Mesenchymal Transition Cell Research Articles

Transcription factor specificity protein 1-mediated Serine/threonine kinase 39 upregulation promotes the proliferation, migration, invasion and epithelial–mesenchymal transition of hepatocellular carcinoma cells by activating the transforming growth factor-β1 /Smad2/3 pathway

ABSTRACT Bioinformatics analysis showed that Serine/threonine kinase 39 (STK39), which was testified to play an important role in human cancers, may be a hub gene in diagnosing hepatocellular carcinoma (HCC). This study aimed to explore whether STK39 could be regulated by specificity protein 1 (SP1) to affect HCC cells malignant processes. Firstly, STK39 expression in tissues of HCC patients and several cell lines was analyzed. After STK39 silencing, cell proliferation was evaluated by methyl thiazolyl tetrazolium and colony formation assay. Tunel staining was used to detect cell apoptosis. Then, the abilities of cell migration and invasion were determined with wound healing and transwell assays. The expression of epithelial–mesenchymal transition (EMT)-related proteins and transforming growth factor-β1 (TGF-β1)/Smad2/3 pathway proteins was tested by western blot analysis. Thereafter, cells were overexpressed with SP1 under the circumstance of STK39 knockdown, and then the above cellular processes were under observation. Results revealed that the increased expression of STK39, which was found in both HHC patients and HCC cell lines, exhibited poor HCC prognosis. STK39 silencing inhibited Hep3b cell proliferation, migration, invasion, EMT and TGF-β1/Smad2/3 expression but promoted cell apoptosis. Additionally, SP1 could bind to the STK39 promoter and facilitate STK39 expression. Further studies revealed that the effects of STK39 silencing on Hep3b cells were blocked by SP1 overexpression. In conclusion, SP1-mediated STK39 up-regulation leads to the increased proliferation, migration, invasion and EMT of HCC cells via activating TGF-β1/Smad2/3 pathway. Therapies that target SP1 to knockdown STK39 expression may contribute to the inhibition of HCC progression.

LncRNA HOXA11-AS regulates the proliferation and epithelial to mesenchymal transition of human skin cancer cells

LncRNA HOXA11-AS functions as regulator of tumorigenesis of several human cancers. The present study was intended to explore its regulatory control in human skin cancer with emphasis on understanding the underlying molecular mechanism. The results showed significant (P < 0.05) upregulation of lncRNA HOXA11-AS transcript levels in human skin cancer tissues and cell lines. The knockdown of HOXA11-AS significantly (P < 0.05) inhibited the proliferation and colony formation of A375 and HMCB skin cancer cells. Flow cytometry showed that HOXA11-AS knockdown triggered arrest of the A375 and HMCB cells at G2/M check point of cell cycle by inhibiting the expression of cyclin B1. Additionally, western blot analysis showed that HOXA11-AS knockdown resulted in the deactivation of PI3K/AKT/mTOR signaling pathway. The silencing of HOXA11-AS significantly (P < 0.05) inhibited the migration and invasion of the A375 and HMCB skin cancer cells. This was also accompanied by increase in E-cadherin and decrease in N-cadherin expression. Collectively, the results indicate that lncRNA HOXA11-AS regulates the proliferation, migration and invasion of human skin cancer and may exhibit therapeutic potential in the treatment of skin cancer.

Krüppel-like factor 1 (KLF1) promoted the proliferation, migration and invasion of human lens epithelial cells by enhancing the expression of Zinc Finger and BTB Domain Containing 7A (ZBTB7A) and activating Wnt/β-catenin pathway

ABSTRACT The epithelial–mesenchymal transition (EMT) of lens epithelial cells enhanced their proliferation and migration and therefore induced the occurrence of posterior capsule opacity (PCO). Some studies revealed that Krüppel-like factor 1 (KLF1) promoted the proliferation and invasion of multiple types of cancer cells. Besides, the expression of KLF1 was elevated in the crystalline lens of cataract patients. However, the effect of KLF1 on the development of PCO remains unclear. In this study, TGF-β2 was used for the stimulation of human lens epithelial cell line to establish EMT (SRA01/04). The KLF1 was overexpressed and knocked down in SRA01/04 cells, the proliferation, migration and invasion of which were detected by clone formation assay, wound healing and transwell assay. In addition, ZBTB7A was overexpressed in KLF1-knocked down SRA01/04 cells, the proliferation and invasion of which were also measured by clone formation assay and transwell assay. KLF1 overexpression promoted the proliferation, migration and invasion of SRA01/04 cells. Moreover, KLF1 also promoted the expression of Vimentin, snail and α-SMA in SRA01/04 cells. KLF1 enhanced the expression of ZBTB7A and β-catenin, resulting in activation of ZBTB7A and Wnt/β-catenin signaling, while overexpression of ZBTB7A abolished the inhibitory effect of knocking down KLF1 on proliferation and invasion of SRA01/04 cells. These results indicated that KLF1 promoted the proliferation, migration and invasion of human lens epithelial cells by activating ZBTB7A and Wnt/β-catenin signaling pathway.

