Epithelial-mesenchymal Transition Proteins Research Articles

GDF-15 Attenuates the Epithelium-Mesenchymal Transition and Alleviates TGFβ2-Induced Lens Opacity.

We sought to evaluate the efficacy of growth differentiation factor (GDF)-15 treatment for suppressing epithelial-mesenchymal transition (EMT) and alleviating transforming growth factor β2 (TGFβ2)-induced lens opacity. To test whether GDF-15 is a molecule that prevents EMT, we pretreated the culture with GDF-15 in neural progenitor cells, retinal pigment epithelial cells, and lens epithelial cells and then treated with factors that promote EMT, GDF-11, and TGFβ2, respectively. To further investigate the efficacy of GDF-15 on alleviating lens opacity, we used mouse lens explant culture to mimic secondary cataracts. We pretreated the lens culture with GDF-15 and then added TGFβ2 to develop lens opacity (n = 3 for each group). Western blot and quantitative reverse transcription polymerase chain reaction (qRT-PCR) were used to measure EMT protein and gene expression, respectively. In cell culture, GDF-15 pretreatment significantly attenuated EMT marker expression in cultured cells induced by treatment with GDF-11 or TGFβ2. In the lens explant culture, GDF-15 pretreatment also reduced mouse lens opacity induced by exposure to TGFβ2. Our results indicate that GDF-15 could alleviate TGFβ2-induced EMT and is a potential therapeutic agent to slow or prevent posterior capsular opacification (PCO) progression after cataract surgery. Cataracts are the leading cause of blindness worldwide, with the only current treatment involving surgical removal of the lens and replacement with an artificial lens. However, PCO, also known as secondary cataract, is a common complication after cataract surgery. The development of an adjuvant that slows the progression of PCO will be beneficial to the field of anterior complications.

Epithelial-to-mesenchymal transition and NF-kB pathways are promoted by a mutant form of DDB2, unable to bind PCNA, in UV-damaged human cells

BackgroundDNA-Damaged Binding protein 2 (DDB2) is a protein involved in the early step of Nucleotide Excision Repair. Recently, it has been reported that DDB2 is involved in epithelial-to-mesenchymal transition (EMT), key process in tumour invasiveness and metastasis formation. However, its role is not completely known.MethodsBoyden chamber and cell adhesion assays, and ICELLigence analysis were performed to detect HEK293 adhesion and invasion. Western blotting and gelatine zymography techniques were employed to assess the EMT protein levels and MMP enzymatic activity. Immunofluorescence analysis and pull-down assays facilitated the detection of NF-kB sub-cellular localization and interaction.ResultsWe have previously demonstrated that the loss of DDB2-PCNA binding favours genome instability, and increases cell proliferation and motility. Here, we have investigated the phenotypic and molecular EMT-like changes after UV DNA damage, in HEK293 clones stably expressing DDB2Wt protein or a mutant form unable to interact with PCNA (DDB2PCNA−), as well as in HeLa cells transiently expressing the same DDB2 constructs. Cells expressing DDB2PCNA− showed morphological modifications along with a reduced expression of E-cadherin, an increased activity of MMP-9 and an improved ability to migrate, in concomitance with a significant upregulation of EMT-associated Transcription Factors (TFs), whose expression has been reported to favour tumour invasion. We observed a higher expression of c-Myc oncogene, NF-kB, both regulating cell proliferation and metastatic process, as well as ZEB1, a TF significantly associated with tumorigenic potential and cell migratory ability. Interestingly, a novel interaction of DDB2 with NF-kB was detected and found to be increased in cells expressing the DDB2PCNA−, suggesting a direct modulation of NF-kB by DDB2.ConclusionThese results highlight the role of DDB2-PCNA interaction in counteracting EMT since DDB2PCNA− protein induces in HEK293 transformed cells a gain of function contributing to the acquisition of a more aggressive phenotype.

MiR-141-3p attenuates RSL3-induced ferroptosis and intestinal epithelial-mesenchymal transition via directly inhabits ZEB1 in intestinal Behçet's syndrome.

