Role of Periostin Expression in Non-Small Cell Lung Cancer: Periostin Silencing Inhibits the Migration and Invasion of Lung Cancer Cells via Regulation of MMP-2 Expression

The involvement of periostin (POSTN) in non-small-cell lung cancer (NSCLC) migration, invasion, and its underlying mechanisms has not been well established. The present study aims to determine epithelial POSTN expression in NSCLC and to assess associations with clinicopathological factors and prognosis as well as to explore the effects of POSTN knockdown on tumor microenvironment and the migration and invasion of lung cancer cells. Immunohistochemistry was used to evaluate epithelial POSTN expression in NSCLC. POSTN mRNA expression in the dissected lung cancer cells was confirmed by laser capture microdissection and real-time PCR. A549 cells were used for transfecting shRNA-POSTN lentiviral particles. Wound healing and Transwell invasion assays were used to assess the migratory and invasive abilities of A549 cells transfected with POSTN-specific short hairpin (sh)RNA. The results demonstrated significantly higher cytoplasmic POSTN expression in the whole NSCLC group compared to non-malignant lung tissue (NMLT). POSTN expression in cancer cells may be considered to be an independent prognostic factor for survival in NSCLC. POSTN knockdown significantly inhibited A549 cell migration and invasion capabilities in vitro. The activity and the expression level of matrix metalloproteinase-2 (MMP-2) were significantly decreased in A549.shRNA compared to control cells. In summary, POSTN may regulate lung cancer cell invasiveness by modulating the expression of MMP-2 and may represent a potential target for novel therapeutic intervention for NSCLC.     

CRISPR-Cas9–Mediated TIM3 Knockout in Human Natural Killer Cells Enhances Growth Inhibitory Effects on Human Glioma Cells

Glioblastoma (GBM) is the most common and aggressive primary malignant brain tumor in adults. Natural Killer (NK) cells are potent cytotoxic effector cells against tumor cells inducing GBM cells; therefore, NK cell based- immunotherapy might be a promising target in GBM. T cell immunoglobulin mucin family member 3 (TIM3), a receptor expressed on NK cells, has been suggested as a marker of dysfunctional NK cells. We established TIM3 knockout in NK cells, using the clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9). Electroporating of TIM3 exon 2- or exon 5-targeting guide RNA- Cas9 protein complexes (RNPs) inhibited TIM3 expression on NK cells with varying efficacy. T7 endonuclease I mutation detection assays showed that both RNPs disrupted the intended genome sites. The expression of other checkpoint receptors, i.e., programmed cell death 1 (PD1), Lymphocyte-activation gene 3 (LAG3), T cell immunoreceptor with Ig and ITIM domains (TIGIT), and TACTILE (CD96) were unchanged on the TIM3 knockout NK cells. Real time cell growth assays revealed that TIM3 knockout enhanced NK cell–mediated growth inhibition of GBM cells. These results demonstrated that TIM3 knockout enhanced human NK cell mediated cytotoxicity on GBM cells. Future, CRISPR-Cas9 mediated TIM3 knockout in NK cells may prove to be a promising immunotherapeutic alternative in patient with GBM.     

Cleavage of LOXL1 by BMP1 and ADAMTS14 Proteases Suggests a Role for Proteolytic Processing in the Regulation of LOXL1 Function

Members of the lysyl oxidase (LOX) family catalyze the oxidative deamination of lysine and hydroxylysine residues in collagen and elastin in the initiation step of the formation of covalent cross-links, an essential process for connective tissue maturation. Proteolysis has emerged as an important level of regulation of LOX enzymes with the cleavage of the LOX isoform by metalloproteinases of the BMP1 (bone morphogenetic protein 1) and ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) families as a model example. Lysyl oxidase-like 1 (LOXL1), an isoform associated with pelvic organ prolapse and pseudoexfoliation (PEX) glaucoma, has also been reported to be proteolytically processed by these proteases. However, precise molecular information on these proteolytic events is not available. In this study, using genetic cellular models, along with proteomic analyses, we describe that LOXL1 is processed by BMP1 and ADAMTS14 and identify the processing sites in the LOXL1 protein sequence. Our data show that BMP1 cleaves LOXL1 in a unique location within the pro-peptide region, whereas ADAMTS14 processes LOXL1 in at least three different sites located within the pro-peptide and in the first residues of the catalytic domain. Taken together, these results suggest a complex regulation of LOXL1 function by BMP1- and ADAMTS14-mediated proteolysis where LOXL1 enzymes retaining variable fragments of N-terminal region may display different capabilities.     