Thymoquinone Suppresses the Proliferation, Migration and Invasiveness through Regulating ROS, Autophagic Flux and miR-877-5p in Human Bladder Carcinoma Cells.

Bladder carcinoma is among the top 10 most frequently diagnosed cancer types in the world. As a phytochemical active metabolic, thymoquinone (TQ) is extracted from seeds of Nigella sativa, possessing various biological properties in a wide range of diseases. Moreover, the outstanding anti-cancer effect of TQ is attracting increasing attentions. In certain circumstances, moderate autophagy is regarded to facilitate the adaptation of malignant cells to different stressors. Conversely, closely linked with the mitochondrial membrane potential (MMP) loss, the upregulation of intracellular reactive oxygen species (ROS) is reported to activate the cell apoptosis in many cancer types. Furthermore, the vital effects of microRNAs in the pathological processes of cancer cells have also been confirmed by previous studies. The present research confirms that TQ restrains the viability, proliferation, migration and invasion through activating caspase-dependent apoptosis in bladder carcinoma cells, which is mediated by TQ induced ROS increase in bladder carcinoma cells. Furthermore, TQ is proved to block the fusion of autophagosomes and lysosomes, causing the accumulation of autophagosomes and subsequent cell apoptosis. In addition, TQ is also found to initiate the miR-877-5p/PD-L1 axis, which suppresses the epithelial mesenchymal transition (EMT) and invasion of bladder carcinoma cells. Taken together, TQ induces the apoptosis through upregulating ROS level and impairing autophagic flux, and inhibiting the EMT and cell invasion via activating the miR-877-5p/PD-L1 axis in bladder carcinoma cells.

Human umbilical cord mesenchymal stem cells-derived exosomal microRNA-451a represses epithelial–mesenchymal transition of hepatocellular carcinoma cells by inhibiting ADAM10

ABSTRACT Exosomes derived from human umbilical cord mesenchymal stem cells (hucMSCs) expressing microRNAs (miRNAs) have been highlighted in human cancers. However, the detailed molecular mechanism of hucMSCs-derived exosomal miR-451a on hepatocellular carcinoma (HCC) remains further investigation. Our study aims to explore the impact of exosomal miR-451a on the progression of HCC. Expression of miR-451a and a disintegrin and metalloprotease 10 (ADAM10) in HCC tissues and adjacent normal tissues were determined. The exosomes were extracted from hucMSCs and co-cultured with Hep3B and SMMC-7721 cell lines. After the treatment of relative exosomes or exosome inhibitor GW4869 in Hep3B and SMMC-7721 cells, the paclitaxel resistance and malignant phenotypes of HCC cells were measured. Moreover, the effect of hucMSCs-derived exosomes on the expression of miR-451a and ADAM10 in HCC cells was assessed. The targeting relationship between miR-451a and ADAM10 was verified to detect the impact of ADAM10-wild type and ADAM10-mutant type (MUT) on HCC cell processes. Low expression of miR-451a and high expression of ADAM10 indicated a poor prognosis of HCC patients. MiR-451a was up-regulated while ADAM10 was down-regulated in HCC cells after co-culture with HucMSC-derived exosomes. The exosomes elevated miR-451a and inhibited ADAM10 to suppress the paclitaxel resistance, cell cycle transition, proliferation, migration and invasion, and promote apoptosis of HCC cells. ADAM10 was verified to be a target gene of miR-451a. ADAM10-MUT promoted HCC process independent of miR-451a mimic. HucMSC-derived exosomal miR-451a could restrict the epithelial–mesenchymal transition of HCC cells by targeting ADAM10, which might provide new targets for HCC treatment.

