Increased Cell Invasion Research Articles

TROP2 Down-regulated DSG2 to Promote Gastric Cancer Cell Invasion and Migration by EGFR/AKT and DSG2/PG/β-Catenin Pathways.

Gastric cancer (GC) is the fourth most commonly found cancer and the second- highest cause of cancer-related death worldwide. TROP2 overexpression is closely related to many cancers, including gastrointestinal tumors. DSG2 is an important protein in cell adhesion, and its loss affects cell migration. This study aimed to explore the specific mechanism of TROP2 in promoting gastric cancer and provide a basis for the prevention and treatment of gastric cancer. DSG2 was identified as an interacting protein of TROP2 in GC cells by coimmunoprecipitation and mass spectrometry. The regulated behavior of TROP2 on DSG2 expression was investigated with TROP2 over-expressure or knockdown. Cell-cell adhesion capacity mediated by DSG2 was evaluated by adhesion-related assays. Electron microscope observation was made for accessing GC tumor desmosome assembly. Proteins in EGFR/AKT and DSG2/PG/β-catenin pathways were evaluated by western blotting. This study suggests that abundant expression of TROP2 in GC cells lessened DSG2 levels as well as desmosome adhesion, increased cell invasion and migration, and promoted malignant progression through EGFR/AKT and DSG2/PG/β-catenin pathways. TROP2 promotes cell invasion and migration in gastric cancer by decreasing DSG2 expression through EGFR/AKT and DSG2/PG/β-catenin pathways.

GRK3 is a poor prognosticator and serves as a therapeutic target in advanced gastric adenocarcinoma

BackgroundG protein-coupled receptor (GPCR) is the most targeted protein family by the FDA-approved drugs. GPCR-kinase 3 (GRK3) is critical for GPCR signaling. Our genomic analysis showed that GRK3 expression correlated with poor prognosis of gastric adenocarcinoma (GAC) patients. However, GRK3’s functions and clinical utility in GAC progression and metastases are unknown.MethodsWe studied GRK3 expression in normal, primary, and metastatic GAC tissues. We identified a novel GRK3 inhibitor, LD2, through a chemical-library screen. Through genetic and pharmacologic modulations of GRK3, a series of functional and molecular studies were performed in vitro and in vivo. Impact of GRK3 on YAP1 and its targets was determined.ResultsGRK3 was overexpressed in GAC tissues compared to normal and was even higher in peritoneal metastases. Overexpression (OE) of GRK3 was significantly associated with shorter survival. Upregulation of GRK3 in GAC cells increased cell invasion, colony formation, and proportion of ALDH1+ cells, while its downregulation reduced these attributes. Further, LD2 potently and specifically inhibited GRK3, but not GRK2, a very similar kinase to GRK3. LD2 highly suppressed GAC cells’ malignant phenotypes in vitro. Mechanistically, GRK3 upregulated YAP1 in GAC tissues and its transcriptional downstream targets: SOX9, Birc5, Cyr61 and CTGF. Knockdown (KD) YAP1 rescued the phenotypes of GRK3 OE in GAC cells. GRK3 OE significantly increased tumor growth but LD2 inhibited tumor growth in the PDX model and dramatically suppressed peritoneal metastases induced by GRK3 OE.ConclusionsGRK3, a poor prognosticator for survival, conferred aggressive phenotype. Genetic silencing of GRK3 or its inhibitor LD2 blunted GRK3-conferred malignant attributes, suggesting GRK3 as a novel therapeutic target in advanced GAC.

IDH1 mutation activates mTOR signaling pathway, promotes cell proliferation and invasion in glioma cells.

