Epithelial-mesenchymal Transition Markers Research Articles

Targeting Fibroblast-Derived Interleukin 6: A Strategy to Overcome Epithelial-Mesenchymal Transition and Radioresistance in Head and Neck Cancer

Background: Cancer-associated fibroblasts have been reported to play a central role in driving cancer progression, promoting metastasis, and conferring resistance to therapy in HNSCC. Methods: Indirect and direct co-culture models of HPV-positive and HPV-negative HNSCC cells with fibroblasts were developed to study the effect of fibroblasts on cancer cells. ELISA was used to measure IL-6 secretion in these models. To dissect the underlying signalling mechanisms, the effects of IL-6, an IL-6 receptor (IL-6R) inhibitor, a MAPK/ERK inhibitor, and a JAK/STAT inhibitor were evaluated. Epithelial-to-mesenchymal transition (EMT) was assessed by measuring EMT markers and conducting scratch assays and spheroid assays. Radioresistance was evaluated using clonogenic assays. Additionally, radioresistant (RR) cell lines were established from parental cells to examine the correlation between radioresistance and EMT. Results: Fibroblasts were found to drive EMT-like changes and heightened radioresistance in HNSCC cells through IL-6 secretion. Remarkably, these Fb-driven effects were robustly reversed using IL-6R and MAPK/ERK inhibitors in both HPV-positive and HPV-negative cell lines, whereas JAK/STAT inhibitors proved effective only in HPV-negative cells. RR cell lines exhibit a more aggressive phenotype than their parental counterparts, marked by pronounced EMT features and heightened resistance to radiotherapy. Importantly, these aggressive characteristics were substantially attenuated by targeting IL-6R or MAPK/ERK pathways. Conclusions: This study highlights the critical role of fibroblast-secreted IL-6 in driving and maintaining EMT and radioresistance in HNSCC, resulting in a more aggressive tumour phenotype. Targeting the IL-6/IL-6R/ERK pathway emerges as a promising therapeutic approach for combating CAF-driven tumour progression and improving clinical outcomes in patients with aggressive, therapy-resistant HNSCC.

Knockdown of the EEF1A2 gene reduces lung cancer brain metastasis by downregulating the BCL10/NFκB pathway

Aim: Brain metastases (BM) in patients with lung cancer (LC) are linked to unfavorable outcomes. The eukaryotic translation elongation factor 1 alpha 2 (EEF1A2) is notably overexpressed across various cancer types and plays a role in promoting tumor initiation and progression. This research aimed to clarify the function of EEF1A2 in the context of lung cancer brain metastasis (LCBM) and to explore the mechanisms underlying its effects. Methods: To identify genes with differential expression between LC and LCBM samples, transcriptomic microarray analyses were conducted, confirming that EEF1A2 expression is elevated in LCBM. EEF1A2 expression levels were validated in multiple LC cell lines. PC9 and SPCA1 cells were transfected with lentiviral vectors carrying siRNAs targeting EEF1A2 to assess its role both in vitro and in vivo . Tandem mass tag proteomics was employed to identify proteins regulated by EEF1A2. The expression of EEF1A2, BCL10, and phosphorylated NF-κB in tumor tissues from LC and LCBM patients was analyzed. Results: Compared to the LC samples, the LCBM samples exhibited significantly higher levels of EEF1A2 expression. EEF1A2 knockdown in PC9 and SPCA1 cells resulted in substantial reductions in cell proliferation, migration, and invasion. Proteomic profiling revealed that BCL10 protein levels were markedly reduced in EEF1A2-knockdown cells. Additionally, there was a decrease in phosphorylated NF-κB, EGFR, and mesenchymal markers (N-cadherin, Twist, Snail, Slug, and Cdc42), along with an increase in E-cadherin expression. In a mouse model, EEF1A2 knockdown in PC9 cells significantly inhibited brain metastasis. Furthermore, patient samples presented elevated levels of EEF1A2, BCL10, and phosphorylated NF-κB in LCBM tissues than in LC tissues. Conclusion: Our research revealed that EEF1A2 is upregulated in LCBM, and that its knockdown suppresses brain metastasis by decreasing BCL10 expression, inhibiting NF-κB signaling, and reducing epithelial-mesenchymal transition markers. These results suggest that targeting EEF1A2 may be a promising therapeutic approach for preventing and treating brain metastasis in lung cancer patients.

