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1.  Polycefin, a New Prototype of a Multifunctional Nanoconjugate Based on Poly(β-l-malic acid) for Drug Delivery 
Bioconjugate chemistry  2006;17(2):317-326.
A new prototype of nanoconjugate, Polycefin, was synthesized for targeted delivery of antisense oligonucleotides and monoclonal antibodies to brain tumors. The macromolecular carrier contains: 1. biodegradable, nonimmunogenic, nontoxic β-poly(l-malic acid) of microbial origin; 2. Morpholino antisense oligonucleotides targeting laminin α4 and β1 chains of laminin-8, which is specifically overexpressed in glial brain tumors; 3. monoclonal anti-transferrin receptor antibody for specific tissue targeting; 4. oligonucleotide releasing disulfide units; 5. l-valine containing, pH-sensitive membrane disrupting unit(s), 6. protective poly(ethylene glycol); 7. a fluorescent dye (optional). Highly purified modules were conjugated directly with N-hydroxysuccinimidyl ester-activated β-poly-(l-malic acid) at pendant carboxyl groups or at thiol containing spacers via thioether and disulfide bonds. Products were chemically validated by physical, chemical, and functional tests. In vitro experiments using two human glioma cell lines U87MG and T98G demonstrated that Polycefin was delivered into the tumor cells by a receptor-mediated endocytosis mechanism and was able to inhibit the synthesis of laminin-8 α4 and β1 chains at the same time. Inhibition of laminin-8 expression was in agreement with the designed endosomal membrane disruption and drug releasing activity. In vivo imaging showed the accumulation of intravenously injected Polycefin in brain tumor tissue via the antibody-targeted transferrin receptor-mediated endosomal pathway in addition to a less efficient mechanism known for high molecular mass biopolymers as enhanced permeability and retention effect. Polycefin was nontoxic to normal and tumor astrocytes in a wide range of concentrations, accumulated in brain tumor, and could be used for specific targeting of several biomarkers simultaneously.
PMCID: PMC3487710  PMID: 16536461
2.  Nanoconjugate based on polymalic acid for tumor targeting 
Chemico-biological interactions  2007;171(2):195-203.
A new prototype of polymer-derived drug delivery system, the nanoconjugate Polycefin, was tested for its ability to accumulate in tumors based on enhanced permeability and retention (EPR) effect and receptor mediated endocytosis. Polycefin was synthesized for targeted delivery of Morpholino antisense oligonucleotides into certain tumors. It consists of units that are covalently conjugated with poly(β-L-malic acid) (Mw 50,000, Mw/Mn 1.3) highly purified from cultures of myxomycete Physarum polycephalum. The units are active in endosomal uptake, disruption of endosomal membranes, oligonucleotide release in the cytoplasm, and protection against enzymatic degradation in the vascular system. The polymer is biodegradable, non-immunogenic and non-toxic. Polycefin was also coupled with AlexaFluor 680 C2-maleimide dye for in vivo detection.
Nude mice received subcutaneous injections of MDA-MB 468 human breast cancer cells into the left posterior mid-dorsum or intracranial injections of human glioma cell line U87MG. Polycefin at concentration of 2.5 mg/kg was injected via the tail vein. In vivo fluorescence tumor imaging was performed at different time points, 0–180 min up to 24 h after the drug injection. The custom-made macro-illumination imaging MISTI system was used to examine the in vivo drug accumulation in animals bearing human breast and brain tumors. In breast tumors the fluorescence signal in large blood vessels and in the tumor increased rapidly until 60 min and remained in the tumor at a level 6 times higher than in non-tumor tissue (180 min) (p < 0.003). In brain tumors drug accumulated selectively in 24 h without any detectable signal in non-tumor areas. The results of live imaging were corroborated histologically by fluorescence microscopic examination of various organs. In addition to tumors, only kidney and liver showed some fluorescent signal. © 2007 Elsevier Ireland Ltd. All rights reserved.
PMCID: PMC2329596  PMID: 17376417
Brain glioma; Breast cancer; EPR effect; Fluorescence imaging; Drug delivery system; Poly(malic acid)
3.  Poly(malic acid) nanoconjugates containing various antibodies and oligonucleotides for multitargeting drug delivery 
Nanomedicine (London, England)  2008;3(2):247-265.
Nanoconjugates are emerging as promising drug-delivery vehicles because of their multimodular structure enabling them to actively target discrete cells, pass through biological barriers and simultaneously carry multiple drugs of various chemical nature. Nanoconjugates have matured from simple devices to multifunctional, biodegradable, nontoxic and nonimmunogenic constructs, capable of delivering synergistically functioning drugs in vivo. This review mainly concerns the Polycefin family of natural-derived polymeric drug-delivery devices as an example. This type of vehicle is built by hierarchic conjugation of functional groups onto the backbone of poly(malic acid), an aliphatic polyester obtained from the microorganism Physarum polycephalum. Particular Polycefin variants target human brain and breast tumors implanted into animals specifically and actively and could be detected easily by noninvasive imaging analysis. Delivery of antisense oligonucleotides to a tumor-specific angiogenic marker using Polycefin resulted in significant inhibition of tumor angiogenesis and increase of animal survival.
PMCID: PMC2734385  PMID: 18373429
biodegradable; brain cancer; breast cancer; imaging analysis; multiple antibodies; multiple drug delivery; multitargeting; Polycefin; poly(malic acid); tumor angiogenesis
4.  Brain tumor tandem targeting using a combination of monoclonal antibodies attached to biopoly(β-L-malic acid) 
Tumor-specific targeting using achievements of nanotechnology is a mainstay of increasing efficacy of anti-tumor drugs. To improve drug targeting we covalently conjugated for the first time two different monoclonal antibodies, an anti-mouse transferrin receptor antibody and a mouse autoimmune anti-nucleosome antibody 2C5, onto the drug delivery nanoplatform, poly(β-L-malic acid). The active anti-tumor drug components attached to the same carrier molecule were antisense oligonucleotides to vascular protein laminin-8. The resulting drug, a new Polycefin variant, was administered intravenously into glioma-bearing xenogeneic animals. The drug delivery system was targeted across mouse endothelial system by the anti-mouse transferring receptor antibody and to the tumor cell surface by the anti-nucleosome antibody 2C5. The targeting efficacies of the Polycefin variants bearing either two antibodies or each single antibody were compared in vitro and in vivo. ELISA confirmed the co-existence of two antibodies on the same nanoplatform molecule and their functional activities. Fluorescence imaging analysis after 24 h of intravenous injection demonstrated significantly higher tumor accumulation of Polycefin variants with the tandem configuration of antibodies than with single antibodies. The results suggest improved efficacy for tandem configuration of antibodies than for single configurations carried by a drug delivery vehicle.