Dynamic changes in endoglin expression in the developing mouse heart

Endoglin (ENG) is essential for cardiovascular development and is expressed in the heart from its earliest developmental stages. ENG expression has been reported in the cardiac crescent, endocardium, valve mesenchyme and coronary vascular endothelial cells. However, its expression in these cell types is non-uniform and the dynamic changes in ENG expression during heart development have not been systematically studied.Using immunofluorescent staining we tracked ENG protein expression in mouse embryonic hearts aged from 11.5 to 17.5 days, and in postnatal and adult hearts. ENG is expressed in the endocardium and in venous endothelial cells throughout these developmental stages. ENG protein is down-regulated by approximately two-fold as a subset of early coronary veins reprogram to form arteries within the developing myocardium from E13.5. This two-fold higher ratio of ENG protein in veins versus arteries is maintained throughout cardiac development and in the adult heart.ENG is also down-regulated two-fold following mesenchymal transition of endocardial cells to form cardiac valve mesenchyme, whilst expression of the pan-endothelial marker CD31 is completely lost. A subset of epicardial cells (which do not express ENG protein) delaminate and undergo a similar mesenchymal transition to form epicardially derived cells (EPDCs). This transient intra-myocardial mesenchymal cell population expresses low levels of ENG protein, similar to valve mesenchyme.In conclusion, ENG shows dynamic changes of expression in vascular endothelial cells, endocardial cells and mesenchymal cells in the developing heart that vary according to cardiovascular cell type.

Exosomal long non-coding RNAs in the diagnosis and oncogenesis of pancreatic cancer.

Extracellular vesicles, specifically exosomes, play a significant role as an extracellular messenger through their transporting cargo. Of particular interest are the potential roles they play in pancreatic cancer, one of the leading causes of cancer-related mortality worldwide. Pancreatic Ductal Adenocarcinoma displays high chemo-resistance and metastatic ability, which may be influenced by cancer-derived exosomes carrying proteins, lipids and RNA. To date, among the most extensively examined exosomal molecular cargo there are long non-coding RNAs (lncRNAs) that, despite the increasing interest in their role and functions, are relatively poorly understood compared to other RNA transcripts. Nevertheless, we have witnessed an increasing interest for lncRNAs roles and functions in the past decade. For example, lncRNAs have been investigated as potential biomarkers for diagnosing pancreatic cancer and may have a role as therapeutics targets for precision medicine, but may also directly intervene in tumour progression features such as metastasis, epithelial to mesenchymal transition and resistance of cancer cells towards chemotherapy agents. The function of lncRNAs within various cancer exosomes is still undefined. In this review, we summarize the current knowledge on pancreatic cancer-derived exosome specific lncRNAs having prominent roles in genome integrity, pancreatic cancer progression and in other oncogenic hallmarks.

LncRNA-MALAT1/miRNA-204-5p/Smad4 Axis Regulates Epithelial–Mesenchymal Transition, Proliferation and Migration of Lens Epithelial Cells

ABSTRACT Purpose: Posterior capsular opacification (PCO), a common complication after cataract surgery, primarily originated from the epithelial–mesenchymal transition (EMT), proliferation, and migration of human lens epithelial cells (LECs). This study aimed to explore whether the long noncoding RNA metastasis-associated lung adenocarcinoma transcript 1 (MALAT1)/miR-204-5p/Smad4 axis are involved in EMT of LECs. Materials and methods LECs were cultured and induced with TGF-β2 (10 ng/mL). SiRNA against MALAT1 (Si‐MALAT1) was transfected into LECs to knockdown the expression of MALAT1. To overexpress or knockdown miR‐204-5p, miR-204-5p mimics (miR-204-5p mimics) and anti-miR-204-5p (miR-204-5p inhibitor) were transfected into LECs. We used RNA FISH to identify the location of MALAT1. RNA levels of MALAT1 and miR-204-5p were analyzed by RT-qPCR. Additionally, target protein levels of Smad4, epithelial differentiation and mesenchymal markers were analyzed with Western blot. We employed EdU Labeling to measured cell proliferation and performed Transwell Assay to analyze the cell migration. Dual-luciferase reporter assays in LECs were conducted to verify whether miRNA-204-5p was negatively regulated by MALAT1 and Smad4 was a direct target of miR-204-5p. Results The expression of MALAT1 was upregulated in PCO specimens. MALAT1 was overexpressed in TGF-β2 induced LECs, and the knockdown of MALAT1 could attenuate TGF-β2 induced EMT. Besides, the upregulation of MALAT1 was correlated with the downregulation of miR-204-5p and upregulation of Smad4. Importantly, MALAT1 was revealed to be located in the cytoplasm of LECs. Furthermore, luciferase reporter assays confirmed that MALAT1 could negatively regulate the expression of miR-204-5p and then regulate its direct target Smad4. Finally, the knockdown of MALAT1 could inhibit the EMT, proliferation, and migration of LECs; however, those can be reversed by anti-miR-204-5p. Conclusions Our findings reveal that MALAT1 may regulate EMT, proliferation, and migration of LECs as a ceRNA by “sponging” miR-204-5p and targeting Smad4, and serve as a promising therapeutic target in preventing PCO.