Mebendazole Exerts Anticancer Activity in Ovarian Cancer Cell Lines via Novel Girdin-Mediated AKT/IKKα/β/NF-κB Signaling Axis

Mebendazole (MBZ), a benzimidazole anthelmintic and cytoskeleton-disrupting compound, exhibits antitumor properties; however, its action on ovarian cancer (OC) is not clearly understood. This study evaluates the effect of MBZ on OC cell lines OVCAR3 and OAW42, focusing on cell proliferation, migration, invasion, and cancer stemness. The underlying mechanisms, including cytoskeletal disruption, epithelial–mesenchymal transition (EMT), and signaling pathways, were explored. MBZ inhibited OVCAR3 and OAW42 cell proliferation in a dose- and time-dependent manner. Additionally, MBZ significantly impedes migration, spheroid invasion, colony formation, and stemness. In addition, it reduced actin polymerization and down-regulated CSC markers (e.g., CD24, CD44, EpCAM). Moreover, MBZ suppressed MMP-9 activity and inhibited the EMT marker as judged by decreased N-Cadherin and Vimentin and increased E-Cadherin. Furthermore, MBZ induced G2/M cell cycle arrest by modulating Cyclin B1, CDC25C, and WEE1. Also, it triggered apoptosis by disrupting mitochondrial membrane potential. Mechanistic studies revealed a significant downregulation of Girdin, an Akt modulator, along with reduced p-Akt, p-IKKα/β, and p-NF-κB, indicating MBZ’s novel mechanism of action through the Girdin-mediated Akt/IKKα/β/NF-κB signaling axis. Thus, by targeting Girdin, MBZ presents a promising repurposed therapeutic strategy to inhibit cancer cell proliferation and metastasis in ovarian cancer.

Potential Modulatory Role of Phoenixin-14 in Epithelial–Mesenchymal Transition of Endometriotic 12Z Cells

Background/Objectives: Endometriosis is a painful chronic condition in which the endometrium grows outside the uterus. The epithelial–mesenchymal transition (EMT) is critical to endometriosis progression, where cells lose epithelial traits and gain invasiveness. Methods: This study investigates the effects of phoenixin-14 (PNX-14), a neuropeptide found at reduced levels in endometriosis patients, on the expression of two molecular EMT markers, CDH1 (E-cadherin) and THBS2 (thrombospondin 2), as well as cell viability in the endometriosis-derived 12Z cell line. Cells were treated with physiological (0.2 nM) and endometriosis-relevant (0.05 nM) concentrations of PNX-14. Gene expression was analyzed using RT-qPCR, while protein localization was assessed by immunocytochemistry. Cell viability was measured using an XTT assay. Results: THBS2 gene expression was significantly decreased, and CDH1 remained unchanged in cells stimulated by 0.05 nM PNX-14. Immunolocalization indicates a weaker THBS2 and CDH1 protein immunosignal reaction for 0.05 nM PNX-14. PNX-14 treatment also exhibited a biphasic effect on cell viability. Lower concentration initially decreased viability at 48 h but then significantly increased it at 72 h. This increase coincided with the decrease in THBS2 expression, suggesting a potential link between PNX-14, THBS2, and cell viability. Conclusions: A negative correlation between cell viability and the expression of both EMT markers further highlights their possible involvement in the survival and adaptability of ectopic epithelial cells. Our findings suggest a complex interplay between PNX-14, EMT markers, and cell viability in ectopic epithelial cells. PNX-14’s ability to modulate these factors warrants further investigation to elucidate its role in endometriosis.

Anti-Inflammatory and Anti-Migratory Effects of Morin on Non-Small-Cell Lung Cancer Metastasis via Inhibition of NLRP3/MAPK Signaling Pathway