PMCID: PMC2394675  PMID: 17630012
Enhanced tumor targeting; Antibody tandem configuration; Nanobiopolymer; Brain tumor; Poly(β-L-malic acid)
5.  Polymalic acid nanobioconjugate for simultaneous inhibition of tumor growth and immunostimulation in HER2/neu-positive breast cancer✩ 
Breast cancer remains the second leading cause of cancer death among women in the United States. The breast cancer prognosis is particularly poor in case of tumors overexpressing the oncoprotein HER2/neu. A new nanobioconjugate of the Polycefin family of anti-cancer drugs based on biodegradable and non-toxic polymalic acid (PMLA) was engineered for a multi-pronged attack on HER2/neu-positive breast cancer cells. An antibody cytokine fusion protein consisting of the immunostimulatory cytokine interleukin-2 (IL-2) genetically fused to an antibody specific for human HER2/neu [anti-HER2/neu IgG3-(IL-2)] was covalently attached to the PMLA backbone to target HER2/neu expressing tumors and ensuring the delivery of IL-2 to the tumor microenvironment. Antisense oligonucleotides (AON) were conjugated to the nanodrug to inhibit the expression of vascular tumor protein laminin-411 in order to block tumor angiogenesis. It is shown that the nanobioconjugate was capable of specifically binding human HER2/neu and retaining the biological activity of IL-2. We also showed the uptake of the nanobioconjugate by HER2/neu-positive breast cancer cells and enhanced tumor targeting in vivo. In addition, the nanobioconjugate was capable of eliciting anti-tumor activity in immunocompetent mice bearing D2F2/E2 murine mammary tumors that express human HER2/neu. Both IgG1 and IgG2a levels were significantly increased in animals treated with the PMLA-fusion nanobioconjugate compared to animals treated with the antibody–cytokine fusion protein alone or control animals, indicative of the induction of a humoral (TH2) and cell-mediated (TH1) immune responses. Animal survival in vivo was significantly longer after treatment with leading nanobioconjugate with fusion [anti-HER2/neu IgG3-(IL-2)] antibody, p < 0.05. The combination of these molecules on a single polymeric platform is expected to act through direct elimination of cancer cells, inhibition of tumor angiogenesis, and orchestration of a potent immune response against tumor.
PMCID: PMC3971991  PMID: 23770212
Polymalic acid; Nanobioconjugate; Nanopolymer; HER2/neu; Antibody fusion protein; IL-2; Laminin-411; Breast cancer
6.  Overexpression of β1-chain-containing laminins in capillary basement membranes of human breast cancer and its metastases 
Breast Cancer Research  2005;7(4):R411-R421.
Laminins are the major components of vascular and parenchymal basement membranes. We previously documented a switch in the expression of vascular laminins containing the α4 chain from predominantly laminin-9 (α4β2γ1) to predominantly laminin-8 (α4β1γ1) during progression of human brain gliomas to high-grade glioblastoma multiforme. Here, differential expression of laminins was studied in blood vessels and ductal epithelium of the breast.
In the present study the expressions of laminin isoforms α1–α5, β1–β3, γ1, and γ2 were examined during progression of breast cancer. Forty-five clinical samples of breast tissues including normal breast, ductal carcinomas in situ, invasive ductal carcinomas, and their metastases to the brain were compared using Western blot analysis and immunohistochemistry for various chains of laminin, in particular laminin-8 and laminin-9.
Laminin α4 chain was observed in vascular basement membranes of most studied tissues, with the highest expression in metastases. At the same time, the expression of laminin β2 chain (a constituent of laminin-9) was mostly seen in normal breast and carcinomas in situ but not in invasive carcinomas or metastases. In contrast, laminin β1 chain (a constituent of laminin-8) was typically found in vessel walls of carcinomas and their metastases but not in those of normal breast. The expression of laminin-8 increased in a progression-dependent manner. A similar change was observed from laminin-11 (α5β2γ1) to laminin-10 (α5β1γ1) during breast tumor progression. Additionally, laminin-2 (α2β1γ1) appeared in vascular basement membranes of invasive carcinomas and metastases. Chains of laminin-5 (α3β3γ2) were expressed in the ductal epithelium basement membranes of the breast and diminished with tumor progression.
These results suggest that laminin-2, laminin-8, and laminin-10 are important components of tumor microvessels and may associate with breast tumor progression. Angiogenic switch from laminin-9 and laminin-11 to laminin-8 and laminin-10 first occurs in carcinomas in situ and becomes more pronounced with progression of carcinomas to the invasive stage. Similar to high-grade brain gliomas, the expression of laminin-8 (and laminin-10) in breast cancer tissue may be a predictive factor for tumor neovascularization and invasion.
PMCID: PMC1175051  PMID: 15987446
7.  Toxicity and efficacy evaluation of multiple targeted polymalic acid conjugates for triple-negative breast cancer treatment 
Journal of drug targeting  2013;21(10):956-967.