Hypoxia Modulates Cellular Endocytic Pathways and Organelles with Enhanced Cell Migration and 3D Cell Invasion**

Hypoxia, a decrease in cellular or tissue level oxygen content, is characteristic of most tumors and has been shown to drive cancer progression by altering multiple subcellular processes. We hypothesized that the cancer cells in a hypoxic environment might have slower proliferation rates and increased invasion and migration rates with altered endocytosis compared to the cancer cells in the periphery of the tumor mass that experience normoxic conditions. We induced cellular hypoxia by exposing cells to cobalt chloride, a chemical hypoxic mimicking agent. This study measured the effect of hypoxia on cell proliferation, migration, and invasion. Uptake of fluorescently labeled transferrin, galectin3, and dextran that undergo endocytosis through major endocytic pathways (Clathrin-mediated pathway (CME), Clathrin-independent pathway (CIE), Fluid phase endocytosis (FPE)) were analyzed during hypoxia. Also, the organelle changes associated with hypoxia were studied with organelle trackers. We found that the proliferation rate decreased, and the migration and invasion rate increased in cancer cells in hypoxic conditions compared to normoxic cancer cells. A short hypoxic exposure increased galectin3 uptake in hypoxic cancer cells, but a prolonged hypoxic exposure decreased clathrin-independent endocytic uptake of galectin 3. Subcellular organelles, such as mitochondria, increased to withstand the hypoxic stress, while other organelles, such as Endoplasmic reticulum (ER), were significantly decreased. These data suggest that hypoxia modulates cellular endocytic pathways with reduced proliferation and enhanced cell migration and invasion.     

Enhancing Anti-Tumoral Potential of CD-NHF by Modulating PI3K/Akt Axis in U87 Ex Vivo Glioma Model

Background: In the latest years, there has been an increased interest in nanomaterials that may provide promising novel approaches to disease diagnostics and therapeutics. Our previous results demonstrated that Carbon-dots prepared from N-hydroxyphthalimide (CD-NHF) exhibited anti-tumoral activity on several cancer cell lines such as MDA-MB-231, A375, A549, and RPMI8226, while U87 glioma tumor cells were unaffected. Gliomas represent one of the most common types of human primary brain tumors and are responsible for the majority of deaths. In the present in vitro study, we expand our previous investigation on CD-NHF in the U87 cell line by adding different drug combinations. Methods: Cell viability, migration, invasion, and immunofluorescent staining of key molecular pathways have been assessed after various treatments with CD-NHF and/or K252A and AKTVIII inhibitors in the U87 cell line. Results: Association of an inhibitor strongly potentiates the anti-tumoral properties of CD-NHF identified by significant impairment of migration, invasion, and expression levels of phosphorylated Akt, p70S6Kinase, or by decreasing expression levels of Bcl-2, IL-6, STAT3, and Slug. Conclusions: Using simultaneously reduced doses of both CD-NHF and an inhibitor in order to reduce side effects, the viability and invasiveness of U87 glioma cells were significantly impaired.     

GCN5 Potentiates Glioma Proliferation and Invasion via STAT3 and AKT Signaling Pathways

The general control of nucleotide synthesis 5 (GCN5), which is one kind of lysine acetyltransferases, regulates a number of cellular processes, such as cell proliferation, differentiation, cell cycle and DNA damage repair. However, its biological role in human glioma development remains elusive. In the present study, we firstly reported that GCN5 was frequently overexpressed in human glioma tissues and GCN5 was positively correlated with proliferation of cell nuclear antigen PCNA and matrix metallopeptidase MMP9. Meanwhile, down-regulation of GCN5 by siRNA interfering inhibited glioma cell proliferation and invasion. In addition, GCN5 knockdown reduced expression of p-STAT3, p-AKT, PCNA and MMP9 and increased the expression of p21 in glioma cells. In conclusion, GCN5 exhibited critical roles in glioma development by regulating cell proliferation and invasion, which suggested that GCN5 might be a potential molecular target for glioma treatment.     

Microglia-Derived Olfactomedin-like 3 Promotes Pro-Tumorigenic Microglial Function and Malignant Features of Glioma Cells

Under the influence of transforming growth factor-beta (TGFβ), glioma-associated microglia produce molecules that promote glioma growth and invasion. Olfactomedin-like 3 (Olfml3), a novel, secreted glycoprotein, is known to promote several non-CNS cancers. While it is a direct TGFβ1 target gene in microglia, the role of microglia-derived OLFML3 in glioma progression is unknown. Here, we tested the hypotheses that microglial Olfml3 is integral to the pro-tumorigenic glioma-associated microglia phenotype and promotes glioma cell malignancy. Using an Olfml3 knockout microglial cell line (N9), we demonstrated that Olfml3 is a direct target gene of all TGFβ isoforms in murine microglia. Moreover, loss of Olfml3 attenuated TGFβ-induced restraint on microglial immune function and production of cytokines that are critical in promoting glioma cell malignancy. Importantly, microglia-derived OLFML3 directly contributes to glioma cell malignancy through increased migration and invasion. While exposure to conditioned medium (CM) from isogenic control microglia pre-treated with TGFβ increased mouse glioma cell (GL261) migration and invasion, this effect was abolished with exposure to CM from TGFβ-treated Olfml3^-/- microglia. Taken together, our data suggest that Olfml3 may serve as a gatekeeper for TGFβ-induced microglial gene expression, thereby promoting the pro-tumorigenic microglia phenotype and glioma cell malignancy.     