Non-small-cell lung cancer (NSCLC) remains the leading cause of cancer-related deaths globally, with a persistently low five-year survival rate of only 14–17%. High rates of metastasis contribute significantly to the poor prognosis of NSCLC, in which inflammation plays an important role by enhancing tumor growth, angiogenesis, and metastasis. Targeting inflammatory pathways within cancer cells may thus represent a promising strategy for inhibiting NSCLC metastasis. This study evaluated the anti-inflammatory and anti-metastatic properties of morin, a bioactive compound derived from a Thai medicinal herb, focusing on its effects on NLRP3 inflammasome-mediated pathways in an in vitro NSCLC model. The A549 and H1299 cell lines were stimulated with lipopolysaccharide (LPS) and adenosine triphosphate (ATP) to activate the NLRP3 pathway. The inhibition effects exhibited by morin in reducing pro-inflammatory secretion in LPS- and ATP-stimulated NSCLC cells were assessed by ELISA, while wound healing and trans-well invasion assays evaluated its impact on cell migration and invasion. RT-qPCR measurement quantified the expression of inflammatory genes, and zymography and Western blotting were used to examine changes in invasive protein levels, epithelial-to-mesenchymal transition (EMT) markers, and underlying molecular mechanisms. Our findings demonstrated the significant ability of morin to decrease the production of IL-1β, IL-18, and IL-6 in a dose-dependent manner (p < 0.05), as well as suppress NSCLC cell migration and invasion. Morin downregulated invasive proteins (MMP-2, MMP-9, u-PAR, u-PA, MT1-MMP) and EMT markers (fibronectin, N-cadherin, vimentin) (p < 0.01) while also reducing the mRNA levels of NLRP3, IL-1β, IL-18, and IL-6. Mechanistic investigations revealed that morin suppressed NLRP3 inflammasome activity and inactivated MAPK pathways. Specifically, it decreased the expression of NLRP3 and ASC proteins and reduced caspase-1 activity, while reducing the phosphorylation of ERK, JNK, and p38 proteins. Collectively, these findings suggest that morin’s inactivation of the NLRP3 inflammasome pathway could offer a novel therapeutic strategy for counteracting pro-tumorigenic inflammation and metastatic progression in NSCLC.

Telocinobufagin suppresses malignant metastasis of undifferentiated thyroid carcinoma via modulation of the LARP1-mTOR pathway.

Metastasis is the trigger of death in anaplastic thyroid cancer (ATC) patients, yet the specific mechanisms at play are still largely enigmatic. While the involvement of LARP1 in the metastatic process of various cancers has been documented, there is a noticeable gap in the literature regarding its potential influence on ATC metastasis. Molecular studies probed LARP1 expression within ATC cells, with subsequent in vitro experiments examining the effects of LARP1 on ATC cell metastasis and the mTOR signaling cascade. A suite of assays, including colony formation, scratch wound healing, transwell invasion, and cell adhesion, was used to assess cell growth, movement, invasion, and attachment. Western Blot determined the expression levels of epithelial-mesenchymal transition (EMT) markers (E-cadherin, Vimentin, N-cadherin) and proteins implicated in metastasis (MMP-2, MMP-9), along with mTOR and p-mTOR. The affinity of Telocinobufagin (TBG) from Yuanhua Toad Essence for LARP1 was investigated through molecular docking, with CETSA assays providing subsequent validation. Further cellular experiments substantiated the influence of TBG on ATC cell metastasis and modulation in the mTOR pathway. LARP1 levels were heightened in ATC cells, and its depletion effectively curbs their proliferative, migratory, invasive, and adhesive activities. With LARP1 knockdown, we also observed that the onset of EMT and metastatic processes was thwarted, as was the mTOR pathway. Subsequent research has uncovered that TBG formed a physical complex with LARP1, allowing it to target and suppress the mTOR pathway, thus preventing the metastasis of ATC. The simultaneous overexpression of LARP1, however, lessened the ability of TBG to inhibit ATC metastasis. This study highlights the importance of TBG binding to LARP1 in the mediation of the mTOR signaling pathway, a key process in the inhibition of ATC cell metastasis. This discovery introduces a new target for the diagnosis of ATC and enlightens the consideration of TBG as a treatment for ATC metastasis.

Peroxidasin is associated with a mesenchymal-like transcriptional phenotype and promotes invasion in metastatic melanoma.