Glioma is the most common type of brain tumors and isocitrate dehydrogenase (IDH1) gene is the most prominent molecular marker about the disease prognosis, response to therapy and patient survival. There are conflicting data about the effect of IDH1 mutation on glial cell proliferation, invasion and migration characteristics. The effect of IDH1 mutation on mTOR signaling pathway, which has key roles in tumorigenesis process, is limited and previous data is controversial. We aimed to explore the effect of wild type and mutant IDH1 overexpression on glioma cells and investigated the correlation with mTOR signaling pathway associated genes. U87-MG and A172 cells were transfected with different IDH1 mutant gene overexpressing (R132H, R132L, R132S, R132C) viral vectors. Cell proliferation, cell invasion and migration analysis as well as quantitative PCR analysis with the mutant glioma cell lines were performed. Forty-two patient derived glioma cells were obtained from patients with different glioma subtypes and cancer cells were enriched by culturing cells. Overexpression of both mutant and wild type IDH1 gene promoted the cell proliferation, but only IDH1 mutation increased cell invasion and migration. The expression of IDH1 mutation activated mTOR signaling via upregulation of WNTA, PRKAA2, GSK3B and MTOR genes as well as phosphorylated mTOR protein level. Our results highlighted IDH1 mutation upregulate mTOR signaling pathway and promote cell proliferation, invasion and migration.

FOXA1 inhibits hypoxia programs through transcriptional repression of HIF1A.

Intratumoral hypoxia is associated with castration-resistant prostate cancer (CRPC), a lethal disease. FOXA1 is an epithelial transcription factor that is down-regulated in CRPC. We have previously reported that FOXA1 loss induces epithelial-mesenchymal transition (EMT) and cell motility through elevated TGFβ signaling. However, whether FOXA1 directly regulates hypoxia pathways of CRPC tumors has not been previously studied. Here we report that FOXA1 down-regulation induces hypoxia transcriptional programs, and FOXA1 level is negatively correlated with hypoxia markers in clinical prostate cancer (PCa) samples. Mechanistically, FOXA1 directly binds to an intragenic enhancer of HIF1A to inhibit its expression, and HIF1A, in turn, is critical in mediating FOXA1 loss-induced hypoxia gene expression. Further, we identify CCL2, a chemokine ligand that modulates tumor microenvironment and promotes cancer progression, as a crucial target of the FOXA1-HIF1A axis. We found that FOXA1 loss leads to immunosuppressive macrophage infiltration and increased cell invasion, dependent on HIF1A expression. Critically, therapeutic targeting of HIF1A-CCL2 using pharmacological inhibitors abolishes FOXA1 loss-induced macrophage infiltration and PCa cell invasion. In summary, our study reveals an essential role of FOXA1 in controlling the hypoxic tumor microenvironment and establishes the HIF1A-CCL2 axis as one mechanism of FOXA1 loss-induced CRPC progression.

CD146 increases stemness and aggressiveness in glioblastoma and activates YAP signaling

Glioblastoma (GBM), a highly malignant and lethal brain tumor, is characterized by diffuse invasion into the brain and chemo-radiotherapy resistance resulting in poor prognosis. In this study, we examined the involvement of the cell adhesion molecule CD146/MCAM in regulating GBM aggressiveness. Analyses of GBM transcript expression databases revealed correlations of elevated CD146 levels with higher glioma grades, IDH-wildtype and unmethylated MGMT phenotypes, poor response to chemo-radiotherapy and worse overall survival. In a panel of GBM stem cells (GSCs) variable expression levels of CD146 were detected, which strongly increased upon adherent growth. CD146 was linked with mesenchymal transition since expression increased in TGF-ß-treated U-87MG cells. Ectopic overexpression of CD146/GFP in GG16 cells enhanced the mesenchymal phenotype and resulted in increased cell invasion. Conversely, GSC23-CD146 knockouts had decreased mesenchymal marker expression and reduced cell invasion in transwell and GBM-cortical assembloid assays. Moreover, using GSC23 xenografted zebrafish, we found that CD146 depletion resulted in more compact delineated tumor formation and reduced tumor cell dissemination. Stem cell marker expression and neurosphere formation assays showed that CD146 increased the stem cell potential of GSCs. Furthermore, CD146 mediated radioresistance by stimulating cell survival signaling through suppression of p53 expression and activation of NF-κB. Interestingly, CD146 was also identified as an inducer of the oncogenic Yes-associated protein (YAP). In conclusion, CD146 carries out various pro-tumorigenic roles in GBM involving its cell surface receptor function, which include the stimulation of mesenchymal and invasive properties, stemness, and radiotherapy resistance, thus providing an interesting target for therapy.

The NFAT3/RERG Complex in Luminal Breast Cancers Is Required to Inhibit Cell Invasion and May Be Correlated With an Absence of Axillary Lymph Nodes Colonization.