The aims of this study were to investigate whether the ferroptosis is involved in intestinal Behçet's syndrome (IBS), and to identify if miR-141-3p could attenuate RAS-selective lethal 3 (RSL3)-induced ferroptosis and intestinal epithelial to mesenchymal transition (EMT) via directly inhabits zinc fnger E-box binding homeobox 1 (ZEB1). The expressions of ferroptosis-related proteins in the intestinal tissues of patients with IBS were investigated by immunohistochemistry and quantitative real-time PCR (qRT-PCR). Malondialdehyde (MDA) contents of the intestinal tissues and cells were detected. Serum from IBS patients and RSL3 were co-cultured with intestinal epithelial cells in vitro. In order to investigate whether RSL3-induced ferroptosis can be ameliorated by miR-141-3p, the intestinal epithelial cells were firstly stimulated with RSL3 and then incubated with miR-141-3p mimics. Western blot was used to measure the expression of EMT and ferroptosis-related proteins. Expression of GPX4 (22.51% ± 2.05%, 51.75% ± 3.47%, t = - 7.77, p = 0.000) and xCT (17.49% ± 1.57%, 28.73% ± 1.75%, t = - 4.38, p = 0.003) were significantly lower in intestinal mucosal tissues of patients with IBS compared with HC group. Compared with the HC samples, the IBS specimens had significantly higher MDA (t = 4.32, p = 0.01). Moreover, the relative mRNA levels of ferritin light chain (FTL) (t = 4.07, p = 0.02) and ferritin heavy chain (FTH) (t = 8.82, p = 0.001) in the intestinal tissues were significant higher in IBS patients than in HC group. Serum from IBS patients could induce intestinal epithelial cell ferroptosis in vitro. Moreover, miR-141-3p could attenuate intestinal epithelial cell ferroptosis-induced by RSL3 and intestinal EMT via targeting ZEB1 in vitro. Ferroptosis were induced in patients with IBS. Moreover, the serum from IBS patients could induce ferroptosis in vitro. miR-141-3p could attenuate intestinal epithelial cell ferroptosis and intestinal EMT via targeting ZEB1. Therefore, miR-141-3p may open new avenues for the treatment of IBS in the future. Key Points • Ferroptosis in IBS is first reported in this study. • In this study, we explored that the serum from IBS patients could induce ferroptosis in vitro and miR-141-3p could attenuate intestinal epithelial cell ferroptosis and intestinal EMT via targeting ZEB1.

Abstract 5909: Sulindac exhibits anti-proliferative effects in pre-clinical models of ovarian cancer

Abstract Objectives: Given the intricate relationship between inflammation and ovarian cancer (OC) development and progression, anti-inflammatory drugs may have potential in the prevention and treatment of OC. Sulindac exhibits potent anti-inflammatory effects through inhibition of COX1/COX2 pathways and promising anti-tumor activity in pre-clinical models of colorectal and endometrial cancer. Thus, we investigated the efficacy of sulindac as an anti-tumorigenic agent in OC pre-clinical cell and mouse models. Methods: The human OC cell lines, MES and OVCAR5, and the K18-gT121+/-;p53fl/fl;Brca1fl/fl (KpB) mouse model of high-grade serous OC were used. Cellular proliferation was assessed by MTT and colony count assays. Apoptosis was assessed using cleaved caspase 3, 8, and 9 assays. To measure cellular stress, production of reactive oxygen species was measured by DCFH-DA assay, and the JC1 assay was used to assess changes in mitochondrial membrane potential. Cellular adhesion was evaluated by laminin assay, and cellular migration was determined by wound healing assay. Western immunoblotting was used to assess the effects of sulindac on downstream targets related to cellular stress, apoptosis, cellular invasion potential, cell cycle control, and inflammation. The KpB mice were treated with sulindac (7.5 mg/kg, oral gavage, daily) or placebo for four weeks. Results: Sulindac inhibited cellular proliferation in a dose-dependent manner in the MES and OVCAR5 cells, with IC50s of 75.3 and 75.69 μM, respectively. Sulindac (100 μM) reduced colony formation by 81% in the MES cells and by 49% in the OVCAR5 cells (p<0.01). Treatment of MES and OVCAR5 cells with sulindac increased ROS production and decreased mitochondrial membrane potential, in a dose-dependent manner (p<0.01). Sulindac induced activity of cleaved caspase 3, 8, and 9, in MES and OVCAR5 cells (p<0.01). In addition, sulindac (100 μM) effectively decreased cellular adhesion by 31% in MES cells and by 23% in OVCAR5 cells (p<0.01). These results were confirmed by Western immunoblotting demonstrating that sulindac downregulated the anti-apoptotic proteins Bcl-xl and Mcl-1 and upregulated the pro-apoptotic proteins Bax and cellular stress proteins Bip, ATF-4, and PDI, in both cell lines. Sulindac downregulated expression of several epithelial-mesenchymal transition proteins in both cell lines, including beta-catenin, snail, and slug. Sulindac also downregulated expression of cell cycle proteins CDK4, CDK5, and Cyclin D1 and inflammatory protein COX-2 in both cell lines. After 4 wks of treatment, sulindac significantly inhibited tumor growth by 71% (1.89 g vs 0.54 g, p<0.01) in KpB mice. Conclusions: Our results find that sulindac exhibited anti-tumorigenic effects in OC cell lines and the KpB OC mouse model. Given these promising pre-clinical results, further studies on the repurposing of sulindac for the prevention and treatment of OC is warranted. Citation Format: Nikita Sinha, Shuning Chen, Jennifer Haag, Xiaochang Shen, Boer Deng, Ziyi Zhao, Chunxiao Zhou, Victoria Bae-Jump. Sulindac exhibits anti-proliferative effects in pre-clinical models of ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5909.

Lipopolysaccharide Stimulates A549 Cell Migration through p-Tyr 42 RhoA and Phospholipase D1 Activity.