Silencing Neuropilin 1 gene reverses TGF-β1-induced epithelial mesenchymal transition in HGC-27 gastric cancer cell line*

Abstract Objective The aim of this study was to determine Neuropilin 1 (NRP1) contribution to transforming growth factor β1 (TGF-β1)-induced epithelial mesenchymal transition (EMT) of HGC-27 gastric cancer cells and study its mechanism. Methods In this study, TGF-β1 was used to induce EMT in HGC-27 cells. Further, these cells were stably transfected with siRNA targeting NRP1. Wound healing and transwell assays were used to measure cell migration and invasion, respectively. NRP1 and EMT markers were measured using quantitative real time reverse transcription polymerase chain reaction and western blotting. Results Exposure of TGF-β1 conferred a fibroblastic-like shape to cancer cells and significantly increased the expression of NRP1 in HGC-27 cells. TGF-β1 subsequently promoted migration and invasion of HGC-27 cells. Furthermore, silencing NRP1 inhibited the invasion and migration of TGF-β1-induced cells undergoing EMT. Conclusion Silencing NRP1 can inhibit cell migration, invasion, and metastasis and reverse the TGF-β1-induced EMT process of gastric cancer.

LINC01089 Inhibits Tumorigenesis and Epithelial-Mesenchymal Transition of Non-small Cell Lung Cancer via the miR-27a/SFRP1/Wnt/β-catenin Axis.

Long noncoding RNAs (lncRNAs) have emerged as regulators of gene expression and play critical regulatory roles in diverse biological functions and diseases, including cancer. In this study, we report the downregulation of LINC01089 in non-small cell lung cancer (NSCLC) samples, relative to adjacent non-tumor tissues, and demonstrate its role in the inhibition of proliferation, migration, and epithelial–mesenchymal transition (EMT) of NSCLC cells. Mechanistic analysis indicates that LINC01089 acts as a sponge for miR-27a, regulating its expression in NSCLC. Interestingly, LINC01089 mediated the upregulation of SFRP1 expression by inhibiting the Wnt/β-catenin–EMT pathway and inhibiting the epithelial–mesenchymal transition of NSCLC via sponging miR-27a. Overall, our findings highlight LINC01089’s tumorigenic role and regulatory mechanism in NSCLC, thereby suggesting its potential as a therapeutic target for managing NSCLC.

Matrix Metalloproteinases (MMPs) in Targeted Drug Delivery: Synthesis of a Potent and Highly Selective Inhibitor against Matrix Metalloproteinase- 7.

Matrix metalloproteinases (MMPs) are a family of zinc endopeptidases that play a key role in both physiological and pathological tissue degradation. MMPs have reportedly shown great potentials in the degradation of the Extracellular Matrix (ECM), have shown great potentials in targeting bioactive and imaging agents in cancer treatment. MMPs could provoke Epithelial to Mesenchymal Transition (EMT) of cancer cells and manipulate their signaling, adhesion, migration and invasion to promote cancer cell aggressiveness. Therefore, targeting and particularly inhibiting MMPs within the tumor microenvironment is an effective strategy for cancer treatment. Based on this idea, different MMP inhibitors (MMPIs) have been developed to manipulate the tumor microenvironment towards conditions appropriate for the actions of antitumor agents. Studies are ongoing to improve the selectivity and specificity of MMPIs. Structural optimization has facilitated the discovery of selective inhibitors of the MMPs. However, so far no selective inhibitor for MMP-7 has been proposed. This study aims to comprehensively review the potentials and advances in applications of MMPs particularly MMP-7 in targeted cancer treatment approaches with the main focus on targeted drug delivery. Different targeting strategies for manipulating and inhibiting MMPs for the treatment of cancer are discussed. MMPs are upregulated at all stages of expression in cancers. Different MMP subtypes have shown significant targeting applicability at the genetic, protein, and activity levels in both physiological and pathophysiological conditions in a variety of cancers. The expression of MMPs significantly increases at advanced cancer stages, which can be used for controlled release in cancers in advance stages. Moreover, this study presents the synthesis and characteristics of a new and highly selective inhibitor against MMP-7 and discusses its applications in targeted drug delivery systems for therapeutics and diagnostics modalities. Our findings showed that the structure of the inhibitor P3' side chains play the crucial role in developing an optimized MMP-7 inhibitor with high selectivity and significant degradation activities against ECM. Optimized NDC can serve as a highly potent and selective inhibitor against MMP-7 following screening and optimization of the P3' side chains, with a Ki of 38.6 nM and an inhibitory selectivity of 575 of MMP-7 over MMP-1.

