Ezrin Modulates the Cell Surface Expression of Programmed Cell Death Ligand-1 in Human Cervical Adenocarcinoma Cells

Cancer cells employ programmed cell death ligand-1 (PD-L1), an immune checkpoint protein that binds to programmed cell death-1 (PD-1) and is highly expressed in various cancers, including cervical carcinoma, to abolish T-cell-mediated immunosurveillance. Despite a key role of PD-L1 in various cancer cell types, the regulatory mechanism for PD-L1 expression is largely unknown. Understanding this mechanism could provide a novel strategy for cervical cancer therapy. Here, we investigated the influence of ezrin/radixin/moesin (ERM) family scaffold proteins, crosslinking the actin cytoskeleton and certain plasma membrane proteins, on the expression of PD-L1 in HeLa cells. Our results showed that all proteins were expressed at mRNA and protein levels and that all ERM proteins were highly colocalized with PD-L1 in the plasma membrane. Interestingly, immunoprecipitation assay results demonstrated that PD-L1 interacted with ERM as well as actin cytoskeleton proteins. Furthermore, gene silencing of ezrin, but not radixin and moesin, remarkably decreased the protein expression of PD-L1 without affecting its mRNA expression. In conclusion, ezrin may function as a scaffold protein for PD-L1; regulate PD-L1 protein expression, possibly via post-translational modification in HeLa cells; and serve as a potential therapeutic target for cervical cancer, improving the current immune checkpoint blockade therapy.     

Role of mTOR signaling in tumor cell motility, invasion and metastasis

Tumor cell migration and invasion play fundamental roles in cancer metastasis. The mammalian target of rapamycin (mTOR), a highly conserved and ubiquitously expressed serine/threonine (Ser/Thr) kinase, is a central regulator of cell growth, proliferation, differentiation and survival. Recent studies have shown that mTOR also plays a critical role in the regulation of tumor cell motility, invasion and cancer metastasis. Current knowledge indicates that mTOR functions as two distinct complexes, mTORC1 and mTORC2. mTORC1 phosphorylates p70 S6 kinase (S6K1) and eukaryotic initiation factor 4E (eIF4E) binding protein 1 (4E-BP1), and regulates cell growth, proliferation, survival and motility. mTORC2 phosphorylates Akt, protein kinase C α (PKCα) and the focal adhesion proteins, and controls the activities of the small GTPases (RhoA, Cdc42 and Rac1), and regulates cell survival and the actin cytoskeleton. Here we briefly review recent knowledge of mTOR complexes and the role of mTOR signaling in tumor cell migration and invasion. We also discuss recent efforts about the mechanism by which rapamycin, a specific inhibitor of mTOR, inhibits cell migration, invasion and cancer metastasis.     

Synthesis and biological evaluation of quinocarcin derivatives

Cyanation of quinocarcin readily opened the oxazolidine ring to provide DX-52-1 (2), which was a key compound in the synthesis of quinocarcin derivatives. Various electrophilic reactions toward aromatic ring of DX-52-1 were examined, and 10-substituted (e.g., halogen, nitro, formyl, cyano, hydroxy, etc.) analogs were prepared. Dehydrocyanation of the derivatives could be achieved to reproduce the oxazolidine ring upon treatment with HCl or AgNO3. 10-Chloride 10 and 10-bromide 11 were the most promising among the derivatives prepared. Antitumor activity of 10 was extended to B-16 melanoma.     

MiR-126-5p Promotes Tumor Cell Proliferation,Metastasis and Invasion by Targeting TDO2 in Hepatocellular Carcinoma

TDO2 is a key enzyme in the kynurenine metabolic pathway, which is the most important pathway of tryptophan metabolism. It has been shown that miRNAs are involved in cell metastasis through interaction with target mRNAs. In this study, we found 645 miRNAs that could be immunoprecipitated with TDO2 through the RNA-immunoprecipitation experiment. miR-126-5p was selected as the research target, which was also confirmed by dual-luciferase reporter assay. Through qRT-PCR analysis, it was verified that the overexpression of miR-126-5p promoted the expression of TDO2, PI3K/AKT and WNT1. Meanwhile, it was verified that overexpression of miR-126-5p can promote intracellular tryptophan metabolism by HPLC. We also verified the effects of miR-126-5p on cell proliferation, migration, and invasion by cck-8, cell colony formation and trans-well assay in both HCCLM3 cells and HepG2 cells. In vivo experiments were also conducted to verify that miR-126-5p promoted tumor formation and growth via immunohistochemical detection of cell infiltration and proliferation to generate markers Ki-67, BAX, and VEGF. In conclusion, our results suggest that miR-126-5p is a biomarker and a potential new treatment target in the progression of HCC via promoting the expression of TDO2.     