Cell migration is a crucial contributor to metastasis, a critical process associated with the mortality of cancer patients. The initiation of metastasis is triggered by epithelial-mesenchymal transition (EMT), along with the changes in the expression of EMT marker proteins. Inflammation plays a significant role in carcinogenesis and metastasis. Lipopolysaccharide (LPS), a typical inflammatory agent, promoted the generation of superoxide through the activation of p-Tyr42 RhoA, Rho-dependent kinase 2 (ROCK2), and the phosphorylation of p47phox. In addition, p-Tyr42 RhoA activated phospholipase D1 (PLD1), with PLD1 and phosphatidic acid (PA) being involved in superoxide production. PA also regulated the expression of EMT proteins. Consequently, we have identified MHY9 (Myosin IIA, NMIIA) as a PA-binding protein in response to LPS. MYH9 also contributed to cell migration and the alteration in the expression of EMT marker proteins. Co-immunoprecipitation revealed the formation of a complex involving p-Tyr42 RhoA, PLD1, and MYH9. These proteins were found to be distributed in both the cytosol and nucleus. In addition, we have found that p-Tyr42 RhoA PLD1 and MYH9 associate with the ZEB1 promoter. The suppression of ZEB1 mRNA levels was achieved through the knockdown of RhoA, PLD1, and MYH9 using si-RNAs. Taken together, we propose that p-Tyr42 RhoA and PLD1, responsible for producing PA, and PA-bound MYH9 are involved in the regulation of ZEB1 expression, thereby promoting cell migration.

PTC596-Induced BMI-1 Inhibition Fights Neuroblastoma Multidrug Resistance by Inducing Ferroptosis.

Neuroblastoma (NB) is a paediatric cancer with noteworthy heterogeneity ranging from spontaneous regression to high-risk forms that are characterised by cancer relapse and the acquisition of drug resistance. The most-used anticancer drugs exert their cytotoxic effect by inducing oxidative stress, and long-term therapy has been demonstrated to cause chemoresistance by enhancing the antioxidant response of NB cells. Taking advantage of an in vitro model of multidrug-resistant (MDR) NB cells, characterised by high levels of glutathione (GSH), the overexpression of the oncoprotein BMI-1, and the presence of a mutant P53 protein, we investigated a new potential strategy to fight chemoresistance. Our results show that PTC596, an inhibitor of BMI-1, exerted a high cytotoxic effect on MDR NB cells, while PRIMA-1MET, a compound able to reactivate mutant P53, had no effect on the viability of MDR cells. Furthermore, both PTC596 and PRIMA-1MET markedly reduced the expression of epithelial-mesenchymal transition proteins and limited the clonogenic potential and the cancer stemness of MDR cells. Of particular interest is the observation that PTC596, alone or in combination with PRIMA-1MET and etoposide, significantly reduced GSH levels, increased peroxide production, stimulated lipid peroxidation, and induced ferroptosis. Therefore, these findings suggest that PTC596, by inhibiting BMI-1 and triggering ferroptosis, could be a promising approach to fight chemoresistance.

Phenomenon of suppression of estrogen signaling in breast cancer cells under ultraviolet irradiation: role of Snail proteins

Introduction. The study of the effect of irradiation or any other DNA-damaging agents on the sensitivity of tumors to conservative therapy, drug or hormonal, is among the most imporant tasks that determine the efficiency of combined therapy of cancer patients.Aim. To investigate the effect of irradiation on the activity of key signaling proteins and the level of hormone dependence of breast cancer cells.Materials and methods. The experiments were performed on in vitro cultured estrogen-dependent MCF-7 breast cancer cells. Ultraviolet (UV) irradiation in the range of 254 nm with the intensity of 25–50 J/m2 was used as an experimental model to study the response of tumor cells to DNA damage. Cell growth rate was determined using the MTT test, cell survival after irradiation was analyzed using the colony-forming test. Estrogen receptor transcriptional activity was determined by reporter assay; cellular protein expression was determined by immunoblotting.Results. Single UV irradiation of MCF-7 cells leads to a marked increase in the level of apoptotic markers: p53, cPARP, suppression of expression of growth signaling proteins: CDK4/6 and estrogen receptor α (ERα). The above changes are accompanied with an increase in phosphorylation of Akt protein kinase and a marked increase in the expression of Snail1, one of the key proteins of epithelial-mesenchymal transition. In UV-resistant MCF-7/UVR cell subline obtained under repeated irradiation cycles, the levels of apoptotic and growth signaling proteins (p53, cPARP, CDK4/6) return to control levels, except for the phosphorylated form of Akt and Snail1, whose content remains high. Transfection of Snail1-expressing plasmid into MCF-7 cells is accompanied by activation of apoptotic signaling, suppression of ERα activity, and development of partial hormone resistance; however, the sensitivity of cells to irradiation is practically unchanged. Transfection of microRNA-181a-2, one of the microRNAs associated with cell resistance, simultaneously activates Akt and Snail1 and leads to the development of cross-resistance of cells to irradiation and hormonal drugs.Conclusion. The obtained data allow us to consider irradiation-induced Snail1 activation as one of the factors involved in deregulation of estrogen signaling and formation of cell resistance to hormonal drugs, while simultaneous activation of Akt and Snail1 is accompanied by the development of cross-resistance to irradiation and hormonal drugs.