Luminal breast cancers represent 70% of newly diagnosed breast cancers per annum and have a relatively good prognosis compared with triple-negative breast cancers. Luminal tumors that are responsive to hormonal therapy are particularly associated with a favorable prognosis. Nonetheless, the absolute number of metastatic relapses in luminal cancers is larger than in triple-negative breast cancers. A better understanding of the biology of luminal cancers, control of metastases formation, and identification of predictive markers of their evolution are therefore still necessary. In this context, we previously disclosed the key role of NFAT3 in regulating luminal breast cancer invasion. We have now identified a specific inhibitory region, in the C-terminal part of NFAT3, required for the inhibition of invasion of the human luminal breast cancer cell line T-47D. Indeed, we showed that this 85 amino acid C-terminal region acts as a dominant negative form of NFAT3 and that its overexpression in the T-47D cell line led to increased cell invasion. Mechanistically, we have revealed that this region of NFAT3 interacts with the small Ras GTPase RERG (RAS like estrogen regulated growth inhibitor) and shown that RERG expression is required for NFAT3 to impede T-47D cell invasion. We have validated the association of NFAT3 with RERG in human luminal breast cancer tissues. We have shown an increase of the quantity of the NFAT3/RERG complexes in patients without axillary lymph node colonization and therefore proposed that the detection of this complex may be a non-invasive marker of axillary lymph node colonization.

Abstract LB036: Molecular insight into ERG transcriptional activity to unravel novel therapeutic options for ERG-positive castration resistant prostate cancer

Abstract Gene fusions of the AR-regulated gene TMPRSS2 with the ETS family transcription factor ERG characterize about 40% of castration resistant prostate cancer (CRPC) - an advanced and lethal stage of prostate cancer. AR-mediated overexpression of ERG hijacks AR inhibiting lineage-specific differentiation and increases cell invasion and migration. In vivo work revealed a causal role of ERG in initiating prostate cancer. Notably, in vitro and in vivo models of CRPC endogenously bearing T2ERG depend on ERG activity for proliferation and survival. Thus, pursuing the development of ERG-based therapeutic strategies represents a promising route for CRPC treatment, although major challenges in targeting transcription factors such as ERG have been reported. Yet, synthetic lethality approaches including genome-wide loss-of-function screens can be employed to avoid direct ERG targeting. Here, we used metastatic PCa cell lines (LNCaP, 22Rv1 and DU145) engineered with inducible systems to overexpress ERG to show that high levels of ERG expression lead to an apparent reduction in the fitness of AR-positive and negative cells. Prolonged overexpression of a mutant form of ERG, which is not able to bind DNA, does not affect cell viability indicating that ERG DNA binding and transcriptional activity are necessary for the induction of this phenotype. The ERG-mediated reduction in cellular fitness is influenced by cell adhesion and is characterized by cell cycle arrest mediated by several cell cycle inhibitors induced by ERG. As revealed by gene expression analyses and functional assays, ERG induces a less proliferative, more dedifferentiated and invasive phenotype, which lead to an apparent reduction of cellular fitness in vitro, but is characteristic of more aggressive tumor cells in vivo. Altogether, these results underscore the reliability of the developed in vitro models for ERG activity, which will be used for screening to identify therapeutic vulnerabilities unlocked by ERG in CRPC. Importantly, we also uncovered confounding factors (i.e. loss of ERG-positive cells not due to screen selection) that could impinge on the results arising from in vitro screens. Citation Format: Francesca Lorenzin, Giulia Fracassi, Yari Ciani, Paola Gasperini, David Rickman, Francesca Demichelis. Molecular insight into ERG transcriptional activity to unravel novel therapeutic options for ERG-positive castration resistant prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB036.