Induction of interleukin-6 by irradiation and its role in epithelial mesenchymal transition and radioresistance of nasopharyngeal carcinoma cells.

Epithelial mesenchymal transition (EMT) participates in the radioresistance of cancer cells. In this study, we evaluated whether interleukin-6 (IL-6) regulates EMT and subsequent radioresistance in nasopharyngeal carcinoma (NPC) cells. Western blot was used to analyze the expression of proteins. Immunofluorescent assay was performed to detect the position and content of specific proteins. Colony formation assay was applied to measure the proliferation ability of NPC cells. Flow cytometry was adopted to measure cell apoptosis. NPC cells seen with typical morphological transition and unique proteins change of EMT when exposed to irradiation. IL-6 and its downstream signal molecules elevated in irradiated cells. Blocking IL-6 with anti-IL-6 receptor antibody hampered EMT and radioresistance. Cell radiosensitivity and apoptosis rates in irradiated NPC cells declined significantly upon IL-6 stimulation. Irradiation could induce EMT and activate IL-6 signaling in NPC cell lines. Blocking IL-6 was able to inhibit EMT and cellular radioresistance.

Prp19 Is an Independent Prognostic Marker and Promotes Neuroblastoma Metastasis by Regulating the Hippo-YAP Signaling Pathway

Pre-mRNA processing factor 19 (Prp19) was previously reported to be involved in tumor progression. However, Prp19 expression and its functions remain elusive in neuroblastoma. Here, we aim to identify the functions and mechanisms of Prp19 in neuroblastoma. Neuroblastic tumor tissue microarrays and two independent validation data sets indicate that Prp19 is associated with high-risk markers and bone marrow metastasis and serves as a prognostic marker for worse clinical outcomes with neuroblastoma. Gain- and loss-of-expression assays reveal that Prp19 promotes invasion, migration, and epithelial–mesenchymal transition (EMT) of neuroblastoma cells in vitro. Bioinformatics analysis of RNA-seq data shows that the expressions of YAP and its downstream genes are significantly inhibited after downregulation of Prp19. Prp19 and YAP expression in metastatic lymph nodes is higher than in situ neuroblastoma tissue. Further experiments show that Prp19 regulates YAP expression and consequently affects cell invasion, migration, and EMT in neuroblastoma by pre-mRNA splicing of YAP. In conclusion, our findings provide the first evidence that Prp19 is a potential therapeutic target and prognostic biomarker for patients with neuroblastoma.

The role of noncoding RNAs during aortic valve stenosis

Abstract Background Aortic valve stenosis (AVS) is the most common valve disease worldwide. Thought to be a purely degenerative disease, it is now clear that shear stress/endothelial dysfunction, lipid deposition and inflammation lead to calcification and stenosis of the valve. There is evidence, that extracellular vesicles (EVs) are actively involved in calcification processes. Practically all cells, including endothelial cells, can generate EVs, which can be shed into the blood stream and into the interstitial space. EVs contain lipids, proteins and nucleic acids, including noncoding RNAs (ncRNAs). EVs can be taken up by acceptor cells and their cargo, especially the ncRNA content, can change the phenotype of these cells. NcRNAs have been shown to have protective and damaging properties in AVS, which can lead to disease progression. EVs are actively involved in atherosclerosis and vascular calcification, but their role during AVS formation remains largely unknown. Purpose We hypothesize, that EV-derived ncRNAs play a crucial role during calcification of the aortic valve through regulation of endothelial to mesenchymal transition (EndMT) and calcification of valvular interstitial cells. Methods and results In initial screening experiments, we investigated ncRNA (micro RNA, miRNA and long noncoding RNA, lncRNA) content in aortic valve tissue from explanted human aortic valves from patients undergoing surgical aortic valve replacement. There is a differential expression of miRNAs and lncRNAs in aortic valve tissue from patients with AVS and patients without AVS. We could also show a differential packaging of ncRNAs into EVs generated from patient aortic valve tissues. Furthermore, ncRNA expression in aortic valve tissue is altered in a “wire-injury” mouse model of AVS. We can demonstrate in vitro that EVs and their content can be transferred from valvular endothelial cells (VECs) to valvular interstitial cells (VICs) and vice versa. Additionally, we have established an isolation method of VECs and VICs from human samples. To identify ncRNAs involved in EndMT, we are investigating the effect of laminar and pulsatile flow on the expression of ncRNAs in vitro. We can demonstrate that different flow patterns lead to a vast change in ncRNA expression in primary VECs. Downstream effects of identified ncRNAs are currently under investigation in our in vitro calcification and EndMT models. Transfection of VICs with miRNA mimics and inhibitors and lentiviral transduction of our identified targets lead to an altered calcification potential of valve cells. Conclusion The analysis of the cell type specific expression of ncRNAs and the intercellular communication via EVs will greatly help our understanding of the pathomechanisms leading to valvular calcification. Pathway analysis will generate new targets that could be used to develop therapeutics to ameliorate disease progression. EV-based miRNA mimics and inhibitors could be used to treat valvular calcification. Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): Deutsche Forschungsgemeinschaft, TRR259; Else-Kröner-Fresenius