Aridanin and oleanane-3- O-β-D-glucoside-2′-acetamide obtained from Tetrapleura tetraptera (Schumach. & Thonn) Taub. (Fabaceae) induces potent apoptotic activity in human prostate cancer cells

Ethnopharmacological relevanceTetrapleura tetraptera (Schumach. and Thonn.) Taub. (Fabaceae) is a tropical plant that is used in Cameroon pharmacopeia for the treatment of many cancers including prostate cancer (PCa), which is a major cause of men's death worldwide. The objective of this study was to evaluate the anticancer properties as well as underlying mechanisms of isolates from T. tetraptera on DU145, PC3 and LNCaP cancer cell lines. Materials and methodsEight (8) compounds were purified from T. tetraptera stem bark extract through silica gel column chromatography (CC) and characterized using spectroscopic techniques (1D and 2D NMR), HRESIMS. Cell growth was assessed by a well-characterized MTT assay, while BrdU and clonogenicity assays provided information on the cell proliferation index. Further, the impact of the compounds on cell cycle progression and cell death were performed through Flow cytometry. Cell adhesion, cell migration and chemotaxis along with some proteins of epithelial-mesenchymal transition (EMT) were assayed. ResultsOut of the eight (1–8) isolates from T. tetraptera only oleanane-3-O-β-D-glucoside-2′-acetamide and aridanin showed potent cell growth arrest with an estimated CC50 of 15, 23, 16 and 17, 26, 16 μg/mL on DU145, PC3 and LNCaP cells, respectively. A 15% (DU145) and 25% (LNCaP) increase in apoptotic cells induced by oleanane-3-O-β-D-glucoside-2′-acetamide and aridanin at 10 μg/mL were noticed. Oleanane-3-O-β-D-glucoside-2′-acetamide and aridanin at 2.5 and 10 μg/mL reduced the number of cells in S-phase and raised cells in G2/M phase. At the same concentrations, they decreased the number of invading DU145 cells and increased the adherence of DU145 cells to fibronectin and collagen matrix at tested concentrations, accompanied by an increase in integrin β-1 (10 μg/mL) and integrin β-4 (2.5 μg/mL) expression. Furthermore, a down-regulation of pcdk1, cdk2, Bcl-2, N-Cad, vimentin and cytokeratine 8–18 was noticed while, p19, p27, p53 pAKT, Bax, caspase-3 and E-Cad were up-regulated. ConclusionsThis study outlines for the first time, the anticancer ability of compounds oleanane-3-O-β-D-glucoside-2′-acetamide (4) and aridanin (6) from Tetrapleura tetraptera and proposes their putative mechanisms of action.

Isolation of tumor stem-like cells from primary laryngeal squamous cell carcinoma cells (FD-LS-6).

The development of efficient treatments for laryngeal squamous cell carcinoma (LSCC) is hindered by the lack of applicable tumor cell lines and animal models of the disease, especially those related to cancer stem-like cells (CSCs). CSCs play critical roles in tumor propagation and pathogenesis whereas no CSCs lines have been developed to date. In this study, we establish an LSCC cell line (FD-LS-6) from primary LSCC tumor tissue (not experienced single-cell cloning) and adapted a culturing condition for the expansion of potential stem cells (EPSCs) to isolate CSCs from FD-LS-6. We successfully derived novel CSCs and named them as LSCC sphere-forming cells (LSCSCs) which were subsequently characterized for their CSC properties. We showed that LSCSCs shared many properties of CSCs, including CSC marker, robust self-renewal capacity, tumorigenesis ability, potential to generate other cell types such as adipocytes and osteoblasts, and resistance to chemotherapy. Compared to parental cells, LSCSCs were significantly more potent in forming tumors in vivo in mice and more resistant to chemotherapy. LSCSCs have higher expressions of epithelial-mesenchymal transition proteins and chemotherapy resistance factors, and exhibit an activated COX2/PEG2 signaling pathway. Altogether, our work establishes the first CSCs of LSCC (FD-LS-6) and provides a tool to study tumorigenesis and metastasis of LSCC and help the development of anticancer therapies.

M6A-modified miR-143-3p inhibits epithelial mesenchymal transition in bronchial epithelial cells and extracellular matrix production in lung fibroblasts by targeting Smad3

BackgroundAirway epithelial cells epithelial mesenchymal transition (EMT) and lung fibroblasts extracellular matrix (ECM) production are the key steps in airway remodeling. Our previous study demonstrated that miR-143-3p has the ability to impede airway smooth muscle cell proliferation and ECM deposition. However, the function of miR-143-3p in airway epithelial cells and lung fibroblasts remains unclear. MethodsCell viability was determined using MTT method, while cell migration was evaluated through scratch assay. EMT and ECM proteins were detected by western blot, RT-qPCR, and ELISA. To determine the level of miR-143-3p m6A methylation, we employed the meRIP-qPCR assay. Additionally, the binding of miR-143-3p with Smad3 were projected by bioinformatics and validated by dual luciferase reporter assays. ResultsIt was discovered that the expression of miR-143-3p were lower in both asthma patients and TGF-β1-treated human bronchial epithelial 16HBE cells and human lung fibroblast HPF cells. Upregulation of miR-143-3p restrained 16HBE cell migration, and decreased EMT mesenchymal markers and increased epithelial markers. And upregulation of miR-143-3p impaired cell viability and ECM protein production in HPF cells. Mechanistically, interfering with METTL3 resulted in decreased m6A modification of miR-143-3p and led to lower levels of miR-143-3p. Moreover, miR-143-3p were verified to directly target and downregulate Smad3. Upregulation of Smad3 attenuated the effects of miR-143-3p on cell EMT and ECM production. ConclusionMiR-143-3p inhibits airway epithelial cell EMT as well as lung fibroblast ECM production by downregulating Smad3. Therefore, miR-143-3p may be a promising target to reduce airway remodeling in asthma.