Abstract 3167: The scaffolding protein DLG5 is necessary to maintain Sonic Hedgehog signaling in glioblastoma stem cells and promote tumor growth

Abstract Proteins complexes that maintain the apico-basal polarity of epithelial cells and organize cell-signaling domains are necessary for normal tissue organization and therefore regarded as tumor suppressors. Accordingly, the scaffolding proteins that form the Scribble polarity complex (SCRIB, LLGL1, and DLG members) are usually downregulated in cancer, in particular in highly aggressive or invasive tumors. Because gliomas arise from non-epithelial precursors that are likely motile before transformation, the expression and functions of these scaffolding proteins in malignant brain tumors has remained almost entirely unexplored. Surprisingly, we have found that one member of the DLG family, DLG5, is highly upregulated in malignant gliomas compared to other solid tumors. Indeed, DLG5 was the predominant DLG member found in all molecular subtypes of glioblastoma (GBM). Knockdown of DLG5 reduced the viability, invasiveness, and self-renewal of GBM stem cells belonging to the proneural and mesenchymal molecular subtypes. As a result, intracranial tumors defficient in DLG5 were smaller and less proliferative than controls, resulting in extended animal survival. At the molecular level, DLG5 knockdown decreased the expression of stemness-related genes (SOX2, NANOG, POU5F1) but increased the expression of LLGL1, a component of the Scribble complex that is associated with neural precursor differentiation. These changes were accompanied by downregulation of Sonic Hedgehog (SHh) signaling, both in vitro and in vivo, which drives GBM cell stemness and proliferation. Moreover, DLG5-deficient GBM stem cells became insensitive to exogenous SHh and were unable to upregulate Gli1 or increase cell invasion in presence of added SHh, compared to control cells. We further confirmed that DLG5 downregulation increased the ubiquitination of Gli1-containing complexes as well as Gli1 proteasomal degradation, providing a suitable mechanism for the loss of SHh signaling and tumor stemness. These results describe for the first time the expression of a DLG family member in malignant gliomas and reveal a novel, polarity-independent, pro-tumoral function of DLG5, which differs from its proposed tumor-suppressor role in other solid tumors. Citation Format: Somanath Kundu, Mohan S. Nandhu, Sharon L. Longo, John A. Longo, Shawn Rai, Lawrence S. Chin, Timothy E. Richardson, Mariano S. Viapiano. The scaffolding protein DLG5 is necessary to maintain Sonic Hedgehog signaling in glioblastoma stem cells and promote tumor growth [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3167.

The role of A-kinase interacting protein 1 in regulating progression and stemness as well as indicating the prognosis in glioblastoma

BackgroundA-kinase interacting protein 1 (AKIP1) is recently implicated in the pathogenesis of several solid tumors, while its role in glioblastoma multiforme (GBM) is largely unknown. Therefore, the current study aimed to investigate the effect of AKIP1 on GBM cell malignant behaviors, stemness, and its underlying molecular mechanisms. MethodsU-87 MG and A172 cells were transfected with control or AKIP1 overexpression plasmid; control or AKIP1 siRNA plasmid. Then cell proliferation, apoptosis, invasion, CD133+ cell proportion, and sphere formation assays were performed. Furthermore, RNA-Seq was performed in U-87 MG cells. Besides, AKIP1 expression was detected in 25 GBM and 25 low-grade glioma (LGG) tumor samples. ResultsAKIP1 was increased in several GBM cell lines compared to the control cell line. After transfections, it was found that AKIP1 overexpression increased cell invasion, CD133+ cell proportion, and sphere formation ability while less affecting cell proliferation or cell apoptosis in U-87 MG and A172 cells. Moreover, AKIP1 siRNA achieved the opposite effect in these cells, except that it inhibited cell proliferation but induced cell apoptosis to some extent. Subsequent RNA-Seq assay showed several critical carcinogenetic pathways, such as PI3K/AKT, Notch, EGFR tyrosine kinase inhibitor resistance, Ras, ErbB, mTOR pathways, etc. were potentially related to the function of AKIP1 in U-87 MG cells. Clinically, AKIP1 expression was higher in GBM tissues than in LGG tissues, which was also correlated with the poor prognosis of GBM to some degree. ConclusionsAKIP1 regulates the malignant behaviors and stemness of GBM via regulating multiple carcinogenetic pathways.

Analysis of the subcellular location of FAM230B and its interaction with premature miR-302b in osteosarcoma.