Synthetic mimetics of actin-binding macrolides: rational design of actin-targeted drugs

Actin polymerization and dynamics are involved in a wide range of cellular processes such as cell division and migration of tumor cells. At sites of cell lysis, such as those occurring during a stroke or inflammatory lung diseases, actin is released into the serum where it polymerizes, leading to problems with clot dissolution and sputum viscosity. Therefore, drugs that target these actin-mediated processes may provide one mechanism to treat these conditions. Marine-organism-derived macrolides, such as reidispongiolide A, can bind to, sever, and inhibit polymerization of actin. Our studies show that the function of these complex macrolides resides in their tail region, whereas the head group stabilizes the actin-drug complex. Synthetic compounds derived from this tail region could therefore be used as a mimetic of the natural product, providing a range of designer compounds to treat actin-associated diseases or as probes to study actin polymerization.     

Validation study of a method for assaying DE-310, a macromolecular carrier conjugate containing an anti-tumor camptothecin derivative, and the free drug in tumor tissue by high performance liquid chromatography/atmospheric pressure chemical ionization tandem mass spectrometry

DE-310 is a macromolecular carrier conjugate containing an anti-tumor camptothecin derivative, DX-8951, which is conjugated to a water-soluble polymer via a peptide spacer. Assay methods have been developed for the determination of a polymer-bonded DX-8951 conjugate, DX-8951, and Glycyl-DX-8951 concentrations in murine Meth A tumor tissue. Free DX-8951 and Glycyl-DX-8951 were extracted from tumor tissue homogenates by protein precipitation and analyzed by LC/MS/MS (method I). Conjugated DX-8951 was isolated by solid-phase extraction after digestion with a thermolysin. The productive phenylalanyl-glycyl-DX-8951 was analyzed by LC/MS/MS (method II). The lower limits of quantitation of DX-8951, Glycyl-DX-8951, and conjugated DX-8951 were 1.36, 1.34 and 73.7 ng/g (as DX-8951 equivalent). These two methods showed satisfactory sensitivity, precision and accuracy. To study the pharmacokinetics of DE-310, it would be of great help to assay the polymer-bonded DX-8951 and its released drugs in tumor tissue.     

Asymmetric elastic properties of Dictyostelium discoideum in relation to chemotaxis

In this study we used an AFM to investigate the cytoskeletal properties of live Dictyostelium discoideum cells by measuring the local stiffness across individual living cells. We have examined differences in elastic properties of polarized and unpolarized AX3 wild type and the mutant DAip1- cells, as well as the differences in the front and rear of the cells in relation to organization of the actin cytoskeleton. We found that the average Young's modulus increases upon polarization for the thin regions of the cell and that in polarized cells, the cell front was stiffer than the cell back. We also found that AX3 cells were stiffer than DAip1- cells. This finding suggests that actin polymerization is one of the major determinants of cell motility in Dictyostelium. In addition, a thin agarose film was studied as a model system to examine the influence of the substrate of thin materials probed with the AFM.     

Evaluation of proposed modes of binding of (2S)-2-[4-[[(3S)-1-acetimidoyl-3-pyrrolidinyl]oxy]phenyl]-3-(7-amidino-2-naphthyl)propanoic acid hydrochloride and some analogs to Factor Xa using a comparative molecular field analysis

The binding mode of (2S)-2-[4-[[(3S)-1-acetimidoyl-3-pyrrolidinyl]oxy]phenyl]-3-(7-amidino-2-naphthyl)propanoic acid hydrochloride (DX-9065a, 4) to Factor Xa is examined using inhibition data for a series of analogs that have a hydrophobic group as well as basic or dibasic functionality. Comparative molecular field analysis is utilized on a series of DX-9065a analogs in a series of proposed alternative binding modes. A quantitative measure is provided that distinguishes between the proposed binding modes that describes how well the binding mode explains the structure-activity relationship or the best 3D QSAR agrees with the crystallographically determined binding mode. The best model is in agreement with recently available data [Brandstetter et al., J. Biol. Chem., 271 (1996) 29988]. The highest statistical correlation occurs with the second basic group accommodated in the vicinity of Glu97 and a hydrophobic group accommodated in the pocket defined by Phe174, Tyr99 and Trp215. Also, the best model arises when the conformation of the Glu97 side chain is modified such that an H-bond interaction is maintained with the inhibitor if possible. The model also shows a tightening of the S1 pocket as is shown in the recent data described above.     