Heparanase promotes malignant phenotypes of human oral squamous carcinoma cells by regulating the epithelial-mesenchymal transition-related molecules and infiltrated levels of natural killer cells

ObjectivesThe aim of the present study was to explore the functional role of heparanase (HPSE) and investigate the effect of HPSE on epithelial-mesenchymal transition (EMT) and Tumor-infiltrating activated natural killer cells in oral squamous cell carcinoma (OSCC). Materials and methodshuman oral squamous carcinoma (SCC-25) cells were transfected with HPSE-specific small interfering RNA. Cell Counting Kit-8 assay was performed to examine cell proliferation, while flow cytometry was performed to analyze the cell cycle. Scratch assay was conducted to analyze cell migration, followed by Transwell assay to determine cell invasion. Real-Time Polymerase Chain Reaction and Western-blot assays were performed to measure epithelial-mesenchymal transition protein expression. RNA Sequencing analysis and tumor-infiltrating immune cells estimation were performed to elucidate the effect of HPSE on OSCC. ResultsKnockdown of HPSE expression decreased the proliferation rate of SCC-25 cells resulting in a significant elevation in cell percentage at the Gap phase 0/Gap phase 1 phase by suppressed cell migration and invasion. The E-cadherin messenger RNA and protein expression increased while Snail and Vimentin expression decreased. RNA Sequencing analysis performed between small interfering RNA and negative control groups identified 42 differentially expressed genes, such as syndecan binding protein, RAB11A, member RAS oncogene family, and DDB1 and CUL4 associated factor 15. ConclusionsThese results indicated that knockdown of HPSE suppressed SCC-25 cell proliferation, invasion, migration, and epithelial-mesenchymal transition, possibly via syndecan binding protein and RAB11A, member RAS oncogene family. Moreover, HPSE regulates the infiltrated levels of natural killer cells activated, possibly via DDB1 and CUL4 associated factor 15.

The novel epithelial-mesenchymal transition-related proteins and their therapeutic targets in cholangiocarcinoma: a narrative review.

Cholangiocarcinoma (CCA), a liver cancer of bile duct epithelial cells, is a severe health issue in northeastern Thailand. The epithelial-mesenchymal transition (EMT) is a crucial process in the development of CCA. To comprehend oncogenic EMT in CCA, several newly found EMT factors are being explored in these underlying pathways. This narrative review explained the latest in vitro and in vivo findings on the molecular mechanisms of 21 new EMT-related proteins that affect CCA progression. We evaluated the PubMed database for relevant articles that fulfilled our criteria for investigating the molecular pathways of the novel EMT markers involved in oncogenic EMT and how they contribute to CCA development, including cell proliferation, apoptosis, invasion, migration, and chemoresistance. We discuss the potential of these new EMT markers as diagnostic, prognostic, and therapeutic indicators for CCA and describe their underlying mechanisms in the development of the disease. The discovery of several oncogenic EMT proteins and their key signaling pathways and downstream targets will also broaden novel paths of investigation into the diagnosis and targeted treatment of CCA. The EMT-related proteins that were found are good sources of knowledge and interesting information for future research. The possible ways to treat CCA that could be tested in clinical trials were also discussed.

NQO1 alleviates renal fibrosis by inhibiting the TLR4/NF-κB and TGF-β/Smad signaling pathways in diabetic nephropathy

ObjectiveDiabetic nephropathy (DN) is one of the main complications of diabetes, and inflammation and fibrosis play an important role in its progression. NAD(P)H: quinone oxidoreductase 1 (NQO1) protects cells from oxidative stress and damage caused by toxic quinones. In the present study, we aimed to investigate the protective effects of NQO1 against diabetes-induced renal inflammation and fibrosis and the underlying mechanisms. MethodsIn vivo, the kidneys of type 2 diabetes model db/db mice were infected with adeno-associated virus vectors to induce NQO1 overexpression. In vitro, human renal tubular epithelial (HK-2) cells transfected with NQO1 pcDNA3.1(+) were cultured under high-glucose (HG) conditions. Gene and protein expression was assessed by quantitative real-time PCR, Western blotting, immunofluorescence, and immunohistochemical staining. Mitochondrial reactive oxygen species (ROS) were detected with MitoSOX Red. ResultOur study revealed that the expression of NQO1 was markedly downregulated and that Toll-like receptor (TLR)4 and TGF-β1 expression was upregulated in vivo and in vitro under diabetic conditions. Overexpression of NQO1 suppressed proinflammatory cytokine (IL-6, TNF-α, MCP-1) secretion, extracellular matrix (ECM) (collagen IV, fibronectin) accumulation and epithelial-mesenchymal transition (EMT) (α-SMA, E-cadherin) in the db/db mouse kidneys and HG-cultured HK-2 cells. Furthermore, NQO1 overexpression ameliorated HG-induced TLR4/NF-κB and TGF-β/Smad pathways activation. Mechanistic studies demonstrated that a TLR4 inhibitor (TAK-242) suppressed the TLR4/NF-κB signaling pathway, proinflammatory cytokine secretion, EMT and ECM-related protein expression in HG-exposed HK-2 cells. In addition, we found that the antioxidants N-acetylcysteine (NAC) and tempol increased the expression of NQO1 and decreased the expression of TLR4, TGF-β1, Nox1, and Nox4 and ROS production in HK-2 cells cultured under HG conditions. ConclusionsThese data suggest that NQO1 alleviates diabetes-induced renal inflammation and fibrosis by regulating the TLR4/NF-κB and TGF-β/Smad signaling pathways.