Engineered nanoparticles are widely used for delivery of drugs but frequently lack proof of safety for cancer patient's treatment. All-in-one covalent nanodrugs of the third generation have been synthesized based on a poly(β-L-malic acid) (PMLA) platform, targeting human triple-negative breast cancer (TNBC). They significantly inhibited tumor growth in nude mice by blocking synthesis of epidermal growth factor receptor, and α4 and β1 chains of laminin-411, the tumor vascular wall protein and angiogenesis marker. PMLA and nanodrug biocompatibility and toxicity at low and high dosages were evaluated in vitro and in vivo. The dual-action nanodrug and single-action precursor nanoconjugates were assessed under in vitro conditions and in vivo with multiple treatment regimens (6 and 12 treatments). The monitoring of TNBC treatment in vivo with different drugs included blood hematologic and immunologic analysis after multiple intravenous administrations. The present study demonstrates that the dual-action nanoconju-gate is highly effective in preclinical TNBC treatment without side effects, supported by hematologic and immunologic assays data. PMLA-based nanodrugs of the Polycefin™ family passed multiple toxicity and efficacy tests in vitro and in vivo on preclinical level and may prove to be optimized and efficacious for the treatment of cancer patients in the future.
PMCID: PMC4043297  PMID: 24032759
Hematologic; immunogenicity; in vivo treatment; nanoconjugate drugs; polymalic acid; toxicity; triple-negative breast cancer
8.  Laminin alpha 2 enables glioblastoma stem cell growth 
Annals of neurology  2012;72(5):766-778.
Glioblastomas (GBMs) are lethal cancers that display cellular hierarchies parallel to normal brain. At the apex are GBM stem cells (GSCs), which are relatively resistant to conventional therapy. An important driver of malignancy and self-renewal in GSCs are interactions with the adjacent perivascular niche. Extracellular matrix (ECM) cues instruct neural stem/progenitor cell-niche interactions and the objective of our study was to elucidate its composition and contribution to GSC maintenance in the perivascular niche.
We interrogated human tumor tissue for immunofluorescence analysis and derived GSC from tumor tissues for functional studies. Bioinformatics analyses were conducted by mining publicly available databases.
We find that laminin ECM proteins are localized to the perivascular GBM niche and inform negative patient prognosis. To identify the source of laminins, we characterized cellular elements within the niche and found that laminin α chains were expressed by non-stem tumor cells and tumor associated endothelial cells (ECs). RNA interference targeting laminin α2 inhibited GSC growth and self-renewal. In co-culture studies of GSCs and ECs, laminin α2 knockdown in ECs resulted in decreased tumor growth.
Our studies highlight the contribution of non-stem tumor cell-derived laminin juxtracrine signaling. As laminin α2 has recently been identified as a molecular marker of aggressive ependymoma, we propose that the brain vascular ECM promotes tumor malignancy through maintenance of the GSC compartment providing not only a molecular fingerprint but also a possible therapeutic target.
PMCID: PMC3615417  PMID: 23280793
9.  Key Role of the Cdx2 Homeobox Gene in Extracellular Matrix–mediated Intestinal Cell Differentiation  
The Journal of Cell Biology  1997;139(6):1553-1565.
To explore the role of homeobox genes in the intestine, the human colon adenocarcinoma cell line Caco2-TC7 has been stably transfected with plasmids synthesizing Cdx1 and Cdx2 sense and antisense RNAs. Cdx1 overexpression or inhibition by antisense RNA does not markedly modify the cell differentiation markers analyzed in this study. In contrast, Cdx2 overexpression stimulates two typical markers of enterocytic differentiation: sucrase-isomaltase and lactase. Cells in which the endogenous expression of Cdx2 is reduced by antisense RNA attach poorly to the substratum. Conversely, Cdx2 overexpression modifies the expression of molecules involved in cell–cell and cell–substratum interactions and in transduction process: indeed, E-cadherin, integrin-β4 subunit, laminin-γ2 chain, hemidesmosomal protein, APC, and α-actinin are upregulated. Interestingly, most of these molecules are preferentially expressed in vivo in the differentiated villi enterocytes rather than in crypt cells. Cdx2 overexpression also results in the stimulation of HoxA-9 mRNA expression, an homeobox gene selectively expressed in the colon. In contrast, Cdx2-overexpressing cells display a decline of Cdx1 mRNA, which is mostly found in vivo in crypt cells. When implanted in nude mice, Cdx2-overexpressing cells produce larger tumors than control cells, and form glandular and villus-like structures.
Laminin-1 is known to stimulate intestinal cell differentiation in vitro. In the present study, we demonstrate that the differentiating effect of laminin-1 coatings on Caco2-TC7 cells is accompanied by an upregulation of Cdx2. To further document this observation, we analyzed a series of Caco2 clones in which the production of laminin-α1 chain is differentially inhibited by antisense RNA. We found a positive correlation between the level of Cdx2 expression, that of endogenous laminin-α1 chain mRNA and that of sucrase-isomaltase expression in these cell lines.
Taken together, these results suggest (a) that Cdx1 and Cdx2 homeobox genes play distinct roles in the intestinal epithelium, (b) that Cdx2 provokes pleiotropic effects triggering cells towards the phenotype of differentiated villus enterocytes, and (c) that Cdx2 expression is modulated by basement membrane components. Hence, we conclude that Cdx2 plays a key role in the extracellular matrix–mediated intestinal cell differentiation.
PMCID: PMC2132620  PMID: 9396760
10.  Endothelial Expression of TNF Receptor-1 Generates a Pro-apoptotic Signal Inhibited by Integrin α6β1 in Glioblastoma 
Cancer research  2012;72(6):1428-1437.
Activation of TNF-receptor 1 (TNF-R1) can generate signals that promote either apoptosis or survival. In this study, we show that these signals can be determined by the character of the extracellular matrix in the tumor microenvironment. Specifically, through studies of glioblastoma we showed that TNFα stimulation induced apoptosis of primary brain endothelial cells (ECs) attached to collagen or fibronectin (which engage integrins α2β1/α3β1 and α5β1, respectively), but did not induce apoptosis of ECs attached to laminin (which engages integrins α6β1 and α3β1). TNF-R1 expression was significantly higher in ECs in glioblastoma tumors (GBM) compared to ECs in normal brain specimens. TNFα was also expressed in GBM tumor-associated ECs, which was associated with longer patient survival. ECs plated on anti-integrin α2 or α3 antibody were susceptible to TNFα induced apoptosis, whereas those plated on anti-integrin α6 antibody were not. Moreover, the ECs plated on laminin, but not collagen, expressed cFLIP and TNFα stimulation of laminin-attached cells in which cFLIP had been downregulated resulted in the induction of apoptosis. In contrast, attachment to laminin did not induce cFLIP expression in GBM tumor stem cells. Together, our findings indicate that the laminin receptor integrin α6β1 promotes the survival of brain ECs by inhibiting pro-death signaling by TNF-R1, in part by inducing cFLIP expression.