p53 and DNA-dependent protein kinase catalytic subunit independently function in regulating actin damage-induced tetraploid G1 arrest

We previously reported that the p53 tumor suppressor protein plays an essential role in the induction of tetraploid G1 arrest in response to perturbation of the actin cytoskeleton, termed actin damage. In this study, we investigated the role of p53, ataxia telangiectasia mutated protein (ATM), and catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) in tetraploid G1 arrest induced by actin damage. Treatment with actin- damaging agents including pectenotoxin-2 (PTX-2) increases phosphorylation of Ser-15 and Ser-37 residues of p53, but not Ser-20 residue. Knockdown of ATM and DNA-PKcs do not affect p53 phosphorylation induced by actin damage. However, while ATM knockdown does not affect tetraploid G1 arrest, knockdown of DNA-PKcs not only perturbs tetraploid G1 arrest, but also results in formation of polyploidy and induction of apoptosis. These results indicate that DNA-PKcs is essential for the maintenance of actin damage induced- tetraploid G1 arrest in a p53-independent manner. Furthermore, actin damage-induced p53 expression is not observed in cells synchronized at G1/S of the cell cycle, implying that p53 induction is due to actin damage-induced tetraploidy rather than perturbation of actin cytoskeleton. Therefore, these results suggest that p53 and DNA- PKcs independently function for tetraploid G1 arrest and preventing polyploidy formation.     

Topography guiding the accelerated and persistently directional cell migration induced by vaccinia virus

The rapid transmission of vaccinia virus(VACV)in vivo is thought to be closely related to the cell migration induced by it.Cell migration involved in dynamic changes of cell-substrate adhesion and actin cytoskeleton organization,which can influence by the micro/nano-scale topographic structures that cells are naturally exposed to via contact guidance.However,migration behaviors of VACV-infected cells exposed to topographic cues are still unknown.Herein,we designed an open chip with microgrooved poly(dimethyl siloxane)(PDMS)substrate to explore the topography roles in VACV-induced cell migration.Differed from the random cell migration observed in traditional scratch assay on planar substrate,VACV-infected cells had a tendency to persistently migrate along the axis parallel to microgroove with increased velocity.Moreover,infected cells exhibited a dominant elongated protrusion aligned to the micro-grating axis compare to the shorter lamella extended in any direction on smooth substrate.Interestingly,the Golgi complex preferred to relocate behind the nucleus confined within the micro-grating axis in majority of infected migratory cells.The directional polarization of cells embodied in protrusion formation and Golgi reorientation was responsible for the directionally persistent migration behaviors induced by VACV on microgrooved substrate.Infected cells response to substrate topography,causing the actin-filled stretched protrusion containing numerous virions and accelerated movement is likely to facilitate direct and rapid spread of VACV.This work opens a window for us to understand the migration behaviors of infected cells in vivo,and also provides a cue for revealing the relationship between virus-induced cell migration and virus rapid spread.     

Synergistic induction of cancer cell migration regulated by Gβγ and phosphatidylinositol 3-kinase

Phosphatidylinositol 3-kinase (PI3K) is essential for both G protein-coupled receptor (GPCR)- and receptor tyrosine kinase (RTK)-mediated cancer cell migration. Here, we have shown that maximum migration is achieved by full activation of phosphatidylinositol 3,4,5-trisphosphate-dependent Rac exchanger 1 (P-Rex1) in the presence of Gβγ and PI3K signaling pathways. Lysophosphatidic acid (LPA)- induced migration was higher than that of epidermal growth factor (EGF)-induced migration; however, LPA-induced activation of Akt was lower than that stimulated by EGF. LPA-induced migration was partially blocked by either Gβγ or RTK inhibitor and completely blocked by both inhibitors. LPA-induced migration was synergistically increased in the presence of EGF and vice versa. In correlation with these results, sphingosine-1-phosphate (S1P)-induced migration was also synergistically induced in the presence of insulin-like growth factor-1 (IGF-1). Finally, silencing of P-Rex1 abolished the synergism in migration as well as in Rac activation. Moreover, synergistic activation of MMP-2 and cancer cell invasion was attenuated by silencing of P-Rex1. Given these results, we suggest that P-Rex1 requires both Gβγ and PI3K signaling pathways for synergistic activation of Rac, thereby inducing maximum cancer cell migration and invasion.     