Abstract PO-118: Novel mechanisms of EGFR-TKI resistance and clonal evolution of lung adenocarcinoma by overexpression of a cell adhesion molecule, CADM1

Abstract Evolution of cancer and generation of its heterogeneity are most dynamically demonstrated in the process of cancer cells acquiring resistance to molecular targeted therapy. Lung adenocarcinoma is the best solid tumor to which molecular targeted therapy is established and clinically available. However, tumors resistant against targeted drugs develop frequently, providing the biggest challenge in current treatment of lung adenocarcinoma. The tumors resistant to the initial treatment also show more malignant features, such as invasion and metastasis. Thus, it is interesting to investigate possible mechanisms to link EGFR-TKI resistance to malignant progression in lung adenocarcinoma. A cell adhesion molecule, CADM1, is originally identified as a tumor suppressor in lung adenocarcinoma. CADM1 is involved in the formation and maintenance of an epithelial cell structure. On the other hand, CADM1 is frequently inactivated by promoter methylation in various cancers in their advanced stages. In lung adenocarcinoma, CADM1 was expressed in the cell-cell attachment sites in lepidic growth components, whereas CADM1 expression was frequently lost in invasive lesions, conferring heterogenous features to cancer tissue. We have also reported that CADM1 inhibits HGF-induced epithelial mesenchymal transition of canine kidney cells, MDCK, by associating with MET and intervening with MET signaling on the cell membrane. Here, we demonstrate significant expression of CADM1 in a lung adenocarcinoma cell, HCC827, but not in its gefitinib-resistant derivatives, HCC827GR5 or GR6, which express significant amounts of phosphorylated MET (p-MET). Introducing CADM1 expression into HCC827GR5 restores the sensitivity to gefitinib in association with decreasing amount of pMET. In contrast, CADM1 is not effective at restoring gefitinib sensitivity in PC9 cells carrying T790M mutation of EGFR. Dox-inducible CADM1 expression in HCC827GR5 also showed restoration of gefitinib sensitivity both in vivo and in vitro. Finally, intra-tumoral injection of adenoviruses expressing CADM1, but not control adenoviruses, significantly suppressed subcutaneous tumor formation only when treated with gefitinib to nude mice. These results suggest that exogenous expression of CADM1 could provide a unique approach to overcome EGFR-TKI resistance of lung adenocarcinoma caused by MET overexpression. Inversely, loss of CADM1 and cancer cell adhesion could promote EGFR-TKI resistance caused by MET overexpression. Considering that CADM1 expression is lost selectively and clonally in microinvasive lesions of lung adenocarcinoma, disruption of cancer cell adhesion would be involved in generation of cell clones resistant to molecular targeted drugs and resultant heterogeneity in a subset of lung adenocarcinoma. Citation Format: Takehiro Tsuchiya, Hideki Kuwano, Takeshi Ito, Masayoshi Nagata, Taketo Kawai, Yumi Tsuboi, Daisuke Matsubara, Isami Okamoto, Kenji Tamura, Jun Nakajima, Motoi Oba, Yoshinori Murakami. Novel mechanisms of EGFR-TKI resistance and clonal evolution of lung adenocarcinoma by overexpression of a cell adhesion molecule, CADM1 [abstract]. In: Proceedings of the AACR Virtual Special Conference on Tumor Heterogeneity: From Single Cells to Clinical Impact; 2020 Sep 17-18. Philadelphia (PA): AACR; Cancer Res 2020;80(21 Suppl):Abstract nr PO-118.