PMCID: PMC3428239  PMID: 22396498
Brain endothelial cells; TNFα; TNF-R1; integrins; glioblastoma; cFLIP
Laminins are the major constituents of blood vessel basement membranes (BMs). Each laminin is a trimer consisting of three assembled polypeptide chains, α, β and γ. More than 15 laminin isoforms are known to date and the expression of specific isoforms may change in certain pathological conditions. Here we show that during progression of glial tumors laminin-9 (α4β2γ1) is switched to laminin-8 (α4β1γ1), which is dramatically increased in glial brain tumors. Laminin-8 overproduction by glial tumor cells facilitates spread of glioma. Brain tumors with laminin-8 overexpression recur faster after standard treatment and patients have shorter survival time. Laminin-8 may be thus used as a predictor of tumor recurrence, patient survival and as a potential molecular target for glioma therapy.
PMCID: PMC3506377  PMID: 16146715
Laminin-8; Laminin-9; Basement Membrane; Extracellular Matrix; Angiogenesis; Human; Cancer; Tumor; Neoplasm; Glioma; Glioblastoma Multiforme; Recurrence; Survival; Invasion; Morpholino antisense; Review
12.  Laminin-411 Is a Vascular Ligand for MCAM and Facilitates TH17 Cell Entry into the CNS 
PLoS ONE  2012;7(7):e40443.
TH17 cells enter tissues to facilitate pathogenic autoimmune responses, including multiple sclerosis (MS). However, the adhesion molecules involved in the unique migratory capacity of TH17 cells, into both inflamed and uninflamed tissues remain unclear. Herein, we characterize MCAM (CD146) as an adhesion molecule that defines human TH17 cells in the circulation; following in vitro restimulation of human memory T cells, nearly all of the capacity to secrete IL-17 is contained within the population of cells expressing MCAM. Furthermore, we identify the MCAM ligand as laminin 411, an isoform of laminin expressed within the vascular endothelial basement membranes under inflammatory as well as homeotstatic conditions. Purified MCAM-Fc binds to laminin 411 with an affinity of 27 nM, and recognizes vascular basement membranes in mouse and human tissue. MCAM-Fc binding was undetectable in tissue from mice with targeted deletion of laminin 411, indicating that laminin 411 is a major tissue ligand for MCAM. An anti-MCAM monoclonal antibody, selected for inhibition of laminin binding, as well as soluble MCAM-Fc, inhibited T cell adhesion to laminin 411 in vitro. When administered in vivo, the antibody reduced TH17 cell infiltration into the CNS and ameliorated disease in an animal model of MS. Our data suggest that MCAM and laminin 411 interact to facilitate TH17 cell entry into tissues and promote inflammation.
PMCID: PMC3391262  PMID: 22792325
13.  Reduced Glycosylation of α-Dystroglycans on Carcinoma Cells Contributes to Formation of Highly Infiltrative Histological Patterns in Prostate Cancer 
The Prostate  2011;71(11):1151-1157.
α-Dystroglycan (DG) carries glycan chains that bind to laminin and thus function in homeostasis of not only skeletal muscle but also of various epithelial cells. Loss of glycosylation has been suggested to play important roles in tumor development, particularly in detachment and migration of carcinoma cells. We previously reported that glycosylation of α-DG, but not levels of α-DG core protein itself, is reduced in prostate carcinoma. In this study, we investigate the association between reduction of laminin-binding glycans on α-DG and the degree of tumor cell differentiation and/or infiltrative properties, as assessed by the Gleason grading system.
Immunohistochemical analysis of 146 biopsy specimens of prostate adenocarcinoma with various Gleason scores was carried out employing IIH6 and 6C1 antibodies, which recognize laminin-binding glycans on α-DG and α-DG core proteins, respectively. Double immunofluorescence staining was performed to evaluate colocalization of α-DG and laminin, and to determine which types of epithelial cells express laminin-binding glycans on α-DG.
Reduction of α-DG glycosylation, rather than loss of α-DG core protein, was correlated with higher Gleason patterns. Reduction was most conspicuous at the interface between carcinoma cells and the basement membrane. In addition, in non-neoplastic prostate glands, laminin-binding glycans were expressed predominantly on the basolateral surface of basal cells.
Reduced expression of laminin-binding glycans on α-DG may contribute to formation of highly infiltrative behavior of prostate carcinoma cells. Substantial reduction of laminin-binding glycans in carcinoma tissue could be partly ascribed to disappearance of pre-existing basal cells.
PMCID: PMC3174275  PMID: 21656825
prostatic neoplasms; dystroglycans; glycosylation
14.  Over-expression of BMPR-IB reduces the malignancy of glioblastoma cells by upregulation of p21 and p27Kip1 
In our previous study, we detected decreased expression of phospho-Smad1/5/8 and its upstream signaling molecule, bone morphogenetic protein receptor IB subunit (BMPR-IB), in certain glioblastoma tissues, unlike normal brain tissues. In order to clarify the functional roles and mechanism of BMPR-IB in the development of glioblastoma, we studied the effects of BMPR-IB overexpression on glioblastoma cell lines in vitro and in vivo.
We selected glioblastoma cell lines U251, U87, SF763, which have different expression of BMPR-IB to be the research subjects. Colony formation analysis and FACS were used to detect the effects of BMPR-IB on the growth and proliferation of glioblastoma cells in vivo. Immunofluresence was used to detect the differentiation changes after BMPR-IB overexpression or knocking-down. Then we used subcutaneous and intracranial tumor models to study the effect of BMPR-IB on the growth and differentiation of glioblastoma cells in vivo. The genetic alterations involved in this process were examined by real-time PCR and western blot analysis.ed.