Cutaneous melanoma is a highly invasive, heterogeneous and treatment resistant cancer. It's ability to dynamically shift between transcriptional states or phenotypes results in an adaptive cell plasticity that may drive cancer cell invasion or the development of therapy resistance. The expression of peroxidasin (PXDN), an extracellular matrix peroxidase, has been proposed to be associated with the invasive metastatic melanoma phenotype. We have confirmed this association by analysing the transcriptomes of 70 metastatic melanoma cell lines with variable levels of PXDN expression. This analysis highlighted a strong association between high PXDN expression and the undifferentiated invasive melanoma phenotype. To assess the functional role of PXDN in melanoma invasion, we performed a knockout of PXDN in a highly invasive cell line (NZM40). PXDN knockout decreased the invasive potential by ∼50% and decreased the expression of epithelial-mesenchymal transition and invasive marker genes as determined by RNAseq and substantiated by proteomics analysis. Bioinformatics analysis of differentially expressed genes following PXDN knockout highlighted decreases in genes linked to extracellular matrix formation, organisation and degradation as well as signalling pathways such as the WNT pathway. This study provides compelling evidence that PXDN plays a functional role in melanoma invasion by promoting an invasive, mesenchymal-like transcriptional phenotype.

Downregulation of PAX1 in OSCC Enhances Stemness and Immunosuppression via IFIT1 and PD‐L1 Pathways

ABSTRACTObjectiveOur study investigated how arecoline‐induced extracellular vesicle (EV) secretion suppresses PAX1 protein production through DNA hypermethylation and examined whether PAX1 downregulation enhances cancer stemness and immunosuppression in the tumor microenvironment.Materials and MethodsEVs were isolated from SAS/TW2.6 cancer cell lines using ultracentrifugation and identified using transmission electron microscopy. PAX1 DNA methylation was tested in an ISO17025‐certified lab, with and without EV pretreatment. Stemness and epithelial–mesenchymal transition markers were assessed by western blotting and 3D culture. PAX1, IFIT1, and PD‐L1 co‐expression were examined through immunofluorescence. Flow cytometry detected various T cells.ResultsArecoline‐induced EVs enhanced PAX1 methylation, suppressing its tumor‐suppressive function. Reduced PAX1 mRNA in OSCCs was linked to larger tumors, nodal metastasis, late‐stage disease, areca quid chewing, and poor survival. Downregulated PAX1 protein negatively correlated with IFIT1 and PD‐L1 expression. Reduced PAX1 promoted stemness via the IFIT1 pathway, increasing PD‐L1 secretion and aiding immune evasion. PD‐L1 expression correlated with Treg and CD8+ T cell levels in OSCC tissues, and the CD4+/CD8+ T cell ratio was lower in OSCC patients than in controls.ConclusionArecoline‐induced EV production, which influences PAX1/IFIT1/PD‐L1 function, may serve as a reliable biomarker for targeted therapy in OSCC patients.

ACVR1 mediates renal tubular EMT in kidney fibrosis via AKT activation.

Tubulointerstitial fibrosis in the kidneys is a chronic and progressive process. Although studies suggested that tubular epithelial-mesenchymal transition (EMT) plays a key role in the development of kidney fibrosis, whether ACVR1, a member of the TGFβ superfamily, is involved in the EMT needs to be illustrated. Using bioinformatics analysis of bulk-seq data (GSE23338 and GSE168876), we found that TGF-β1 perhaps activated the PI3K/AKT signaling pathway and induced the mRNA expression of ACVR1, fibronectin, and Collagen I in HK-2 cells (human renal tubular epithelial cell line). Furthermore, qPCR and western blotting results confirmed the high expressions of ACVR1 and EMT markers in TGFβ-induced HK-2 cells. Similar results were also found in the UUO mouse model. Besides, different time-point immunofluorescent staining indicated a positive correlation between the expression of the ACVR1 and EMT marker vimentin in TGF-β1-induced HK-2 cells. Consequently, knockdown ACVR1 effectively inhibited the expression of TGF-β1-induced EMT markers and AKT phosphorylation in HK-2 cells. Moreover, treatment of HK-2 cells with MK2206 (an allosteric inhibitor of AKT) decreased the activation of AKT and the expression of α-SMA while treatment of cells with SC79 (a AKT activator) enhanced the expression of α-SMA. These findings suggest that ACVR1 regulated the EMT of renal tubular epithelial cells through activation of the AKT signaling pathway and that ACVR1 could be considered novel therapeutic targets for renal fibrosis.

RPF2 and CARM1 cooperate to enhance colorectal cancer metastasis via the AKT/GSK-3β signaling pathway.