Cadmium induces epithelial–mesenchymal transition and migration of renal cancer cells by increasing PGE2 through a cAMP/PKA-COX2 dependent mechanism

Environmental or occupational exposure of Cadmium (Cd) is concerned to be a threat to human health. The kidney is main target of Cd accumulation, which increases the risk of renal cell carcinoma (RCC). In addition, low content of Cd had been determined in kidney cancer, however, the roles of presence of Cd in renal tumors progression are still unclear. The present study is proposed to determine the effect of low-dose Cd exposure on the renal cancer cells and aimed to clarify the underlying mechanisms. The cell viability, cytotoxicity, and the migratory effect of low-dose Cd on the renal cancer cells were detected. Moreover, the roles of reactive oxygen species (ROS), Ca2+, and cyclic AMP (cAMP)/protein kinase A (PKA)-cyclooxygenase2 (COX2) signaling, as well as COX2 catalytic product prostaglandin E2 (PGE2) on cell migration and invasion were identified. Our results suggested that low dose Cd exposure promoted migration of renal cancer Caki-1 cells, which was not dependent on Cd-induced ROS and intracellular Ca2+ levels. Cd exposure induced cAMP/PKA-COX2, which mediated cell migration and invasion, and decreased expressions of epithelial-mesenchymal transition (EMT) marker, E-cadherin, but increased expressions of N-cadherin and Vimentin. Moreover, Cd-induced secretion of PGE2 feedback on activation of cAMP/PKA-COX2 signaling, also promoted EMT, migration and invasion of renal cancer Caki-1 cells. This study might contribute to understanding of the mechanism of Cd-induce progression of renal cancer and future studies on the prevention and therapy of renal cell carcinomas.

The Effects of Bone Morphogenetic Protein-4 on Cellular Viability, Osteogenic Potential, and Global Gene Expression on Gingiva-Derived Stem Cell Spheroids

Bone morphogenetic protein-4 (BMP-4) is engaged in the migration ability of mesenchymal stem cells and the transition of mesenchymal stem cells into osteogenic and adipocytic lines. The aim of this study was to evaluate the effects of BMP-4 on the cellular viability, osteogenic differentiation, and genome-wide mRNA levels using three-dimensional cell spheroids composed of stem cells. Stem cell spheroids were formed using concave microwells in the presence of BMP-4 with final concentrations of 0, 2, 6, and 10 ng/mL. Cellular viability was measured qualitatively using a microscope and quantitatively using an assay kit based on water-soluble tetrazolium salt. Osteogenic differentiation was assessed by measuring the level of alkaline phosphatase activity. Global gene expression was assessed using next-generation mRNA sequencing and performing gene ontology and pathway analyses. Spheroids were well-maintained with the addition of BMP-4 up to Day 7. No significant differences were observed in cell viability between each group. There were significantly higher alkaline phosphatase values in the 2 ng/mL BMP-4 groups when compared with the control (p &lt; 0.05). A total of 25,737 mRNAs were differentially expressed. Expression of β-catenin (CTNNB1) was increased with higher dosages of BMP-4. The expression of runt-related transcription factor 2 (RUNX2) was increased up to 6 ng/mL. The phosphoinositide-3-kinase–protein kinase B/Akt signaling pathway was associated with the target genes. This study demonstrates that the application of BMP-4 enhanced alkaline phosphatase activity and the expression of CTNNB1 and RUNX2 without affecting cellular viability.

Hypoxia-Inducible Ubiquitin Specific Peptidase 13 Contributes to Tumor Growth and Metastasis via Enhancing the Toll-Like Receptor 4/Myeloid Differentiation Primary Response Gene 88/Nuclear Factor-κB Pathway in Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is one of the leading causes of cancer death worldwide. The activation of the toll-like receptor 4/myeloid differentiation primary response gene 88/nuclear factor-κB (TLR4/MyD88/NF-κB) pathway contributes to the development and progression of HCC. The ubiquitin–proteasome system regulates TLR4 expression. However, whether ubiquitin specific peptidase 13 (USP13) stabilizes TLR4 and facilitates HCC progression remains unclear. Here, quantitative real-time PCR (qRT-PCR) and immunohistochemistry analysis revealed that USP13 expression in HCC tissues was higher than in non-tumor liver tissues. Moreover, the elevated expression of USP13 was detected in HCC cells (SK-HEP-1, HepG2, Huh7, and Hep3B) compared to LO2 cells. Interestingly, the positive staining of USP13 was closely correlated with tumor size ≥ 5 cm and advanced tumor stage and conferred to significantly lower survival of HCC patients. Next, USP13 knockdown prominently reduced the proliferation, epithelial–mesenchymal transition (EMT), migration, and invasion of Hep3B and Huh7 cells, while USP13 overexpression enhanced these biological behaviors of HepG2 and LO2 cells. The silencing of USP13 significantly restrained the growth and lung metastasis of HCC cells in vivo. Mechanistically, the USP13 depletion markedly inhibited the TLR4/MyD88/NF-κB pathway in HCC cells. USP13 interacted with TLR4 and inhibited the ubiquitin-mediated degradation of TLR4. Significantly, TLR4 re-expression remarkably reversed the effects of USP13 knockdown on HCC cells. USP13 expression was markedly upregulated in HCC cells under hypoxia conditions. Notably, USP13 knockdown repressed hypoxia-induced activation of the TLR4/MyD88/NF-κB pathway in HCC cells. In conclusion, our study uncovered that hypoxia-induced USP13 facilitated HCC progression via enhancing TLR4 deubiquitination and subsequently activating the TLR4/MyD88/NF-κB pathway.