Validation study of assay method for DX-8951 and its metabolite in human plasma and urine by high-performance liquid chromatography/atmospheric pressure chemical ionization tandem mass spectrometry

A new liquid chromatographic/mass spectrometric assay has been developed for the determination of DX-8951, a new anti-tumor drug, and its 4-hydroxymethyl metabolite (UM-1) in human plasma and urine. Solid-phase extractions were used for sample preparation. A gradient reverse-phase HPLC separation was developed with mobile phases consisting of trifluoroacetic acid and methanol. The detection was conducted using atmospheric pressure chemical ionization tandem mass spectrometry in the selected reaction monitoring mode. A structural analog, camptothecin (CPT), was used as the internal standard. The assay was validated for the determination of DX-8951 and UM-1 in human plasma and urine. The lower limits of quantitation of DX-8951 and UM-1 were 0.1 ng/mL in plasma and 1 ng/mL in urine. The method showed a satisfactory sensitivity, precision, accuracy, recovery and selectivity.     

Lysophosphatidic acid induces cell migration through the selective activation of Akt1

Akt plays pivotal roles in many physiological responses including growth, proliferation, survival, metabolism, and migration. In the current studies, we have evaluated the isoform-specific role of akt in lysophosphatidic acid (LPA)-induced cell migration. Ascites from ovarian cancer patients (AOCP) induced mouse embryo fibroblast (MEF) cell migration in a dose-dependent manner. On the other hand, ascites from liver cirrhosis patients (ALCP) did not induce MEF cell migration. AOCP-induced MEF cell migration was completely blocked by pre-treatment of cells with LPA receptor antagonist, Ki16425. Both LPA- and AOCP-induced MEF cell migration was completely attenuated by PI3K inhibitor, LY294002. Furthermore, cells lacking Akt1 displayed defect in LPA-induced cell migration. Re-expression of Akt1 in DKO (Akt1-/-Akt2-/-) cells restored LPA-induced cell migration, whereas re-expression of Akt2 in DKO cells could not restore the LPA-induced cell migration. Finally, Akt1 was selectively phosphorylated by LPA and AOCP stimulation. These results suggest that LPA is a major factor responsible for AOCP-induced cell migration and signaling specificity of Akt1 may dictate LPA-induced cell migration.     

Screening for cell migration inhibitors via automated microscopy reveals a Rho-kinase inhibitor

Small-molecule kinase inhibitors are predominantly discovered in pure protein assays. We have discovered an inhibitor of Rho-kinase (ROCK) through an image-based, high-throughput screen of cell monolayer wound healing. Using automated microscopy, we screened a library of approximately 16,000 compounds finding many that affected cell migration or cell morphology as well as compounds that blocked mitotic progression. We tested approximately 200 compounds in a series of subassays and chose one, 3-(4-pyridyl)indole (Rockout), for more detailed characterization. Rockout inhibits blebbing and causes dissolution of actin stress fibers, phenocopying Rho-kinase inhibitors. Testing Rho-kinase activity in vitro, Rockout inhibits with an IC50 of 25 microM ( approximately 5-fold less potent than Y-27632) but has a similar specificity profile. We also profile the wound healing assay with a library of compounds with known bioactivities, revealing multiple pathways involved in the biology.     

Lysophosphatidic acid receptor 2 and Gi/Src pathway mediate cell motility through cyclooxygenase 2 expression in CAOV-3 ovarian cancer cells

Lysophosphatidic acid (LPA) is a bioactive phospholipids and involves in various cellular events, including tumor cell migration. In the present study, we investigated LPA receptor and its transactivation to EGFR for cyclooxygenase-2 (COX-2) expression and cell migration in CAOV-3 ovarian cancer cells. LPA induced COX-2 expression in a dose-dependent manner, and pretreatment of the cells with pharmacological inhibitors of Gi (pertussis toxin), Src (PP2), EGF receptor (EGFR) (AG1478), ERK (PD98059) significantly inhibited LPA-induced COX-2 expression. Consistent to these results, transfection of the cells with selective Src siRNA attenuated COX-2 expression by LPA. LPA stimulated CAOV-3 cell migration that was abrogated by pharmacological inhibitors and antibody of EP2. Higher expression of LPA2 mRNA was observed in CAOV-3 cells, and transfection of the cells with a selective LPA2 siRNA significantly inhibited LPA-induced activation of EGFR and ERK, as well as COX-2 expression. Importantly, LPA2 siRNA also blocked LPA-induced ovarian cancer cell migration. Collectively, our results clearly show the significance of LPA2 and Gi/Src pathway for LPA-induced COX-2 expression and cell migration that could be a promising drug target for ovarian cancer cell metastasis.     