Results and conclusion
Forced BMPR-IB expression in malignant human glioma cells, which exhibit lower expression of BMPR-IB, induced the phosphorylation and nuclear localization of smad1/5/8 and arrested the cell cycle in G1. Additionally, BMPR-IB overexpression could suppress anchorage-independent growth and promote differentiation of theses glioblastoma cells. Furthermore, overexpression of BMPR-IB inhibited the growth of subcutaneous and intracranial tumor xenografts and prolonged the survival of mice injected intracranially with BMPR-IB-overexpressing glioblastoma cells. Conversely, inhibition of BMPR-IB caused SF763 malignant glioma cells, a line known to exhibit high BMPR-IB expression that does not form tumors when used for xenografts, to show increased growth and regain tumorigenicity in a nude mouse model system, ultimately shortening the survival of these mice. We also observed significant accumulation of p21 and p27kip1 proteins in response to BMPR-IB overexpression. Our study suggests that overexpression of BMPR-IB may arrest and induce the differentiation of glioblastoma cells due to upregulation of p21 and p27kip1 in vitro and that in vivo and decreased expression of BMPR-IB in human glioblastoma cells contributes to glioma tumorigenicity. BMPR-IB could represent a new potential therapeutic target for malignant human gliomas.
PMCID: PMC3408360  PMID: 22650359
1. BMPR-IB; 2. Glioblastoma; 3. Growth inhibition; 4. Differentiation
15.  Laminin Isoforms and Laminin-Producing Cells in Rat Anterior Pituitary 
Acta Histochemica et Cytochemica  2012;45(5):309-315.
Laminin is a key component of the basement membrane and is involved in the structural scaffold and in cell proliferation and differentiation. Research has identified 19 laminin isoforms, which are assemblies of α, β, and γ chains (eg, the α1, β1, and γ1 chains form the laminin 111 isoform). Although laminin is known to be present in the anterior pituitary, the specific laminin isoforms have not been identified. This study used molecular biological and histochemical techniques—namely, RT-PCR, immunohistochemistry, and in situ hybridization—to identify the laminin isoforms and laminin-producing cells in rat anterior pituitary. RT-PCR showed that laminin α1, α3, and α4 genes were expressed in anterior pituitary. Immunohistochemistry revealed laminin α1 in gonadotrophs and laminin α4 in almost all vascular endothelial cells. Laminin α3 was seen in a subset of vascular endothelial cells. We then performed in situ hybridization to localize β and γ chains in these cells and found that laminin β1, β2, and γ1 were expressed in gonadotrophs and that laminin β1 and γ1 were expressed in endothelial cells. In conclusion, we identified gonadotroph-type (laminin 111 and 121) and vascular-type (laminin 411 and 311) laminin isoforms in rat anterior pituitary.
PMCID: PMC3499700  PMID: 23209340
anterior pituitary; laminin; basement membrane; gonadotroph; endothelial cell
16.  Normal and tumor-derived myoepithelial cells differ in their ability to interact with luminal breast epithelial cells for polarity and basement membrane deposition 
Journal of cell science  2002;115(Pt 1):39-50.
The signals that determine the correct polarity of breast epithelial structures in vivo are not understood. We have shown previously that luminal epithelial cells can be polarized when cultured within a reconstituted basement membrane gel. We reasoned that such cues in vivo may be given by myoepithelial cells. Accordingly, we used an assay where luminal epithelial cells are incorrectly polarized to test this hypothesis. We show that culturing human primary luminal epithelial cells within collagen-I gels leads to formation of structures with no lumina and with reverse polarity as judged by dual stainings for sialomucin, epithelial specific antigen or occludin. No basement membrane is deposited, and β4-integrin staining is negative. Addition of purified human myoepithelial cells isolated from normal glands corrects the inverse polarity, and leads to formation of double-layered acini with central lumina. Among the laminins present in the human breast basement membrane (laminin-1, -5 and -10/11), laminin-1 was unique in its ability to substitute for myoepithelial cells in polarity reversal.
Myoepithelial cells were purified also from four different breast cancer sources including a biphasic cell line. Three out of four samples either totally lacked the ability to interact with luminal epithelial cells, or conveyed only correction of polarity in a fraction of acini. This behavior was directly related to the ability of the tumor myoepithelial cells to produce α-1 chain of laminin. In vivo, breast carcinomas were either negative for laminin-1 (7/12 biopsies) or showed a focal, fragmented deposition of a less intensely stained basement membrane (5/12 biopsies). Dual staining with myoepithelial markers revealed that tumor-associated myoepithelial cells were either negative or weakly positive for expression of laminin-1, establishing a strong correlation between loss of laminin-1 and breast cancer. We conclude that the double-layered breast acinus may be recapitulated in culture and that one reason for the ability of myoepithelial cells to induce polarity is because they are the only source of laminin-1 in the breast in vivo. A further conclusion is that a majority of tumor-derived/-associated myoepithelial cells are deficient in their ability to impart polarity because they have lost their ability to synthesize sufficient or functional laminin-1. These results have important implications for the role of myoepithelial cells in maintenance of polarity in normal breast and how they may function as structural tumor suppressors.
PMCID: PMC2933194  PMID: 11801722
Breast cancer; Morphogenesis; Laminin; Reversion; Polarity; Myoepithelial function
17.  Uncoupled responses of Smad4-deficient cancer cells to TNFα result in secretion of monomeric laminin-γ2 
Molecular Cancer  2010;9:65.
Functional loss of the tumor suppressor Smad4 is involved in pancreatic and colorectal carcinogenesis and has been associated with the acquisition of invasiveness. We have previously demonstrated that the heterotrimeric basement membrane protein laminin-332 is a Smad4 target. Namely, Smad4 functions as a positive transcriptional regulator of all three genes encoding laminin-332; its loss is thus implicated in the reduced or discontinuous deposition of the heterotrimeric basement membrane molecule as evident in carcinomas. Uncoupled expression of laminin genes, on the other hand, namely overexpression of the laminin-γ2 chain is an impressive marker at invasive edges of carcinomas where tumor cells are maximally exposed to signals from stromal cell types like macrophages. As Smad4 is characterized as an integrator of multiple extracellular stimuli in a strongly contextual manner, we asked if loss of Smad4 may also be involved in uncoupled expression of laminin genes in response to altered environmental stimuli. Here, we address Smad4 dependent effects of the prominent inflammatory cytokine TNFα on tumor cells.