FAM230B has been characterized as an oncogenic lncRNA in papillary thyroid cancer and gastric cancer, while its role in osteosarcoma (OS) is unclear. This research was conducted to analyze the interaction between FAM230B and miR-302b in OS. Paired OS and non-tumor tissues donated by 58 OS patients were subjected to RNA preparation and RT-qPCR to quantify the expression of FAM230B and miR-302b (both mature and premature). The subcellular location of FAM230B and its interaction with premature miR-320b were analyzed by cellular fractionation assay and RNA pull-down assay, respectively. The roles of FAM230B and miR-302b in OS cell movement were evaluated using Transwell assay. Increased FAM230B and premature miR-302b and decreased mature miR-302b were observed in OS. FAM230B was detected in both nuclear and cytoplasm samples and directly interacted with premature miR-302b. FAM230B overexpression in OS cells 143B and HOS decreased miR-302b maturation and increased cell invasion and migration, while miR-302b overexpression suppressed cell invasion and migration. FAM230B silencing restrained cell invasion and migration, and miR-302b inhibitor accelerated cell invasion and migration. MiR-302b knockdown significantly reversed the suppressive effects of shFAM230B on migration and invasion of 143B and HOS cells. FAM230B is accumulated to high levels in OS and may serve as an endogenous competing RNA for premature miR-302b in the nucleus to promote OS cell invasion and migration.

Multi-Omics Analysis of Glioblastoma and Glioblastoma Cell Line: Molecular Insights Into the Functional Role of GPR56 and TG2 in Mesenchymal Transition.

G protein-coupled receptor 56 (GPR56/ADGRG1) is an adhesion GPCR with an essential role in brain development and cancer. Elevated expression of GPR56 was observed in the clinical specimens of Glioblastoma (GBM), a highly invasive primary brain tumor. However, we found the expression to be variable across the specimens, presumably due to the intratumor heterogeneity of GBM. Therefore, we re-examined GPR56 expression in public domain spatial gene expression data and single-cell expression data for GBM, which revealed that GPR56 expression was high in cellular tumors, infiltrating tumor cells, and proliferating cells, low in microvascular proliferation and peri-necrotic areas of the tumor, especially in hypoxic mesenchymal-like cells. To gain a better understanding of the consequences of GPR56 downregulation in tumor cells and other molecular changes associated with it, we generated a sh-RNA-mediated GPR56 knockdown in the GBM cell line U373 and performed transcriptomics, proteomics, and phospho-proteomics analysis. Our analysis revealed enrichment of gene signatures, pathways, and phosphorylation of proteins potentially associated with mesenchymal (MES) transition in the tumor and concurrent increase in cell invasion and migration behavior of the GPR56 knockdown GBM cells. Interestingly, our analysis also showed elevated expression of Transglutaminase 2 (TG2) - a known interactor of GPR56, in the knockdown cells. The inverse expression of GPR56 and TG2 was also observed in intratumoral, spatial gene expression data for GBM and in GBM cell lines cultured in vitro under hypoxic conditions. Integrating all these observations, we propose a putative functional link between the inverse expression of the two proteins, the hypoxic niche and the mesenchymal status in the tumor. Hypoxia-induced downregulation of GPR56 and activation of TG2 may result in a network of molecular events that contribute to the mesenchymal transition of GBM cells, and we propose a putative model to explain this functional and regulatory relationship of the two proteins.

MiR-140-5p exerts a protective function in pregnancy-induced hypertension via mediating TGF-β/Smad signaling pathway

ABSTRACT Animal experiments showed that PIH rats had increased mean arterial pressure (MAP), systolic blood pressure (SBP), and diastolic blood pressure (DBP), but decreased litter size, number of viable fetuses, fetal weight, and placental weight. The higher Flt-1 and lower VEGF was observed in PIH rats with elevated TNF-α and IL-6 levels and decreased IL-10 levels. Treatment with agomiR-140-5p improved regarding the above indicators. Cell experiments demonstrated that miR-140-5p mimic increased cell invasion and migration abilities and decreased the activity of TGF-β/Smad pathway, while TGFBR1 can reverse the role of miR-140-5p mimic in trophoblasts.