Abstract 428: The role of sphingosine kinase 2 in overcoming EGFR-tyrosine kinase inhibitor resistance, EMT and NSCLC tumor progression

Abstract Background: Tyrosine Kinase Inhibitors (TKIs) such as Erlotinib (ER) and Osimertinib (OR) are being used to treat lung cancer patients with EGFR mutations; however, patients develop resistance to these drugs within 10-18 months. EGFR-TKI resistance may be mediated by epithelial-mesenchymal transition (EMT), which causes a motile and invasive phenotype similar to mesenchymal cells. Sphingosine kinase 2 (SphK2), which is upregulated in Non-Small Cell Lung Cancer (NSCLC), phosphorylates sphingosine to sphingosine-1-phosphate (S1P) and increases cell proliferation, tumor survival and cell migration. The sphingolipid rheostat controls the cell fate by maintaining a balance between pro-survival and pro-apoptotic sphingolipids, which is regulated by SphK2. However, the role of SphK2 in TKI resistance and EMT is not well understood. Hypothesis: We hypothesize that the inhibition/downregulation of SphK2 will reverse EMT, reduce tumorigenicity and overcome ER and OR resistance. Study design and Results: We studied expression of SphK2 in both drug resistant (OR and ER) and drug sensitive (parental) H358, H2170, H3255 and PC9 NSCLC cell lines. In drug resistant cells, qPCR and immunoblotting studies showed that SphK2 was upregulated by 1.5-1.7 and 1.5-3.3 fold in ER and OR resistant cell lines respectively compared to parental cells. 48 hours after SphK2 knockdown with siRNA treatment, SphK2 was downregulated by 4.6-fold and EMT mediator E-cadherin expression was upregulated by 2.9-fold. EMT proteins Vimentin and N-cadherin were downregulated by 3.8 and 2.2 fold respectively (p≤0.01). Using an MTT assay, differences in percentage viability of cells with different treatments were calculated with respect to their viability in the presence of OR. We found that 81.43% of H358OR and 76.35% of the H3255OR cells were inhibited by a combination of Opaganib, a Sphk2 inhibitor, and OR, this is 27.74% and 12.28% higher compared to the additive effect of drug and inhibitor in H358OR and H3255 cells respectively. Furthermore, transwell migration assays, wound healing assays and spheroid assays were performed in OR resistant cells and the results indicated inhibition of the above assays by 94.97%, 82.36%, 88.67% respectively when treated with a combination of Opaganib and OR as compared to the diluent. This indicates the potential of SphK2 inhibition to overcome TKI resistance and EMT. To analyze the role of SphK2 expression in cancer progression, IHC was performed on 63 early and 48 late-stage lung cancer patient sections. We observed upregulation of SphK2 in late-stage lung cancer (p≤0.05), indicating its role in cancer progression. Conclusion: In this study, SphK2 was shown to decrease tumor survival, cancer cell proliferation, and expression of EMT markers. Thus, SphK2 may be a promising clinical target for overcoming EGFR-TKI resistance and NSCLC tumor progression. Citation Format: S M Nasifuzzaman, Namrata Dube, Katie Lam, Robert J. Hillwig, Neelu Puri. The role of sphingosine kinase 2 in overcoming EGFR-tyrosine kinase inhibitor resistance, EMT and NSCLC tumor progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 428.

CircZFR promotes pancreatic cancer progression through a novel circRNA-miRNA-mRNA pathway and stabilizing epithelial-mesenchymal transition protein

Pancreatic cancer (PC) ranks third in incidence and seventh in mortality among cancers worldwide. CircZFR has been implicated in various human cancers. Yet, how they affect PC progression is understudied. Herein, we demonstrated that circZFR was upregulated in PC tissues and cells, a feature that was correlated with the poor performance of patients with PC. Functional analyses elucidated that circZFR facilitated cell proliferation and enhanced tumorigenicity of PC. Moreover, we found that circZFR facilitated cell metastasis by differentially regulating the levels of proteins related to epithelial-mesenchymal transition (EMT). Mechanistic investigations revealed that circZFR sponged miR-375, thereby upregulating the downstream target gene, GREMLIN2 (GREM2). Additionally, circZFR knockdown resulted in attenuation of the JNK pathway, an effect that was reversed by GREM2 overexpression. Collectively, our findings implicate circZFR as a positive regulator of PC progression through the miR-375/GREM2/JNK axis.