Smad4-reconstituted colon carcinoma cells like adenoma cells respond to TNFα with an increased expression of all three chains encoding laminin-332; coincubation with TGFβ and TNFα leads to synergistic induction and to the secretion of large amounts of the heterotrimer. In contrast, in Smad4-deficient cells TNFα can induce expression of the γ2 and β3 but not the α3 chain. Surprisingly, this uncoupled induction of laminin-332 chains in Smad4-negative cells rather than causing intracellular accumulation is followed by the release of γ2 into the medium, either in a monomeric form or in complexes with as yet unknown proteins. Soluble γ2 is associated with increased cell migration.
Loss of Smad4 may lead to uncoupled induction of laminin-γ2 in response to TNFα and may therefore represent one of the mechanisms which underlie accumulation of laminin-γ2 at the invasive margin of a tumor. The finding, that γ2 is secreted from tumor cells in significant amounts and is associated with increased cell migration may pave the way for further investigation to better understand its functional relevance for tumor progression.
PMCID: PMC2853515  PMID: 20307265
18.  Expressions of the γ2 chain of laminin-5 and secreted protein acidic and rich in cysteine in esophageal squamous cell carcinoma and their relation to prognosis 
Chinese Journal of Cancer  2011;30(1):69-78.
Previous studies have shown that the expressions of the γ2 chain of laminin-5 and secreted protein acidic and rich in cysteine (SPARC) play important roles in oncogenesis and the development of carcinoma. To assess the expressions of laminin-5 γ2 chain and SPARC in esophageal squamous cell carcinoma (SCC), and to clarify the prognostic significance of the expressions of laminin-5 γ2 chain and SPARC in esophageal SCC, we detected the expressions of laminin-5 γ2 chain and SPARC in cancer tissue and corresponding normal mucosa from 116 patients with advanced (stages II–IV) esophageal SCC using the tissue microarray-based immunohistochemistry and analyzed the correlation of the expressions with clinicopathologic characteristics and survival. We found that in normal esophageal tissues, laminin-5 γ2 chain was expressed in the basement membrane, whereas in esophageal SCC tissues, laminin-5 γ2 chain was expressed in the cytoplasm of carcinoma cells, with a positive rate of 72.4%. SPARC was not detected in normal esophageal mucosa, but was expressed in stromal fibroblasts in 84.6% of esophageal SCC cases and in cancer cells in 7.8% of esophageal SCC cases. There was a significant correlation between laminin-5 γ2 chain and stromal SPARC expression in esophageal SCC (Spearman' s rho = 0.423, P < 0.001). The expressions of both laminin-5 γ2 chain and stromal SPARC were correlated with survival (P = 0.032 and P = 0.034, respectively). In stage-II esophageal SCC, the expression of laminin-5 γ2 chain was significantly correlated with survival (P = 0.023), while the expression of SPARC was not significantly correlated with survival (P = 0.154). Patients with elevated levels of laminin-5 γ2 chain and SPARC expressions had a poorer prognosis than did those lacking elevated levels of laminin-5 γ2 chain expression and/or elevated levels of SPARC expression (P = 0.001). In stage-II esophageal SCC, patients with elevated levels of laminin-5 γ2 chain and SPARC expressions had a poorer prognosis (P < 0.001). These results suggest that laminin-5 γ2 chain and SPARC may play roles in the progression of esophageal SCC and their simultaneous expression is correlated with poorer prognosis, especially in patients with stage-II SCC.
PMCID: PMC4012265  PMID: 21192846
Laminin-5 γ2; SPARC; esophageal cancer; squamous cell carcinoma; pathology
19.  Keratinocyte-Targeted Expression of Human Laminin γ2 Rescues Skin Blistering and Early Lethality of Laminin γ2 Deficient Mice 
PLoS ONE  2012;7(9):e45546.
Laminin-332 is a heterotrimeric basement membrane component comprised of the α3, ß3, and γ2 laminin chains. Laminin-332 modulates epithelial cell processes, such as adhesion, migration, and differentiation and is prominent in many embryonic and adult tissues. In skin, laminin-332 is secreted by keratinocytes and is a key component of hemidesmosomes connecting the keratinocytes to the underlying dermis. In mice, lack of expression of any of the three Laminin-332 chains result in impaired anchorage and detachment of the epidermis, similar to that seen in human junctional epidermolysis bullosa, and death occurs within a few days after birth. To bypass the early lethality of laminin-332 deficiency caused by the knockout of the mouse laminin γ2 chain, we expressed a dox-controllable human laminin γ2 transgene under a keratinocyte-specific promoter on the laminin γ2 (Lamc2) knockout background. These mice appear similar to their wild-type littermates, do not develop skin blisters, are fertile, and survive >1.5 years. Immunofluorescence analyses of the skin showed that human laminin γ2 colocalized with mouse laminin α3 and ß3 in the basement membrane zone underlying the epidermis. Furthermore, the presence of “humanized” laminin-332 in the epidermal basement membrane zone rescued the alterations in the deposition of hemidesmosomal components, such as plectin, collagen type XVII/BP180, and integrin α6 and ß4 chains, seen in conventional Lamc2 knockout mice, leading to restored formation of hemidesmosomes. These mice will be a valuable tool for studies of organs deficient in laminin-332 and the role of laminin-332 in skin, including wound healing.
PMCID: PMC3445496  PMID: 23029085
20.  Migration by haptotaxis of a Schwann cell tumor line to the basement membrane glycoprotein laminin 
The Journal of Cell Biology  1983;97(3):772-777.