Galangin alleviates rheumatoid arthritis in rats by downregulating the phosphatidylinositol 3-kinase/protein kinase B signaling pathway

ABSTRACT Rheumatoid arthritis (RA) is a chronic autoimmune disease that greatly affect patients’ quality of life. Galangin extract is renowned for its anti-proliferative and anti-oxidative characteristics. However, galangin cytotoxicity studies are presently inadequate. We aimed to investigate the therapeutic potential of galangin on RA by investigating the PI3K/AKT signaling pathway.Fibroblast-like synovial cells (FLSs) were exposed to lipopolysaccharide (LPS) to establish an RA model in vitro. An ELISA assay was used to detect the levels of IL-1β, TNF-α, and IL-6. Cell viability and apoptosis were determined by CCK8/EdU and flow cytometry assays. A western blot assay was used to analyze the protein expression levels. An RA rat model was established to evaluate the function of galangin through histopathological examination. Our results found that galangin induced apoptosis, inhibited cell proliferation, and increased cell invasion of rheumatoid arthritis fibroblast-like synovial cells (RAFLSs). Galangin inactivated the PI3K/AKT signaling pathway and the inflammatory response. An agonist of PI3K signaling, 740Y‐P, restored the cellular functions of RAFLSs. Moreover, galangin suppressed the development of RA in vivo. Galangin effected its anti-arthritic influence through the PI3K/AKT signaling pathway. Galangin has potential as an alternative treatment for RA.

MAGEA6 positively regulates MSMO1 and promotes the migration and invasion of oesophageal cancer cells.

The melanoma antigen gene family A (MAGEA) family of proteins comprises of cancer-testis antigens that are highly expressed in a number of tumours but are minimally expressed in normal cells. Due to its expression characteristics, this protein family has become a popular target for anti-cancer drugs and immunotherapy research over recent years. Although, elevated expression levels of MAGEA6 has been found in different types of tumours, there remains to be insufficient information on the function of MAGEA6 and its associated gene regulation pathways. The present study used Transwell, Cell Counting Kit-8 and wound healing assays to analyse the effects of MAGEA6 on Eca109 cell invasion, migration and proliferation. The main functions and pathways involved in MAGEA6 were predicted by Illumina Hiseq screening for mutually regulated genes and core genes. Eca109 cell line with a high expression of MAGEA6 was a stable cell line obtained by transfection in the early stage, and this cell line was used in subsequent experiments. Transcriptome sequencing was performed on this cell line and the Eca109 cell line that normally expressed MAGEA6. It was revealed that a high expression of MAGEA6 conferred a significant stimulating effect on cell proliferation whilst also significantly increasing cell invasion and migration. Transcriptomic analysis identified 14 differentially expressed genes and 13 core regulatory genes closely associated with MAGEA6 expression regulation, such as methylsterol monooxygenase 1 (MSMO1). The present study suggest that MAGEA6 positively regulated MSMO1 expression, which may serve an oncogenic role in cells through this regulatory effect. Overall, this provided a novel route of investigation for an in-depth study of the regulatory function of MAGEA6.

Nitric oxide regulates adhesiveness, invasiveness, and migration of anoikis-resistant endothelial cells.

Anoikis is a type of apoptosis that occurs in response to the loss of adhesion to the extracellular matrix (ECM). Anoikis resistance is a critical mechanism in cancer and contributes to tumor metastasis. Nitric oxide (NO) is frequently upregulated in the tumor area and is considered an important player in cancer metastasis. The aim of this study was to evaluate the effect of NO on adhesiveness, invasiveness, and migration of anoikis-resistant endothelial cells. Here, we report that anoikis-resistant endothelial cells overexpress endothelial nitric oxide synthase. The inhibition of NO release in anoikis-resistant endothelial cells was able to decrease adhesiveness to fibronectin, laminin, and collagen IV. This was accompanied by an increase in cell invasiveness and migration. Furthermore, anoikis-resistant cell lines displayed a decrease in fibronectin and collagen IV protein expression after L-NAME treatment. These alterations in adhesiveness and invasiveness were the consequence of MMP-2 up-regulation observed after NO release inhibition. The decrease in NO levels was able to down-regulate the activating transcription factor 3 (ATF3) protein expression. ATF3 represses MMP-2 gene expression by antagonizing p53-dependent trans-activation of the MMP-2 promoter. We speculate that the increased release of NO by anoikis-resistant endothelial cells acted as a response to restrict the MMP-2 action, interfering in MMP-2 gene expression via ATF3 regulation. The up-regulation of nitric oxide by anoikis-resistant endothelial cells is an important response to restrict tumorigenic behavior. Without this mechanism, invasiveness and migration potential would be even higher, as shown after L-NAME treatment.