Directional Persistence of Cell Migration in Schizophrenia Patient-Derived Olfactory Cells

Cell migration is critical for brain development and linked to several neurodevelopmental disorders, including schizophrenia. We have shown previously that cell migration is dysregulated in olfactory neural stem cells from people with schizophrenia. Although they moved faster than control cells on plastic substrates, patient cells were insensitive to regulation by extracellular matrix proteins, which increase the speeds of control cells. As well as speed, cell migration is also described by directional persistence, the straightness of movement. The aim of this study was to determine whether directional persistence is dysregulated in schizophrenia patient cells and whether it is modified on extracellular matrix proteins. Directional persistence in patient-derived and control-derived olfactory cells was quantified from automated live-cell imaging of migrating cells. On plastic substrates, patient cells were more persistent than control cells, with straighter trajectories and smaller turn angles. On most extracellular matrix proteins, persistence increased in patient and control cells in a concentration-dependent manner, but patient cells remained more persistent. Patient cells therefore have a subtle but complex phenotype in migration speed and persistence on most extracellular matrix protein substrates compared to control cells. If present in the developing brain, this could lead to altered brain development in schizophrenia.     

Synaptic Cell Adhesion Molecule 3 (SynCAM3) Deletion Promotes Recovery from Spinal Cord Injury by Limiting Glial Scar Formation

Synaptic cell adhesion molecules (SynCAMs) play an important role in the formation and maintenance of synapses and the regulation of synaptic plasticity. SynCAM3 is expressed in the synaptic cleft of the central nervous system (CNS) and is involved in the connection between axons and astrocytes. We hypothesized that SynCAM3 may be related to the astrocytic scar (glial scar, the most important factor of CNS injury treatment) through extracellular matrix (ECM) reconstitution. Thus, we investigated the influence of the selective removal of SynCAM3 on the outcomes of spinal cord injury (SCI). SynCAM3 knock-out (KO) mice were subjected to moderate compression injury of the lower thoracic spinal cord using wild-type (WT) (C57BL/6JJc1) mice as controls. Single-cell RNA sequencing analysis over time, quantitative real-time polymerase chain reaction (qRT-PCR) analysis, and immunohistochemistry (IHC) showed reduced scar formation in SynCAM3 KO mice compared to WT mice. SynCAM3 KO mice showed improved functional recovery from SCI by preventing the transformation of reactive astrocytes into scar-forming astrocytes, resulting in improved ECM reconstitution at four weeks after injury. Our findings suggest that SynCAM3 could be a novel therapeutic target for SCI.     

Compensatory Protection of Thioredoxin-Deficient Cells from Etoposide-Induced Cell Death by Selenoprotein W via Interaction with 14-3-3

Selenoprotein W (SELENOW) is a 9.6 kDa protein containing selenocysteine (Sec, U) in a conserved Cys-X-X-Sec (CXXU) motif. Previously, we reported that SELENOW regulates various cellular processes by interacting with 14-3-3β at the U of the CXXU motif. Thioredoxin (Trx) is a small protein that plays a key role in the cellular redox regulatory system. The CXXC motif of Trx is critical for redox regulation. Recently, an interaction between Trx1 and 14-3-3 has been predicted. However, the binding mechanism and its biological effects remain unknown. In this study, we found that Trx1 interacted with 14-3-3β at the Cys32 residue in the CXXC motif, and SELENOW and Trx1 were bound at Cys191 residue of 14-3-3β. In vitro binding assays showed that SELENOW and Trx1 competed for interaction with 14-3-3β. Compared to control cells, Trx1-deficient cells and SELENOW-deficient cells showed increased levels of both the subG1 population and poly (ADP-ribose) polymerase (PARP) cleavage by etoposide treatment. Moreover, Akt phosphorylation of Ser473 was reduced in Trx1-deficient cells and was recovered by overexpression of SELENOW. These results indicate that SELENOW can protect Trx1-deficient cells from etoposide-induced cell death through its interaction with 14-3-3β.     