RPF2 plays a crucial role in promoting epithelial-mesenchymal transition (EMT) and regulating metastasis in colorectal cancer (CRC). By analyzing data from the TCGA and GEO databases, we observed significantly elevated RPF2 expression in CRC, which correlated with EMT markers. Further investigations using stable RPF2 overexpression and knockdown cell lines demonstrated that RPF2 facilitates EMT activation through the AKT/GSK-3β signaling pathway. Notably, CARM1 was identified as a key downstream effector of RPF2. Selective inhibition of CARM1 effectively suppressed the activation of the AKT/GSK-3β pathway and EMT induced by RPF2 overexpression. Both in vitro and in vivo experiments confirmed that RPF2 expression levels positively correlate with the metastatic potential of CRC cells. Moreover, treatment with a CARM1 inhibitor significantly reduced the invasive and migratory capabilities of RPF2-overexpressing cells. These findings suggest that RPF2 drives CRC metastasis by modulating EMT via the AKT/GSK-3β pathway, with CARM1 serving as a critical mediator, offering potential therapeutic targets for CRC.

Morusin Reverses Epithelial-Mesenchymal Transition in Gallbladder Cancer Cells by Regulating STAT3/HIF-1α Signaling.

Gallbladder cancer is the most prevalent malignancy of the biliary tract and has a dismal overall survival even in the present day. The development of new drugs holds promise for improving the prognosis of this lethal disease. The possible anti-neoplastic role of morusin was investigated both invitro and invivo. Through cell viability and colony formation assays, we observed that morusin inhibited the proliferation of gallbladder cancer cells invitro. Wound healing and transwell assays revealed that morusin impeded the migration and invasion of gallbladder cancer cells. Given the observed morphological changes, we examined epithelial-mesenchymal transition (EMT) markers. Subsequent investigations demonstrated that morusin treatment, both invitro and invivo, downregulated the expression of phospho-STAT3 (Signal transducer and activator of transcription 3) and HIF-1α (Hypoxia-inducible factor 1α) in gallbladder cancer cells. Furthermore, morusin effectively reversed EMT induced by phospho-STAT3 or HIF-1α. Morusin has a reversing effect on the EMT of gallbladder cancer cells by modulating STAT3/HIF-1α signaling.

Silencing of Epidermal Growth Factor-like Domain 8 Promotes Proliferation and Cancer Aggressiveness in Human Ovarian Cancer Cells by Activating ERK/MAPK Signaling Cascades.

Ovarian cancer (OC) is the second most common female reproductive cancer and the most lethal gynecological malignancy worldwide. Most human OCs are characterized by high rates of drug resistance and metastasis, leading to poor prognosis. Improving the outcomes of patients with relapsed and treatment-resistant OC remains a challenge. This study aimed to investigate the role of epidermal growth factor-like domain 8 (EGFL8) in human OC by examining the effects of siRNA-mediated EGFL8 knockdown on cancer progression. EGFL8 knockdown in human OC cells promoted aggressive traits associated with cancer progression, including enhanced proliferation, colony formation, migration, invasion, chemoresistance, and reduced apoptosis. Additionally, knockdown upregulated the expression of epithelial-mesenchymal transition (EMT) markers (Snail, Twist1, Zeb1, Zeb2, and vimentin) and cancer stem cell biomarkers (Oct4, Sox2, Nanog, KLF4, and ALDH1A1), and increased the expression of matrix metallopeptidases (MMP-2 and MMP-9), drug resistance genes (MDR1 and MRP1), and Notch1. Low EGFL8 expression also correlated with poor prognosis in human OC. Overall, this study provides crucial evidence that EGFL8 inhibits the proliferation and cancer aggressiveness of human OC cells by suppressing ERK/MAPK signaling. Therefore, EGFL8 may serve as a valuable prognostic biomarker and a potential target for developing novel human OC therapies.