HNF4A Negatively Regulated Posterior Capsular Opacification via Transcriptional Inhibition of MMP2

Purpose Posterior capsular opacification is the most common complication after cataract surgery. Abnormal proliferation, migration, epithelial-mesenchymal transition, and extracellular matrix synthesis of residual lens epithelial cells are considered to be the main pathogenic mechanisms. Hepatocyte nuclear factor 4α has been reported to regulate epithelial-mesenchymal transition in different tumors. Our objective was to investigate the role and mechanism of hepatocyte nuclear factor 4α in posterior capsular opacification. Methods Hepatocyte nuclear factor 4α expression was tested in posterior capsular opacification rat lens capsules and cell models. Hepatocyte nuclear factor 4α was knocked down using small hairpin RNA. Cell viability was measured by Cell Counting Kit-8 assay. Cell migration ability was evaluated by wound healing and Transwell assays. Epithelial-mesenchymal transition markers were detected by Western blotting. Transcriptome sequencing was used to screen for downstream effectors of hepatocyte nuclear factor 4α. Chromatin immunoprecipitation and a dual luciferase reporter assay were used to determine the binding of hepatocyte nuclear factor 4α to the MMP2 promoter region. Results Hepatocyte nuclear factor 4α was downregulated in posterior capsular opacification tissue and cell models. In vitro studies showed that hepatocyte nuclear factor 4α deletion facilitated cell proliferation, migration, and epithelial-mesenchymal transition protein marker expression in lens epithelial cells. Hepatocyte nuclear factor 4α knockdown promoted epithelial-mesenchymal transition and migration of lens epithelial cells via MMP2. Mechanistically, hepatocyte nuclear factor 4α decreased MMP2 expression by binding to the MMP2 promoter region. Hepatocyte nuclear factor 4α deletion also promoted epithelial-mesenchymal transition in rat lens capsules. Conclusions We demonstrated that hepatocyte nuclear factor 4α inhibited epithelial-mesenchymal transition of lens epithelial cells by directly binding to the MMP2 promoter region and inhibiting the expression of MMP2, thus leading to retardation of posterior capsular opacification formation and development, suggesting that hepatocyte nuclear factor 4α is a potential therapeutic target for posterior capsular opacification.

QiHuangYiShen Granules Modulate the Expression of LncRNA MALAT1 and Attenuate Epithelial-Mesenchymal Transition in Kidney of Diabetic Nephropathy Rats.

QiHuangYiShen granules (QHYS), a traditional Chinese herbal medicine formula, have been used in clinical practice for treating diabetic kidney disease for several years by our team. The efficacy of reducing proteinuria and delaying the decline of renal function of QHYS has been proved by our previous studies. However, the exact mechanism by which QHYS exerts its renoprotection remains largely unknown. Emerging evidence suggests that lncRNA MALAT1 is abnormally expressed in diabetic nephropathy (DN) and can attenuate renal fibrosis by modulating podocyte epithelial-mesenchymal transition (EMT). In the present study, we aimed to explore whether QHYS could modulate lncRNA MALAT1 expression and attenuate the podocyte EMT as well as the potential mechanism related to the Wnt/β-catenin signal pathway. SD rats were fed with the high-fat-high-sucrose diet for 8 weeks and thereafter administered with 30 mg/kg streptozotocin intraperitoneally to replicate the DN model. Quality control of QHYS was performed using high-performance liquid chromatography. QHYS were orally administered at 1.25, 2.5, and 5 g/kg doses, respectively, to the DN model rats for 12 weeks. Body weight, glycated haemoglobin, blood urea nitrogen, serum creatinine, 24-h proteinuria, and kidney index were measured. The morphologic pathology of the kidney was evaluated by Hematoxylin-eosin and Masson's trichrome staining. The expression level of lncRNA MALAT1 was determined by quantitative real-time polymerase chain reaction. In addition, the expression levels of podocyte EMT protein markers and Wnt/β-catenin pathway proteins in renal tissues were evaluated by Western blotting and immunohistochemistry. The results showed that QHYS significantly reduced 24-h proteinuria, blood urea nitrogen, kidney index, and ameliorated glomerular hypertrophy and collagen fiber deposition in the kidney of DN rats. Importantly, QHYS significantly downregulated the expression level of lncRNA MALAT1, upregulated the expression of nephrin, the podocyte marker protein, downregulated the expression of desmin and FSP-1, and mesenchymal cell markers. Furthermore, QHYS significantly downregulated the expression levels of Wnt1, β-catenin, and active β-catenin. Conclusively, our study revealed that QHYS significantly reduced proteinuria, alleviated renal fibrosis, and attenuated the podocyte EMT in DN rats, which may be associated with the downregulation of lncRNA MALAT1 expression and inhibition of the Wnt/β-catenin pathway.