Norcantharidin Induces Immunogenic Cell Death of Bladder Cancer Cells through Promoting Autophagy in Acidic Culture

The acidic tumor microenvironment stands as a major obstacle to the efficient elimination of tumor cells. Norcantharidin (NCTD) is a powerful antitumor agent with multiple bioactivities. However, the effect of NCTD under acidic conditions is still unclear. Here, we report that NCTD can efficiently kill bladder cancer (BC) cells in acidic culture, and more intriguingly, NCTD can induce immunogenic cell death (ICD), thereby promoting antitumor immunity. In NCTD-treated BC cells, the surface-exposed calreticulin (ecto-CALR) was significantly increased. Consistently, co-culture with these cells promoted dendritic cell (DC) maturation. The NCTD-induced ICD is autophagy dependent, as autophagy inhibition completely blocked the NCTD-induced ecto-CALR and DC maturation. In addition, the DC showed a distinct maturation phenotype (CD80^high CD86^low) in acidic culture, as compared to that in physiological pH (CD⁸⁰ high CD86^high). Finally, the NCTD-induced ICD was validated in a mouse model. NCTD treatment significantly increased the tumor-infiltrating T lymphocytes in MB49 bladder cancer mice. Immunizing mice with NCTD-treated MB49 cells significantly increased tumor-free survival as compared to control. These findings demonstrate that NCTD could induce ICD in an acidic environment and suggest the feasibility to combine NCTD with anticancer immunotherapy to treat BC.     

MT1-MMP Cooperates with TGF-β Receptor-Mediated Signaling to Trigger SNAIL and Induce Epithelial-to-Mesenchymal-like Transition in U87 Glioblastoma Cells

Epithelial-to-mesenchymal transition (EMT) recapitulates metastasis and can be induced in vitro through transforming growth factor (TGF)-β signaling. A role for MMP activity in glioblastoma multiforme has been ascribed to EMT, but the molecular crosstalk between TGF-β signaling and membrane type 1 MMP (MT1-MMP) remains poorly understood. Here, the expression of common EMT biomarkers, induced through TGF-β and the MT1-MMP inducer concanavalin A (ConA), was explored using RNA-seq analysis and differential gene arrays in human U87 glioblastoma cells. TGF-β triggered SNAIL and fibronectin expressions in 2D-adherent and 3D-spheroid U87 glioblastoma cell models. Those inductions were antagonized by the TGF-β receptor kinase inhibitor galunisertib, the JAK/STAT inhibitors AG490 and tofacitinib, and by the diet-derived epigallocatechin gallate (EGCG). Transient gene silencing of MT1-MMP prevented the induction of SNAIL by ConA and abrogated TGF-β-induced cell chemotaxis. Moreover, ConA induced STAT3 and Src phosphorylation, suggesting these pathways to be involved in the MT1-MMP-mediated signaling axis that led to SNAIL induction. Our findings highlight a new signaling axis linking MT1-MMP to TGF-β-mediated EMT-like induction in glioblastoma cells, the process of which can be prevented by the diet-derived EGCG.     

基质金属蛋白酶在肿瘤侵袭转移作用中的研究

肿瘤细胞的侵袭与转移是肿瘤的恶性生物学行为,其作用机制多样且复杂。基底膜是机体的重要组织屏障,多种基质蛋白酶纷纷参与了肿瘤细胞降解基底膜和细胞外基质的过程,其中以基质金属蛋白酶类的作用最为突出。该文对基质金属蛋白酶类在肿瘤细胞侵袭与转移中的作用机制、相关转录因子以及国内外研发的抑制剂做了综述。     

Increased Activity of Cell Surface Peptidases in HeLa Cells Undergoing UV-Induced Apoptosis Is Not Mediated by Caspase 3

We have previously shown that in HeLa cells treated with a variety of agents there is an increase in cell surface peptidase (CSP) activity in those cells undergoing apoptosis. The increase in CSP activity observed in UVB-irradiated cells undergoing apoptosis was unaffected when the cultures were treated with the aminopeptidase inhibitor bestatin, and matrix metalloprotease inhibitor BB3103, but greatly enhanced when treated with the caspase 3 inhibitor-DEVD, and reduced in the presence of the poly(ADP-ribose) polymerase (PARP) inhibitor-3-aminobenzamide (3AB). Neither 3AB nor DEVD had an effect on the gross morphology of the apoptotic cells observed under electron microscopy, nor did they have an effect on phosphatidylserine eversion on the cell membrane, or that of PARP cleavage. All the agents except for DEVD had no effect on the level of caspase 3 activity in the cells. The results suggest that other caspases may cleave PARP in these cells. Both 3AB and DEVD treatment reduced the level of actin cleavage seen in the apoptotic cells. The increase in CSP activity observed in cells undergoing UVB-induced apoptosis appears to involve PARP but not caspase 3.