LncRNA DNM1P35 sponges hsa-mir-326 to promote ovarian cancer progression

Long non-coding RNAs (lncRNAs) have emerged as crucial regulators in cancer progression. We found lncRNA DNM1P35 is elevated in ovarian tumors compared to normal tissues, and demonstrated that lncRNA DNM1P35 promoted cancer cell proliferation, migration and invasion in SK-OV-3 and OVCAR-3 cell lines. Furthermore, lncRNA DNM1P35 also facilitated the epithelial-mesenchymal transition (EMT) of ovarian cancer cells. Mechanistic studies identified microRNA-326 (miR-326) as a target of lncRNA DNM1P35. Overexpression of miR-326 diminished the tumor-promoting activity of lncRNA DNM1P35, resulting in reduction of Zinc finger E-box-binding homeobox 1 (ZEB1) expression and EMT features. We further revealed that ZEB1, a master transcription factor for EMT that is negatively regulated by miR-326, was essential for lncRNA DNM1P35-mediated cancer cell progression and EMT. Loss of ZEB1 led to compromised pro-tumoral activity of lncRNA DNM1P35. In vivo studies using a xenograft mouse model of ovarian cancer revealed that tumors with higher levels of lncRNA DNM1P35 led to shorter survival, increased tumor burden, as well as elevated expression of proliferative marker Ki67 and EMT marker ZEB1. Our comprehensive study underscored the significance of lncRNA DNM1P35 in ovarian cancer progression, elucidating the underlying mechanism through miR-326/ZEB1 axis to promote ovarian cancer progression.

Modification of Fc-fusion protein structures to enhance efficacy of cancer vaccine in plant expression system.

Epithelial cell adhesion molecule (EpCAM) fused to IgG, IgA and IgM Fc domains was expressed to create IgG, IgA and IgM-like structures as anti-cancer vaccines in Nicotiana tabacum. High-mannose glycan structures were generated by adding a C-terminal endoplasmic reticulum (ER) retention motif (KDEL) to the Fc domain (FcK) to produce EpCAM-Fc and EpCAM-FcK proteins in transgenic plants via Agrobacterium-mediated transformation. Cross-fertilization of EpCAM-Fc (FcK) transgenic plants with Joining chain (J-chain, J and JK) transgenic plants led to stable expression of large quaternary EpCAM-IgA Fc (EpCAM-A) and IgM-like (EpCAM-M) proteins. Immunoblotting, SDS-PAGE and ELISA analyses demonstrated that proteins with KDEL had higher expression levels and binding activity to anti-EpCAM IgGs. IgM showed the strongest binding among the fusion proteins, followed by IgA and IgG. Sera from BALB/c mice immunized with these vaccines produced anti-EpCAM IgGs. Flow cytometry indicated that the EpCAM-Fc fusion proteins significantly activated CD8+ cytotoxic Tcells, CD4+ helper Tcells and B cells, particularly with EpCAM-FcKP and EpCAM-FcP (FcKP) × JP (JKP). The induced anti-EpCAM IgGs captured human prostate cancer PC-3 and colorectal cancer SW620 cells. Sera from immunized mice inhibited cancer cell proliferation, migration and invasion; down-regulated proliferation markers (PCNA, Ki-67) and epithelial-mesenchymal transition markers (Vimentin); and up-regulated E-cadherin. These findings suggest that N. tabacum can produce effective vaccine candidates to induce anti-cancer immune responses.

PDGFR-α shRNA-polyplex for uveal melanoma treatment via EMT mediated vasculogenic mimicry interfering

Up to 50% of individuals with uveal melanoma (UM), a frequent cancer of the eye, pass away from metastases. One of the major challenges in treating UM is the role of receptor tyrosine kinases (RTKs), which mediate the epithelial-mesenchymal transition (EMT) of tumors. RTKs are involved in binding multiple growth factors, leading to angiogenesis and vasculogenic mimicry (VM) phenomena. Currently, most anti-angiogenic drugs have shown a tendency to increase the VM of tumors in clinical trials, resulting in limited efficacy. The existing gap in UM treatment lies in the lack of effective strategies to target RTK-mediated EMT and VM. While some approaches have been attempted, there is still a need for novel therapeutic interventions that can specifically interfere with these processes. This research employed the gene vector PEI-g-PEG to interfere with the platelet derived growth factor-alpha receptor (PDGFR-α)-mediated EMT process, thereby retarding the growth of UM. The cell experiments demonstrated that the gene polyplex exhibited favorable cell uptake and lysosome escape properties, effectively suppressing the expression of PDGFR-α protein and EMT marker proteins and the occurrence of VM phenomenon. In vivo animal studies also inhibited the growth of UM, and PAS assays showed that the treatment reduced the generation of VM in tumor tissue. This study broadens the application of PEI-g-PEG while interfering with the RTK-mediated tumor EMT process with the help of RNAi technology, providing a new idea for tumor reduction research.