Circular RNA hsa_circ_0007121 regulates proliferation, migration, invasion, and epithelial–mesenchymal transition of trophoblast cells by miR-182-5p/PGF axis in preeclampsia

BackgroundMounting evidence has revealed that abnormal expression of circular RNAs play pivotal roles in many human diseases including preeclampsia (PE). While human sapiens circular RNA 0007121 (hsa_circ_0007121) has been verified to be downregulated in human placental tissues, the underlying mechanisms were still unclear. This research aims to investigate the effect and underlying mechanisms of hsa_circ_0007121 in preeclampsia.MethodsThe expression of hsa_circ_0007121, microRNA (miR)-182-5p, and placental growth factor (PGF) was assessed by quantitative reverse transcription polymerase chain reaction in PE placentas relative to the expression in normal pregnancy placentas. After transfection, cell counting kit-8 assay was employed to detect cell proliferation. Cell migration and invasion were tested by the transwell assay. The relative level of epithelial–mesenchymal transition (EMT)-related proteins in HTR-8/SVneo cells and PGF in placentas samples were measured by western blot. The relationship between miR-182-5p and hsa_circ_0007121 or PGF was predicated by circular RNA interactome or ENCORI and verified by dual-luciferase reporter assay and RNA immunoprecipitation assay.ResultsThe levels of hsa_circ_0007121 and PGF were significantly declined in PE placental tissues and HTR-8/SVneo cells, whereas miR-182-5p had an opposite result. Downregulation of hsa_circ_0007121 obviously inhibited HTR-8/SVneo cell proliferation, migration, invasion, and EMT, while upregulation of hsa_circ_0007121 promoted this process. Besides, miR-182-5p was a target gene of hsa_circ_0007121 and could target PGF. Further analysis indicated that hsa_circ_0007121 regulated the proliferation, migration, invasion, and EMT of HTR-8/SVneo cells via altering PGF expression by interacting with miR-182-5p.ConclusionHsa_circ_0007121 mediated the progression of PE via miR-182-5p/PGF axis.

Circ_0000043 promotes the proliferation, migration, invasiveness, and epithelial-mesenchymal transition in breast cancer cells via the miR-136-Smad3 axis.

Circular RNAs (circRNAs) are tissue-specific RNAs with a more stable structure than linear RNAs, and their association with breast cancer (BC) is poorly understood. This study examined the biological effects of circ_0000043 in the progression of BC. In this study, expression of circ_0000043 in BC tissue samples was measured using quantitative real-time polymerase chain reaction. Immunohistochemistry and Western blot were used to detect the expression of Smad family member 3 (Smad3). CCK-8, wound healing, and Transwell assays were used to assess the effect of circ_0000043 on the proliferation, migration, and invasiveness of BC cells. Moreover, the binding relationships between circ_0000043 and miR-136, and miR-136 and Smad3 were detected by dual-luciferase reporter assay. Additionally, Western blot was used to detect the expressions of markers related to epithelial-mesenchymal transition, including E-cadherin, N-cadherin, and vimentin. Our results show that the expression of circ_0000043 is up-regulated in BC tissues and cell lines. The proliferation, migration, invasiveness, and epithelial-mesenchymal transition of BC cells were significantly inhibited by knockdown of circ_0000043, and overexpression of circ_0000043 had the opposite effects. Additionally, circ_0000043 up-regulate the expression of Smad3 by sponging miR-136. In conclusion, our study demonstrates that circ_0000043 promote the progression of BC via regulating the miR-136-Smad3 axis.