Activation of platelet-derived growth factor pathway in human asthmatic pulmonary-derived mesenchymal cells

Cell cultures of mesenchymal type were obtained from biopsies taken after bronchoscopy from patients with asthma. It was possible to achieve outgrowth of fibroblast-like cells from these lung biopsies, which stained for alpha-smooth actin indicating that they were of myofibroblast type. Morphologically, two types of myofibroblasts could be observed: one intermediate form with more stretched cell shape and lamellipodia protrusions, and one more differentiated compact form of myofibroblast. The intermediate form was the most dominant type in these patients, indicating an active ongoing remodelling process. Further studies showed that platelet-derived growth factor (PDGF) might be the factor that stimulates the formation of the intermediate type of myofibroblasts, since it enhance migration of normal human lung fibroblasts 4-fold compared to control through an induced formation of stress fibers and lamellipodia protrusions. Additionally, intracellular signalling pathways involved in migration, such as RhoA and MAPkinase were stimulated 1.5-fold and 3.5-fold, respectively. By using two-dimensional (2-D) gel electrophoresis and protein identification by peptide mass finger printing matrix assisted laser desporption/ionization - time of flight - mass spectrometry (MALDI-TOF-MS) it was possible to confirm that PDGF affected the synthesis of proteins involved in the remodelling process, such as collagen VI and post-translational forms thereof. PDGF also stimulated the production of FK506 binding protein of 65 kDa, a protein involved in smooth muscle differentiation, and proteins involved in the rearrangement of the cytoskeleton connected to migration such as the actin related protein ARP3, the T-complex protein and the heat shock protein 60. We demonstrate that PDGF has a potential pathological role in asthma and formation of subepithelial fibrosis by inducing changes in the proteome.     

The role of the alpha4 integrin-paxillin interaction in regulating leukocyte trafficking

The movement of leukocytes from the blood into peripheral tissues is a central feature of immune surveillance, but also contributes to the pathogenesis of inflammatory and autoimmune diseases. Integrins are a family of adhesion and signaling molecules made up of paired a and beta subunits, and the integrin alpha4beta1 plays a prominent role in the trafficking of mononuclear leukocytes. We have previously described the direct interaction of the signaling adaptor molecule paxillin with the cytoplasmic domain of the alpha4 integrin subunit. This interaction is critical for alpha4beta1 integrin dependent cell adhesion under shear flow conditions as it provides a needed connection to the actin cytoskeleton. Furthermore, the alpha4-paxillin interaction is required for effective alpha4beta1 dependent leukocyte migration and does so through the temporal and spatial regulation of the small GTPase Rac. These findings make the alpha4-paxillin interaction a potentially attractive therapeutic target in controlling leukocyte trafficking.     

A chemical inhibitor reveals the role of Raf kinase inhibitor protein in cell migration

Raf kinase inhibitor protein (RKIP) is a modulator of cell signaling that functions as an endogenous inhibitor of multiple kinases. We demonstrate here a positive role for RKIP in the regulation of cell locomotion. We discovered that RKIP is the relevant cellular target of locostatin, a cell migration inhibitor. Locostatin abrogates RKIP's ability to bind and inhibit Raf-1 kinase, and it acts by disrupting a protein-protein interaction, an uncommon mode of action for a small molecule. Small interfering RNA-mediated silencing of RKIP expression also reduces cell migration rate. Overexpression of RKIP converts epithelial cells to a highly migratory fibroblast-like phenotype, with dramatic reduction in the sensitivity of cells to locostatin. RKIP is therefore the compound's valid target and a key regulator of cell motility.     

Polymeric Delivery of siRNA against Integrin‐β1 (CD29) to Reduce Attachment and Migration of Breast Cancer Cells

Cell surface integrins, which play important roles in the survival, proliferation, migration, and invasion of cancer cells, are a viable target for treatment of metastatic breast cancer. This line of therapy still remains challenging due to the lack of proper identification and validation of effective targets as well as the lack of suitable therapeutic agents for treatment. The focus is on one such molecular target for this purpose, namely integrin‐β1, and effective lowering of integrin‐β1 levels on a breast cancer model (MDA‐MB‐231 cells) is achieved by delivering a dicer‐substrate short interfering RNA (siRNA) targeting integrin‐β1 with lipid‐modified low molecular weight polyethylenimine polymers. Reduction of integrin‐β1 levels leads to reduced adhesion of MDA‐MB‐231 cells to extracellular matrix component fibronectin as well as to human bone marrow cells. A reduced migration of the breast cancer cells is also observed after integrin‐β1 silencing in “scratch” and “transwell” migration assays. These results highlight the importance of integrin‐β1 for the migration of metastatic breast cancer cells by effectively silencing this target with a practical dose of siRNA.