Physical Confinement and Cell Proximity Increase Cell Migration Rates and Invasiveness: A Mathematical Model of Cancer Cell Invasion Through Flexible Channels

Physical Confinement and Cell Proximity Increase Cell Migration Rates and Invasiveness: A Mathematical Model of Cancer Cell Invasion Through Flexible Channels

LncRNA HOXC-AS3 overexpression inhibits TGF-β2-induced colorectal cancer cell migration and invasion by sponging miR-1269

Long non-coding RNA (lncRNA) HOXC-AS3 has been characterized as a cancer-related lncRNA in many types of cancer, while its role in colorectal cancer (CRC) is unknown. The expression of HOXC-AS3 and TGF-β2 were detected by RT-qPCR. Overexpression assays were performed to explore the interaction between HOXC-AS3 and TGF-β2. A follow-up study was performed to explore the prognostic value of HOXC-AS3 for CRC. The direct interaction between HOXC-AS3 and miR-1269 was assessed with RNA-RNA pulldown assay. Transwell assays were performed to determine the role of HOXC-AS3 and TGF-β2 in regulating CRC cell invasion and migration. HOXC-AS3 was significantly downregulated in CRC tissues, while TGF-β2 was significantly upregulated in CRC tissues compared to that in adjacent non-cancer tissues of CRC patients. The follow-up study showed that low expression levels of HOXC-AS3 in CRC tissues were closely correlated with poor survival. Correlation analysis showed that HOXC-AS3 and TGF-β2 were inversely correlated across CRC tissues but not non-cancer tissues. Overexpression of HOXC-AS3 in the two cell lines resulted in downregulation of TGF-β2, while the expression of HOXC-AS3 was not affected by TGF-β2. Transwell migration and invasion assay showed that overexpression of TGF-β2 increased cell invasion and migration, while overexpression of HOXC-AS3 decreased cell migration and invasion. In addition, overexpression of HOXC-AS3 attenuated the effects of overexpression of TGF-β2. MiR-1269 increased the expression of TGF-β2. HOXC-AS3 directly interacted with miR-1269 in CRC cells. Upregulation of HOXC-AS3 inhibited TGF-β2-induced colorectal cancer (CRC) cell migration and invasion possibly by sponging miR-1269.

The Tumour Suppressor CYLD Is Required for Clathrin-Mediated Endocytosis of EGFR and Cetuximab-Induced Apoptosis in Head and Neck Squamous Cell Carcinoma.

Simple SummaryEpidermal growth factor receptor (EGFR) is a target for the therapeutic antibody cetuximab (CTX) in head and neck squamous cell carcinoma (HNSCC). Identification of its predictive biomarkers and potentiation of CTX-based therapies are important. In this study, we found that the N-terminal portion of cylindromatosis (CYLD) was required for clathrin-mediated endocytosis (CME) and degradation of EGFR. Loss of CYLD limited EGFR to lipid rafts and inhibited CTX-induced apoptosis. Destruction of lipid rafts by cholesterol removers restored EGFR CME and CTX activity in CYLD-downregulated cells. Our findings provide novel insights into the molecular mechanisms underlying EGFR trafficking and resistance to CTX, and suggest the usefulness of CTX-based therapy combined with cholesterol-lowering drugs in HNSCC.Epidermal growth factor receptor (EGFR) is frequently overexpressed in head and neck squamous cell carcinoma (HNSCC) and is a target for the therapeutic antibody cetuximab (CTX). However, because only some patients have a significant clinical response to CTX, identification of its predictive biomarkers and potentiation of CTX-based therapies are important. We have recently reported a frequent downregulation of cylindromatosis (CYLD) in primary HNSCC, which led to increased cell invasion and cisplatin resistance. Here, we show that CYLD located mainly in lipid rafts was required for clathrin-mediated endocytosis (CME) and degradation of the EGFR induced by EGF and CTX in HNSCC cells. The N-terminus containing the first cytoskeleton-associated protein-glycine domain of CYLD was responsible for this regulation. Loss of CYLD restricted EGFR to lipid rafts, which suppressed CTX-induced apoptosis without impeding CTX’s inhibitory activity against downstream signalling pathways. Disruption of the lipid rafts with cholesterol-removing agents overcame this resistance by restoring CME and the degradation of EGFR. Regulation of EGFR trafficking by CYLD is thus critical for the antitumour activity of CTX. Our findings suggest the usefulness of a combination of cholesterol-lowering drugs with anti-EGFR antibody therapy in HNSCC.