Laminin is a large (greater than 850-kdalton) glycoprotein that is localized within basement membranes. Recent work has indicated that this protein is present within the endoneurium of mouse sciatic nerve. Furthermore, it has been shown that a rat Schwannoma cell line, RN22F, produced laminin and that laminin promoted the attachment of these cells to bacterial plastic. This report presents evidence that RN22F cells migrate in vitro to laminin in a concentration-dependent fashion. Laminin was extracted from the mouse EHS tumor and purified by molecular sieve and heparin-agarose affinity chromatography. The migration of Schwannoma cells to laminin, as assessed in a microwell modified Boyden chamber, was inhibited in a dose-dependent manner by affinity-purified antilaminin antibody. Zigmond-Hirsch checkerboard analysis experiments indicated that laminin stimulated both random and directed movement of RN22F cells. Additionally, reversal of the laminin gradient in the chambers also stimulated RN22F migration in a concentration-dependent manner, suggesting that directed migration of RN22F cells was due to a substratum-bound laminin (haptotaxis) as opposed to cell movement in response to fluid-phase laminin (chemotaxis). Binding studies using [3H]laminin demonstrated that laminin bound to the filter surface under the assay conditions used, and support the contention that cells are migrating to substrate-bound material. Furthermore, RN22F cells were shown to migrate on filters coated with laminin in the absence of additional fluid-phase laminin. The magnitude of this response could be altered by changing the relative density of bound laminin. In contrast, fibronectin promoted only marginal migration of RN22F cells. Collectively, these observations indicate that haptotaxis may be a mechanism by which laminin may guide cells during development and raise the possibility that it may be involved in peripheral nervous system myelination.
PMCID: PMC2112555  PMID: 6885918
21.  Endothelial Cell Laminin Isoforms, Laminins 8 and 10, Play Decisive Roles in T Cell Recruitment across the Blood–Brain Barrier in Experimental Autoimmune Encephalomyelitis 
The Journal of Cell Biology  2001;153(5):933-946.
An active involvement of blood–brain barrier endothelial cell basement membranes in development of inflammatory lesions in the central nervous system (CNS) has not been considered to date. Here we investigated the molecular composition and possible function of the extracellular matrix encountered by extravasating T lymphocytes during experimental autoimmune encephalomyelitis (EAE).
Endothelial basement membranes contained laminin 8 (α4β1γ1) and/or 10 (α5β1γ1) and their expression was influenced by proinflammatory cytokines or angiostatic agents. T cells emigrating into the CNS during EAE encountered two biochemically distinct basement membranes, the endothelial (containing laminins 8 and 10) and the parenchymal (containing laminins 1 and 2) basement membranes. However, inflammatory cuffs occurred exclusively around endothelial basement membranes containing laminin 8, whereas in the presence of laminin 10 no infiltration was detectable. In vitro assays using encephalitogenic T cell lines revealed adhesion to laminins 8 and 10, whereas binding to laminins 1 and 2 could not be induced. Downregulation of integrin α6 on cerebral endothelium at sites of T cell infiltration, plus a high turnover of laminin 8 at these sites, suggested two possible roles for laminin 8 in the endothelial basement membrane: one at the level of the endothelial cells resulting in reduced adhesion and, thereby, increased penetrability of the monolayer; and secondly at the level of the T cells providing direct signals to the transmigrating cells.
PMCID: PMC2174323  PMID: 11381080
laminin; experimental autoimmune encephalomyelitis; endothelium; basement membranes; inflammation
22.  Laminin 411 acts as a potent inducer of umbilical cord mesenchymal stem cell differentiation into insulin-producing cells 
Diabetes mellitus (DM) is an incurable metabolic disease constituting a major threat to human health. Insulin-producing cells (IPCs) differentiated from mesenchymal stem cells (MSCs) hold great promise in the treatment of DM. The development of an efficient IPC induction system is a crucial step for the clinical application of IPCs for DM. Laminin 411 is a key component of the basement membrane and is involved in the regulation of cell differentiation; however, little is known about a role of laminin 411 in the regulation of IPC differentiation from human MSCs.
MSCs were isolated from human umbilical cord (UC-MSCs) and expanded in an in vitro culture system. UC-MSCs were then cultured in the IPC induction and differentiation medium in the presence of laminin 411. Flow cytometry, Quantitative realtime PCR, immunofluorescence staining, ELISA, Western blotting and other techniques were applied to determine IPC generation, insulin expression and related mechanisms. To evaluate potential therapeutic efficacy of IPCs induced from UC-MSCs, a type-1 diabetes (T1DM) rat model was generated using streptozotocin. Blood glucose, insulin levels, and survival of rats were monitored periodically following intravenous injection of the tested cells.
Laminin 411 markedly induced the expression of the genes Foxa2 and Sox17, markers for pancreatic precursor cells, efficiently induced IPC differentiation from MSCs, and up-regulated insulin expression at both mRNA and protein levels. Furthermore, the expression of the genes known to govern insulin expression including Pdx1 and Ngn3 was markedly induced by laminin 411, which suggests that Pdx1 and Ngn3 signaling pathways are involved in laminin 411 induced-insulin expression machinery. More importantly, administration of laminin 411-induced IPCs rapidly and significantly down-regulated fasting blood glucose levels, significantly reduced the HbA1c concentration and markedly improved the symptoms and survival of T1DM rats.
Our results demonstrate that laminin 411 acts as a potent differentiation inducer of IPCs from UC-MSCs via the Pdx1 and Ngn3 signaling pathways. Moreover, transfusion of laminin 411 induced-IPCs more efficiently improves symptoms and survival of T1DM rats. These novel finding highlights a potential clinical application of laminin 411 induced-IPCs in the treatment of T1DM, which calls for further studies.