The Impact of Bevacizumab and miR200c on EMT and EGFR-TKI Resistance in EGFR-Mutant Lung Cancer Organoids.

Objectives: This research aims to investigate the mechanisms of resistance to epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) in non-small-cell lung cancer (NSCLC), particularly focusing on the role of the epithelial-mesenchymal transition (EMT) within the tumor microenvironment (TME). Materials and Methods: We employed an in vitro three-dimensional organoid model that mirrors the physiology of human lung cancer. These organoids consist of lung cancer cells harboring specific EGFR mutations, human mesenchymal stem cells, and human umbilical vein endothelial cells. We analyzed EMT and drug resistance markers, and evaluated the effects of the anti-angiogenic agent Bevacizumab and micro-RNA miR200c. Results: The study identified a significant link between EMT and EGFR-TKI resistance. Notable findings included a decrease in E-cadherin and an increase in Zinc Finger E-Box Binding Homeobox 1 (ZEB1), both of which influenced EMT and resistance to treatment. Bevacizumab showed promise in improving drug resistance and mitigating EMT, suggesting an involvement of the Vascular Endothelial Growth Factor (VEGF) cascade. Transfection with miR200c was associated with improved EMT and drug resistance, further highlighting the role of EMT in TKI resistance. Conclusions: Our research provides significant insights into the EMT-driven EGFR-TKI resistance in NSCLC and offers potential strategies to overcome resistance, including the use of Bevacizumab and miR200c. However, due to the limitations in organoid models in replicating precise human cancer TME and the potential influence of specific EGFR mutations, further in vivo studies and clinical trials are necessary for validation.

Anti-Inflammatory and Anticancer Effects of Kaurenoic Acid in Overcoming Radioresistance in Breast Cancer Radiotherapy.

Background/Objectives: Peroxisome proliferator-activated receptor γ (PPARγ) plays a key role in mediating anti-inflammatory and anticancer effects in the tumor microenvironment. Kaurenoic acid (KA), a diterpene compound isolated from Sphagneticola trilobata (L.) Pruski, has been demonstrated to exert anti-inflammatory, anticancer, and antihuman immunodeficiency virus effects. Methods: In this study, we identified KA as a novel activator of PPARγ with potent anti-inflammatory and antitumor effects both in vitro and in vivo. Given the potential of PPARγ regulators in overcoming radioresistance and chemoresistance in cancer therapies, we hypothesized that KA may enhance the efficacy of breast cancer radiotherapy. Results: In a lipopolysaccharide (LPS)-induced mouse inflammation model, KA treatment reduced the levels of pro-inflammatory cytokines, including COX-2, IL-6, IL-1β, and TNFα. In a xenograft mouse mode of breast cancer, KA treatment inhibited tumor growth. Specifically, KA treatment enhanced caspase-3 activity and cytotoxicity against MDA-MB-231 and MCF-7 breast cancer cells. When KA was co-treated with a caspase inhibitor, Z-VAD-FMK, caspase-dependent apoptosis was suppressed in these cells. KA was found to induce the generation of cytosolic calcium ions (Ca2+) and reactive oxygen species (ROS), triggering endoplasmic reticulum (ER) stress via the PERK-ATF4-CHOP axis. Hence, the ER stressor thapsigargin (TG) synergized with KA treatment to enhance apoptosis in these cells, while the loss of the PERK or CHOP function inhibited this phenomenon. KA treatment was shown to induce oxidative stress via the NADPH oxidase 4 (NOX4) and stimulate ROS production. Specifically, NOX4 knockdown (KD) and antioxidant treatment (N-acetyl cysteine or diphenyleneiodonium) suppressed such ER stress-mediated apoptosis by inhibiting KA-enhanced caspase-3 activity, cytotoxicity, and intracellular ROS production in the treated cells. In radioresistant MDA-MB-231R and MCF-7R cells, KA combined with 2 Gy radiation overcame radioresistance by upregulating PPARγ and modulating epithelial-mesenchymal transition (EMT) markers, such as E-cadherin, N-cadherin, and vimentin. In PPARγ KD MDA-MB-231R and MCF-7R cells, this phenomenon was inhibited due to reduced PPARγ and NOX4 expression. Conclusions: In conclusion, these findings demonstrated KA as a novel PPARγ regulator with promising potential to enhance the efficacy of breast cancer radiotherapy.