Design, synthesis, and biological evaluation of indolin-2-one derivatives as novel cyclin-dependent protein kinase 8 (CDK8) inhibitors

Cyclin-dependent protein kinase 8 (CDK8) plays important roles in regulating fibrotic growth factors and inflammatory signaling pathways. Long-term chronic inflammation of the lungs can lead to idiopathic pulmonary fibrosis (IPF). Abnormal alveolar epithelial regeneration leads to the release of various fibrotic growth factors and the activation of inflammatory cells. CDK8 regulates profibrotic cytokines broadly implicated in the pathogenesis of fibrosis. Therefore, inhibition of CDK8 is considered a promising strategy for treating IPF. Here, CDK8 inhibitors were designed and optimized using a fragment-based drug design strategy. Testing results revealed that 71% of the synthesized compounds inhibited CDK8 activity better than the original compound E966–0530. Of these compounds, compound 4k exhibited the strongest CDK8 enzyme-inhibiting activity (IC50 =129 nM). Notably, it displayed a 13-fold increase in potency when compared to E966–0530. Experiments on toxicity and inhibition of epithelial-mesenchymal transition (EMT) protein expressions showed that compound 4k can inhibit EMT protein expressions, but with no significant cytotoxicity for alveolar epithelial cells. Compound 4k showed a potent inhibitory effect in cell migration assays. Furthermore, compound 4k significantly inhibited the phosphorylation of p-Smad3 and RNA Pol II, which are critical mediators in the fibrotic response signaling pathway. Compound 4k remarkably reduced TGF-β1-induced oxidative stress. The above results reveal optimized CDK8 inhibitors with potential use for IPF therapeutic treatment.

Elevated extracellular calcium ions accelerate the proliferation and migration of HepG2 cells and decrease cisplatin sensitivity.

Hepatoblastoma is the most frequent liver malignancy in children. HepG2 has been discovered as a hepatoblastoma-derived cell line and tends to form clumps in culture. Intriguingly, we observed that the addition of calcium ions reduced cell clumping and disassociated HepG2 cells. The calcium signal is in connection with a series of processes critical in the tumorigenesis. Here, we demonstrated that extracellular calcium ions induced morphological changes and enhanced the epithelial-mesenchymal transition in HepG2 cells. Mechanistically, calcium ions promoted HepG2 proliferation and migration by up-regulating the phosphorylation levels of focal adhesion kinase (FAK), protein kinase B, and p38 mitogen-activated protein kinase. The inhibitor of FAK or Ca 2+/calmodulin-dependent kinase Ⅱ (CaMKⅡ) reversed the Ca 2+-induced effects on HepG2 cells, including cell proliferation and migration, epithelial-mesenchymal transition protein expression levels, and phosphorylation levels of FAK and protein kinase B. Moreover, calcium ions decreased HepG2 cells' sensitivity to cisplatin. Furthermore, we found that the expression levels of FAK and CaMKⅡ were increased in hepatoblastoma. The group with high expression levels of FAK and CaMKⅡ exhibited significantly lower ImmunoScore as well as CD8 + T and NK cells. The expression of CaMKⅡ was positively correlated with that of PDCD1 and LAG3. Correspondingly, the expression of FAK was negatively correlated with that of TNFSF9, TNFRSF4, and TNFRSF18. Collectively, extracellular calcium accelerates HepG2 cell proliferation and migration via FAK and CaMKⅡ and enhances cisplatin resistance. FAK and CaMKⅡ shape immune cell infiltration and responses in tumor microenvironments, thereby serving as potential targets for hepatoblastoma.

β-ionone Inhibits Epithelial-Mesenchymal Transition (EMT) in Prostate Cancer Cells by Negatively Regulating the Wnt/β-Catenin Pathway.

β-ionone is a terminal cyclic analog of beta-carotenoids widely found in plants. In recent years, accumulating evidence has shown that β-ionone exerts antitumor effects on various malignant tumors. However, limited studies have revealed the role of β-ionone in regulating the epithelial-mesenchymal transition (EMT) of prostate cancer (PCa) cells. This study aimed to investigate the effect of β-ionone on the EMT process of PCa, focusing on Wnt/β-catenin signaling pathway. After exposure to β-ionone, cell viability was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and the Brdu proliferation assay. The Transwell and wounding healing were used to investigate the migration and invasion abilities of PCa cells. Expression of proteins involved in the EMT process (E-cadherin, N-cadherin, vimentin) and proteins in the Wnt/β-catenin pathway (β-catenin, GSK3-β, and p-GSK3-β) were explored by western blotting. The effects of β-ionone on β-catenin degradation were explored by cycloheximide tracking assay and in vitro ubiquitination assay. Nude mouse xenograft model was served as the model system in vivo. The migration, invasion, and EMT process of PCa Human PC-3 prostate adenocarcinoma cells (PC3) and Human 22RV1 prostate adenocarcinoma cells (22RV1) cells were significantly inhibited after β-ionone treatment. In addition, β-ionone also inhibited the growth and EMT process of subcutaneous xenograft tumors in nude mice. The study also found that β-catenin, which promotes EMT, was downregulated after β-ionone treatment. Further mechanistic studies revealed that β-ionone inhibited the Wnt/β-catenin pathway by accelerating the ubiquitination and degradation of β-catenin in PCa, thus inhibiting the downstream migration, invasion, and EMT processes. These findings demonstrate that β-ionone may be a potential natural compound targeting the Wnt/β-catenin pathway for the treatment of PCa.