PMCID: PMC4040110  PMID: 24885418
Laminin 411; Mesenchymal stem cell; Insulin-producing cell
23.  Laminin121 – Recombinant expression and interactions with integrins 
Laminin-121, previously referred as to laminin-3, was expressed recombinantly in human embryonic kidney (HEK) 293 cells by triple transfection of full-length cDNAs encoding mouse laminin α1, β2 and γ1 chains. The recombinant laminin-121 was purified using Heparin-Sepharose followed by molecular sieve chromatography and shown to be correctly folded by electron microscopy and circular dichroism (CD). The CD spectra of recombinant laminin-121 were very similar to those of laminin-111 isolated from Engelbreth-Holm Swarm tumor (EHS-laminin) but its Tm value was smaller than EHS-laminin and recombinant lamnin-111 suggesting that the replacement of the β chain reduced the stability of the coiled-coil structure of laminin-121. Its binding to integrins was compared with EHS-laminin, laminin-3A32 purified from murine epidermal cell line and recombinantly expressed laminins-111, -211 and -221. Laminin-121 showed the highest affinity to α6β1 and α7β1 integrins and furthermore, laminin-121 most effectively supported neurite outgrowth. Together, this suggests that the β2 laminins have higher affinity for integrins than the β1 laminins.
PMCID: PMC2939186  PMID: 20566382
extracellular matrix; basement membrane; laminin; recombinant expression; integrin; neurite outgrowth
24.  Connective Tissue Growth Factor (CTGF/CCN2) Is Negatively Regulated during Neuron-Glioblastoma Interaction 
PLoS ONE  2013;8(1):e55605.
Connective-tissue growth factor (CTGF/CCN2) is a matricellular-secreted protein involved in complex processes such as wound healing, angiogenesis, fibrosis and metastasis, in the regulation of cell proliferation, migration and extracellular matrix remodeling. Glioblastoma (GBM) is the major malignant primary brain tumor and its adaptation to the central nervous system microenvironment requires the production and remodeling of the extracellular matrix. Previously, we published an in vitro approach to test if neurons can influence the expression of the GBM extracellular matrix. We demonstrated that neurons remodeled glioma cell laminin. The present study shows that neurons are also able to modulate CTGF expression in GBM. CTGF immnoreactivity and mRNA levels in GBM cells are dramatically decreased when these cells are co-cultured with neonatal neurons. As proof of particular neuron effects, neonatal neurons co-cultured onto GBM cells also inhibit the reporter luciferase activity under control of the CTGF promoter, suggesting inhibition at the transcription level. This inhibition seems to be contact-mediated, since conditioned media from embryonic or neonatal neurons do not affect CTGF expression in GBM cells. Furthermore, the inhibition of CTGF expression in GBM/neuronal co-cultures seems to affect the two main signaling pathways related to CTGF. We observed inhibition of TGFβ luciferase reporter assay; however phopho-SMAD2 levels did not change in these co-cultures. In addition levels of phospho-p44/42 MAPK were decreased in co-cultured GBM cells. Finally, in transwell migration assay, CTGF siRNA transfected GBM cells or GBM cells co-cultured with neurons showed a decrease in the migration rate compared to controls. Previous data regarding laminin and these results demonstrating that CTGF is down-regulated in GBM cells co-cultured with neonatal neurons points out an interesting view in the understanding of the tumor and cerebral microenvironment interactions and could open up new strategies as well as suggest a new target in GBM control.
PMCID: PMC3561339  PMID: 23383241
25.  A Kinome-Wide RNAi Screen in Drosophila Glia Reveals That the RIO Kinases Mediate Cell Proliferation and Survival through TORC2-Akt Signaling in Glioblastoma 
PLoS Genetics  2013;9(2):e1003253.
Glioblastoma, the most common primary malignant brain tumor, is incurable with current therapies. Genetic and molecular analyses demonstrate that glioblastomas frequently display mutations that activate receptor tyrosine kinase (RTK) and Pi-3 kinase (PI3K) signaling pathways. In Drosophila melanogaster, activation of RTK and PI3K pathways in glial progenitor cells creates malignant neoplastic glial tumors that display many features of human glioblastoma. In both human and Drosophila, activation of the RTK and PI3K pathways stimulates Akt signaling along with other as-yet-unknown changes that drive oncogenesis. We used this Drosophila glioblastoma model to perform a kinome-wide genetic screen for new genes required for RTK- and PI3K-dependent neoplastic transformation. Human orthologs of novel kinases uncovered by these screens were functionally assessed in mammalian glioblastoma models and human tumors. Our results revealed that the atypical kinases RIOK1 and RIOK2 are overexpressed in glioblastoma cells in an Akt-dependent manner. Moreover, we found that overexpressed RIOK2 formed a complex with RIOK1, mTor, and mTor-complex-2 components, and that overexpressed RIOK2 upregulated Akt signaling and promoted tumorigenesis in murine astrocytes. Conversely, reduced expression of RIOK1 or RIOK2 disrupted Akt signaling and caused cell cycle exit, apoptosis, and chemosensitivity in glioblastoma cells by inducing p53 activity through the RpL11-dependent ribosomal stress checkpoint. These results imply that, in glioblastoma cells, constitutive Akt signaling drives RIO kinase overexpression, which creates a feedforward loop that promotes and maintains oncogenic Akt activity through stimulation of mTor signaling. Further study of the RIO kinases as well as other kinases identified in our Drosophila screen may reveal new insights into defects underlying glioblastoma and related cancers and may reveal new therapeutic opportunities for these cancers.
Author Summary
Glioblastomas, the most common primary brain tumor, harbor mutations in receptor tyrosine kinases (RTKs), such as EGFR, and components of the Pi-3 kinase (PI3K) signaling pathway. However, the genes that act downstream of RTK and PI3K signaling to drive glioblastoma remain unclear. To investigate the genetic and molecular basis of this disease, we created a glioblastoma model in the fruit fly Drosophila melanogaster. To identify new genes involved in glioblastoma development, we performed a screen for the genes required for tumor cell proliferation using our Drosophila glioblastoma model and then functionally assessed the activity of human versions of novel genes identified in this screen. Our results revealed that the RIO kinases become overexpressed in human glioblastomas but not in normal human glial or neuronal cells. We found that overexpression of the RIO kinases promotes and maintains signals that drive tumor cell proliferation and survival in RTK- and PI3K-dependent human glioblastoma, and reduction of RIO kinase expression decreased proliferation and prompted cell death and chemosensitivity in glioblastoma cells. Therefore, disruption of the RIO kinases may provide new therapeutic opportunities to target glioblastoma and other RTK- or PI3K-dependent cancers.
PMCID: PMC3573097  PMID: 23459592

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