Generation of the MCF-7 cell sublines with CRISPR/Cas9 mediated disruption of estrogen receptor alfa (ESR1) expression

Supported by the literature, our initial hypothesis was that Estrogen Receptor alfa (ESR1) may function as a master regulator by influencing the expression of epithelial-to-mesenchymal transition (EMT)-related genes in cancer cells. To explore this further, we used the CRISPR/Cas9 gene editing system to create MCF-7 sublines with down-regulated ESR1 expression and analyzed its impact on EMT initiation. By applying two distinct types of gRNA for gene editing, we established six MCF-7 cell sublines with either nearly complete or partial down-regulation of the ESR1 isoforms. Unexpectedly, the data obtained revealed no discernible impact of ESR1 down-regulation on EMT manifestation as Western blot and Real-Time qPCR analysis of selected clones revealed no changes in EMT markers expression. We suggested that those of the ESR1 isoforms, the expression of which was not affected by gene editing, could be crucial for the initiation of EMT. The obtained cell models will be used further to evaluate the activity of ESR1 isoforms. Keywords: CRISPR/Cas9, epithelial-to-mesenchymal transition, estrogen receptor alfa, MCF-7 cells

Mefunidone Inhibits Inflammation, Oxidative Stress, and Epithelial-Mesenchymal Transition in Lens Epithelial Cells.

Inflammation, oxidative stress, and epithelial-mesenchymal transition (EMT) play crucial roles in forming posterior capsular opacification (PCO), particularly in fibrotic PCO. Here we investigated the protective effects of mefunidone (MFD), a novel compound with potent antifibrotic properties, which could be useful in preventing PCO. We utilized an extracapsular lens extraction (ECLE) surgery in mice to simulate the development of PCO in vivo. Treatment was performed immediately postsurgery through the intracameral injection of MFD solution. Expression levels of EMT and inflammatory markers were analyzed using Western blot, qRT-PCR, immunofluorescence, and hematoxylin and eosin staining. Additionally, the oxidative stress indicator malondialdehyde and glutathione expression were monitored to assess the oxidative stress response. In vitro experiments, TGF-β2, and H2O2 were used to treat lens epithelial cells to induce EMT and oxidative stress models, respectively. These models were employed to explore the effects of MFD and investigate its underlying mechanisms. Compared to the model group, the group treated with anterior chamber MFD injection effectively suppressed inflammation, oxidative stress, and fibrotic responses within the capsular bag after ECLE and partially inhibited the downregulation of the epithelial marker E-cadherin. To further elucidate the underlying mechanisms, we discovered that MFD treatment in vitro remarkably reduced inflammation, decreased the production of reactive oxygen species, and suppressed the phosphorylation of TGF-β/SMAD as well as MAPK/ERK, thereby inhibiting the occurrence of EMT. Our findings substantiate the efficacy of MFD in treating PCO and provide insights into its potential mechanisms of action.

TAM-Derived Exosomes Promote EMT by Upregulating lncRNA MIR4435-2HG in Head and Neck Cancer.

This study aimed to investigate the impact of tumor-associated macrophage (TAM)-derived exosomes on epithelial-mesenchymal transition (EMT) in head and neck squamous cell carcinoma (HNSCC) and the underlying mechanisms involved. Exosomes were isolated and characterized using transmission electron microscopy, nanoparticle size analysis, and western blotting. The effect on EMT in HNSCC cells was assessed using wound healing, transwell invasion, and EMT marker assays. Bioinformatics analysis was conducted to predict key TAM-related long noncoding RNAs and evaluate their relationship with EMT in HNSCC. We observed that treatment with TAM-derived conditioned medium (CM) promoted EMT in HNSCC cells. Within the CM, we observed abundant exosomes that were taken up by HNSCC cells. Furthermore, TAM-derived exosomes promoted EMT in HNSCC cells. Mechanistically, high MIR4435-2HG expression levels were observed in TAM-derived exosomes and in HNSCC cells after treatment with TAM-derived exosomes. Notably, high MIR4435-2HG expression levels may be closely related to molecules that promote EMT in HNSCC. TAM-derived exosomes promote EMT in HNSCC cells by upregulating MIR4435-2HG expression, suggesting that MIR4435-2HG is a candidate target for HNSCC therapy.