T790M mutation and is approved as a first-line treatment, reducing the incidence of T790M-related resistance. Nonetheless, non-T790M resistance mechanisms such as mesenchymal transformation are on the rise. The molecular targets or exact mechanisms of EGFR TKI resistance with mesenchymal phenotypes remain elusive. We have discovered that ERK reactivation, commonly found in EGFR TKI-resistant cells with a mesenchymal phenotype, is due to activation of an atypical GPCR, CXC chemokine receptor type 7 (CXCR7). CXCR7 is overexpressed in a cell line, an EGFR TKI-resistant tumor model, and in samples from patients that progressed on EGFR TKIs. Currently, there are no effective CXCR7 inhibitors to evaluate in NSCLC; the mechanisms and ligands for CXCR7 are not fully understood. To evaluate ligand-dependent activation of CXCR7, we generated HEK293 cells and EGFR-mutant NSCLC cells ectopically-expressing CXCR7 and stimulated the cells with CXCL12, a well-documented ligand for CXCR7. Exposures of HEK293 cells with CXCR7 to CXCL12 resulted in the internalization of CXCR7 followed by the activation of the MAPK pathway within 30 minutes of EGFR TKI treatment whereas MAPK activation in EGFR mutant NSCLC cells took 8 hours. MIF activation was also delayed in NSCLC compared to the HEK293 model, requiring the presence of an EGFR TKI. We discovered that CXCR7 co-immunoprecipitated with mutant EGFR in NSCLC cells while CXCR7 did not co-immunoprecipitate when EGFR was inhibited. Furthermore, we discovered additional chemokines including GDF15 that activate the CXCR7-MAPK axis and promote cell migration, a key characteristic of the mesenchymal phenotype in EGFR mutant cells. Taken together, we elucidated a unique mechanism of ligand-induced activation of CXCR7 and downstream MAPK pathway by canonical and putative ligands. This complex mechanism of CXCR7 activation in EGFR mutant NSCLC suggests that preventing the receptor-ligand binding may not be feasible in the context of overcoming EGFR TKI resistance. Citation Format: Laura C. Gunder, Ines Pulido, Jeffrey H. Becker, Malek G. Massad, Takeshi Shimamura. Distinct mechanism of CXCR7 activation in EGFR-mutated NSCLC by chemokines [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 LB453.

GGT5 facilitates migration and invasion through the induction of epithelial–mesenchymal transformation in gastric cancer

BackgroundGamma-glutamyltransferase 5 (GGT5), one of the two members in the GGT family (GGT1 and GGT5), plays a crucial role in oxidative regulation, inflammation promotion, and drug metabolism. Particularly in the tumorigenesis of various cancers, its significance has been recognized. Nevertheless, GGT5’s role in gastric cancer (GC) remains ambiguous. This study delves into the function and prognostic significance of GGT5 in GC through a series of in vitro experiments.MethodsEmploying online bioinformatics analysis tools such as The Cancer Genome Atlas (TCGA), Gene Expression Omnibus (GEO), Kaplan–Meier plotter, and cBioPortal, we explored GGT5 characteristics and functions in GC. This encompassed aberrant expression, prognostic value, genomic alterations and mutations, immune cell infiltration, and associated signaling pathways. Immunohistochemistry was conducted to assess GGT5 expression in GC and adjacent normal tissues. Subsequently, univariate and multivariate logistic regression analyses were applied to investigate the associations between GGT5 and clinical characteristics. CCK8, wound healing, and migration assays were utilized to evaluate the impact of GGT5 on cell viability and migration. Additionally, Gene Set Enrichment Analysis (GSEA) and Western blot analysis were performed to scrutinize the activity of the epithelial–mesenchymal transformation (EMT) signaling pathway under GGT5 regulation.ResultsGGT5 exhibits upregulation in gastric cancer, with its overexpression significantly linked to histological differentiation in GC patients (P < 0.05). Multivariate analysis indicates that elevated GGT5 expression is an independent risk factor associated with poorer overall survival in gastric cancer patients (P < 0.05). In vitro experiments reveal that downregulation of GGT5 hampers the proliferation and migration of GC cell lines. Finally, GSEA using TCGA data highlights a significant correlation between GGT5 expression and genes associated with EMT, a finding further confirmed by Western blot analysis.ConclusionsGGT5 emerges as a promising prognostic biomarker and potential therapeutic target for GC.

Inhibition of PD-L1 expression in non-small cell lung cancer may reduce vasculogenic mimicry formation by inhibiting the epithelial mesenchymal transformation process

Non-small cell lung cancer (NSCLC) is a kind of highly malignant tumor. Studies have shown that Vasculogenic mimicry (VM) may be responsible for dismal prognosis in NSCLC. Immunotherapy with programmed death-1 (PD-1) or programmed death ligand-1 (PD-L1) has significantly altered the treatment of assorted cancers, including NSCLC, but its role and mechanism in the formation of Vasculogenic mimicry (VM) in NSCLC remains unclear. This study aimed to investigate the role of the anti-PD-L1 antibody in the formation of VM in NSCLC and its possible mechanisms. The results showed that anti-PD-L1 antibody therapy could inhibit the growth of NSCLC-transplanted tumors and reduce the formation of VMs. In addition, this study found that anti-PD-L1 antibodies could increase the expression of the epithelial-mesenchymal transition (EMT) related factor E-cadherin. zinc finger E-box binding homeobox 1 (ZEB1) is an important transcription factor regulating EMT. Knocking down ZEB1 could significantly inhibit tumor growth, as well as the expression of VE-cadherin and mmp2, while remarkably increase the expression of E-cadherin. During this process, the formation of VM was inhibited by knowing down ZEB1 in both in vitro and in vivo experiments of the constructed ZEB1 knockdown stable transfected cell strains. Therefore, in this study, we found that anti-PD-L1 antibodies may reduce the formation of VMs by inhibiting the EMT process.

The role of PI3K/Akt signaling pathway in chronic kidney disease.

Chronic kidney disease (CKD), including chronic glomerulonephritis, IgA nephropathy and diabetic nephropathy, are common chronic diseases characterized by structural damage and functional decline of the kidneys. The current treatment of CKD is symptom relief. Several studies have reported that the phosphatidylinositol 3 kinases (PI3K)/protein kinase B (Akt) signaling pathway is a pathway closely related to the pathological process of CKD. It can ameliorate kidney damage by inhibiting this signal pathway which is involved with inflammation, oxidative stress, cell apoptosis, epithelial mesenchymal transformation (EMT) and autophagy. This review highlights the role of activating or inhibiting the PI3K/Akt signaling pathway in CKD-induced inflammatory response, apoptosis, autophagy and EMT. We also summarize the latest evidence on treating CKD by targeting the PI3K/Akt pathway, discuss the shortcomings and deficiencies of PI3K/Akt research in the field of CKD, and identify potential challenges in developing these clinical therapeutic CKD strategies, and provide appropriate solutions.