Circ-ATAD1 is overexpressed in osteosarcoma (OS) and suppresses the maturation of miR-154-5p to increase cell invasion and migration

BackgroundCirc-ATAD1 plays an oncogenic role in gastric cancer. However, its roles in other cancers are unclear. We aimed to analyze the role of circ-ATAD1 in osteosarcoma (OS).MethodsThe expression levels of circ-ATAD1, mature miR-154-5p, and premature miR-154-5p in paired OS and non-tumor tissues from 56 OS patients were determined using RT-qPCR. Nuclear fractionation assay was performed to analyze the subcellular location of circ-ATAD1. The interaction between circ-ATAD1 and premature miR-154-5p was analyzed using RNA pull-down assay. The role of circ-ATAD1 in regulating miR-154-5p maturation was analyzed using RT-qPCR in cells with overexpression. Transwell assays were performed to analyze the roles of circ-ATAD1 and miR-154-5p in regulating OS cell invasion and migration.ResultsCirc-ATAD1 was overexpressed in OS compared to non-tumor tissues and was detected in the nuclei of OS cells. Mature miR-154-5p, but not premature miR-154-5p, was downregulated in OS tissues compared to non-tumor tissues and was inversely correlated with circ-ATAD1. In OS cells, circ-ATAD1 overexpression decreased the expression of mature miR-154-5p, but not premature miR-154-5p. Transwell assay analysis showed that circ-ATAD1 overexpression increased cell invasion and migration, and mature miR-154-5p overexpression suppressed these cell behaviors. In addition, circ-ATAD1 overexpression reduced the effects of mature miR-154-5p overexpression on cell behaviors.ConclusionsCirc-ATAD1 is overexpressed in OS and suppresses miR-154-5p maturation to increase cell invasion and migration.

Telmisartan-Induced Cytotoxicity <i>via</i> G<sub>2</sub>/M Phase Arrest in Renal Cell Carcinoma Cell Lines

Renal cell carcinoma (RCC) is the most common type of kidney cancer. Given that stage IV RCC is intractable, there is a need for a novel treatment strategy. We investigated the antitumor effects of telmisartan (TEL) and their underlying mechanisms in RCC, including their impact on apoptosis, Akt/mammalian target of rapamycin (mTOR) pathways, and the cell cycle using two human RCC cell lines: 786-O and Caki-2. Cell viability was detected via fluorescence-based assays. Cells were stained with Hoechst 33342 to observe chromatin condensation, and Western blotting was performed to analyze protein expression. The cell cycle was assessed using flow cytometry. Invasion and migration assays were performed using 24-well chambers. TEL induced cell death in a dose-dependent manner and increased the percentage of cells with high chromatin condensation and Bax/Bcl-2 ratio in both cell lines. TEL-induced cell death was attenuated by neither peroxisome proliferator-activated receptor-γ nor -δ inhibitors. Although TEL elevated c-Jun N-terminal kinase levels and p38 phosphorylation rates in Caki-2 cells, as well as extracellular signal-regulated kinase phosphorylation rates in 786-O cells, their inhibitors did not suppress TEL-induced cell death. TEL decreased Akt phosphorylation in 786-O cells and mTOR phosphorylation in both cell lines, increased the population of cells in the G2/M phase, and altered G2/M-related proteins in both cell lines. TEL moderately suppressed cell invasion and migration in 786-O and Caki-2 cells, respectively, and increased cell invasion in Caki-2 cells, suggesting a potential therapeutic role of TEL in RCC.